Professional Documents
Culture Documents
Edited by
NR Jagannathan
Professor and Head
Department of NMR
All India Institute of Medical Sciences
New Delhi, India
Foreword
Richard R Ernst
Nobel Laureate
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Ackerstaff E Bhujwalla ZM
Department of Radiology Department of Radiology
Johns Hopkins University School of Medicine Johns Hopkins University School of Medicine
Baltimore, USA Baltimore, USA
Baert R Bolan PJ
Institute for Biodiagnostics Centre for Magnetic Resonance Research
National Research Council of Canada University of Minnesota Medical School
Winnipeg, Canada Minneapolis, USA
Barker PB Carmichael D
Department of Radiology Department of Obstetrics and Gynaecology
Johns Hopkins University School of Medicine
University College London
Baltimore, USA
London, UK
Becker ED
Chung HK
National Institutes of Health
G. E. Healthcare Technologies
Bethesda, USA
Waukesha, USA
Bendahan D
Centre de Résonance Magnétique Chung YL
Biologique et Médicale Department of Basic Medical Sciences
Faculté de Médecine de Marseille St. George’s Hospital Medical School
Marseille, France London, UK
Bezabeh T Cozzone PJ
Institute for Biodiagnostics Centre de Résonance Magnétique
National Research Council of Canada Biologique et Médicale
Winnipeg, Canada Faculté de Médecine de Marseille
Marseille, France
Bhandari M
Department of Urology Crawford TO
Sanjay Gandhi Post-Graduate Department of Neurology
Institute of Medical Sciences Johns Hopkins University School of Medicine
Lucknow, India Baltimore, USA
viii Biomedical Magnetic Resonance: Proceedings of the International Workshop
Damon BM Gambini A
Vanderbilt University The Kennedy Kreiger Institute
Institute of Imaging Science Johns Hopkins University School of Medicine
Nashville, Tennessee Baltimore, USA
De Bruhl N Garwood M
Department of Radiological Sciences Centre for Magnetic Resonance Research
David Geffen School of Medicine at UCLA University of Minnesota Medical School
Los Angeles, USA Minneapolis, USA
DelaBarre L Gillies RJ
Centre for Magnetic Resonance Research Arizona Cancer Centre
University of Minnesota Medical School Tucson, USA
Minneapolis, USA
Glunde K
Devasahayam N Department of Radiology
Centre for Cancer Research Johns Hopkins University School of Medicine
National Cancer Institute, NIH Baltimore, USA
Bethesda, USA
Gore JC
Ellermann J Vanderbilt University
Department of Radiology Institute of Imaging Science
University of Minnesota Medical School Nashville, Tennessee
Minneapolis, USA
Govil G
Emory TH Tata Institute of Fundamental Research
Department of Radiology Mumbai, India
University of Minnesota Medical School
Minneapolis, USA Gowda NGA
Centre of Biomedical Magnetic Resonance
Everson LI Sanjay Gandhi Post-Graduate
Department of Radiology Institute of Medical Sciences
University of Minnesota Medical School Lucknow, India
Minneapolis, USA
Griffiths JR
Fatemi SA Department of Basic Medical Sciences
The Kennedy Kreiger Institute St. George’s Hospital Medical School
Johns Hopkins University School of Medicine London, UK
Baltimore, USA
Grodd W
Gadian DG Radiology Clinic
Radiology and Physics Unit Abteilung Fur Neuroradiologie
Institute of Child Health, University College University of Tuebingen
London, UK Tuebingen, Germany
Contributors ix
Gulbahce E Khetrapal CL
Department of Pathology and Centre of Biomedical Magnetic Resonance
Laboratory Medicine Sanjay Gandhi Post-Graduate
University of Minnesota Medical School Institute of Medical Sciences
Minneapolis, USA Lucknow, India
Gullapalli RP Kochupillai N
Department of Radiology All India Institute of Medical Sciences (Rtd.)
University of Maryland Medical System New Delhi, India
Baltimore, USA
Komoroski RA
Gupta A Department of Radiology
Centre of Biomedical Magnetic Resonance University of Arkansas for Medical Sciences
Sanjay Gandhi Post-Graduate Little Rock, USA
Institute of Medical Sciences
Lucknow, India Kossoff EH
Department of Neurology
Han S Johns Hopkins University School of Medicine
Department of Hepatology Baltimore, USA
David Geffen School of Medicine at UCLA
Los Angeles, USA Krishna MC
Centre for Cancer Research
Hennig J
National Cancer Institute
Department of Diagnostic Radiology
National Institutes of Health
University Hospital Freiburg
Bethesda, USA
Freiburg, Germany
Kumar A
Horska A
Department of Psychiatry
Department of Radiology
David Geffen School of Medicine at UCLA
Johns Hopkins University School of Medicine
Los Angeles, USA
Baltimore,USA
Huda A Lean CL
Department of Physics Institute for Magnetic Resonance Research,
California State University St Leonards, NSW
Fresno, USA Australia
Jagannathan NR Lindquist DM
Department of NMR Department of Radiology
All India Institute of Medical Sciences University of Arkansas for Medical Sciences
New Delhi, India Little Rock, USA
x Biomedical Magnetic Resonance: Proceedings of the International Workshop
Mathur R Nandini V
Division of Radiopharmaceuticals Department of Anaesthesiology
and Radiation Biology St Johns Medical College
Institute of Nuclear Medicine Bangalore, India
and Allied Sciences, Delhi, India
Nelson MT
Matsumoto K Department of Radiology
Centre for Cancer Research University of Minnesota Medical School
National Cancer Institute, NIH Minneapolis, USA
Bethesda, USA
Ordidge RJ
McIntosh A Department of Medical Physics and
Centre for Magnetic Resonance Research Bioengineering
University of Minnesota Medical School University College London
Minneapolis, USA London, UK
Meisamy S Pathak AP
Centre for Magnetic Resonance Research Department of Radiology
University of Minnesota Medical School Johns Hopkins University School of Medicine
Minneapolis, USA Baltimore, USA
Mishra AK Peebles D
Division of Radiopharmaceuticals Department of Obstetrics and Gynaecology
and Radiation Biology University College London
Institute of Nuclear Medicine London, UK
and Allied Sciences, Delhi, India
Raghunand N
Mitchell JB Arizona Cancer Centre
Centre for Cancer Research Tucson, USA
National Cancer Institute, NIH
Bethesda, USA Raghunathan P
Fluorosis Research Foundation
New Delhi, India
Mountford CE
Department of Magnetic Resonance in Medicine
University of Sydney, Sydney and Raman V
Institute for Magnetic Resonance Research, Department of Radiology
St Leonard’s, NSW, Australia Johns Hopkins University School of Medicine
Baltimore, USA
Murugesan R
Centre for Cancer Research Röll S
National Cancer Institute, NIH Siemens Medical Solutions
Bethesda, USA Erlangen, Germany
Contributors xi
Russel P Snyder CJ
Institute for Magnetic Resonance Research, Centre for Magnetic Resonance Research
St Leonards, NSW University of Minnesota Medical School
Australia Minneapolis, USA
Saini RS Solaiyappan M
Medical Solutions Division Department of Radiology
Siemens Ltd Johns Hopkins University School of Medicine
New Delhi, India Baltimore, USA
Schenker KV Styczynski A
MRI Division Centre for Magnetic Resonance Research
Bruker Bio-Spin, AG University of Minnesota Medical School
Faellanden, Switzerland Minneapolis, USA
Sharma R Subramanian S
Department of Radiodiagnosis Centre for Cancer Research
All India Institute of Medical Sciences
National Cancer Institute, NIH
New Delhi, India
Bethesda, USA
Sharma RK
Thomas MA
Division of Radiopharmaceuticals
Department of Radiological Sciences
and Radiation Biology
David Geffen School of Medicine at UCLA
Institute of Nuclear Medicine
Los Angeles, USA
and Allied Sciences
Delhi, India
Tuttle TM
Sharma U Department of Surgery
Department of NMR University of Minnesota Medical School
All India Institute of Medical Sciences Minneapolis, USA
New Delhi, India
Van Zijl PCM
Smith ICP Department of Radiology
Institute for Biodiagnostics Johns Hopkins University School of Medicine
National Research Council of Canada Baltimore, USA
Winnipeg, Canada
Smith M Vaughan JT
Department of Radiology Centre for Magnetic Resonance Research
Johns Hopkins University School of Medicine University of Minnesota Medical School
Baltimore, USA Minneapolis, USA
xii Biomedical Magnetic Resonance: Proceedings of the International Workshop
Wang X Zhou J
Department of Obstetrics and Gynaecology Department of Radiology
University College London Johns Hopkins University School of Medicine
London, UK Baltimore, USA
Yee D
Department of Medicine Zhou J
University of Minnesota Medical School Department of Radiology
Minneapolis, USA University of Maryland Medical System
Baltimore, USA
Yue K
Department of Medicine
University of Hawaii
Honolulu, USA
Foreword
What an exciting International Workshop that has been organized by Professor N.R.
Jagannathan! More and more, magnetic resonance imaging (MRI) is becoming the most
versatile and most informative diagnostic tool of medical research and clinical practice. It
was a long pathway from the original conception of magnetic resonance in nuclear
physics in the forties of last century to today’s perfection of MRI in our most advanced
medical institutions, such as the All India Institute of Medical Sciences. Both,
sensitivity and inherent information contents were originally quite meager; and none of
the pioneers could conceive the incredible development that took place during the past 60
years.
Magnetic resonance, or NMR (Nuclear Magnetic Resonance) as it is called by more
knowledgeable scientists, owes its power to the omnipresence of nuclear spins in virtually
all living and dead materials. These nuclear spins serve as natural ‘spies’ for reporting on
their immediate environment. They are interacting very weakly only with the surrounding
material. Thus, they remain ‘unseen’ but are capable nevertheless of gathering most valuable
information. Much technological advancement and sophistication was necessary for reliably
recording the feeble spies’ messages. In addition, advanced digital data processing and
artificial intelligence were required for their interpretation.
Today, NMR is indispensable in chemical and biomolecular research for visualizing
molecular structures, interactions, and processes. Today, MRI is a mature tool in the hands
of clinicians and medical researchers. The teething problems are largely overcome, although
further development and improvements are still needed and are indeed quite feasible.
The involvement of many of the most capable scientists in the instrumental, chemical,
biological, and medical fields gives us hope for further progress.
We scientists, responsible for the development of these marvelous tools, are quite
proud of ‘our’ immortal achievements. Perhaps, we do not realize that we have just been
extremely lucky in our endeavors. Quite accidentally, we found a golden egg, while other
compatriots working extremely hard gain very little compensation, recognition, or joy.
The Indian cities provide plentiful of speaking examples of such tragic injustice.
Many of us might just shrug their shoulders, being used to what they might call
‘eternal fate’. Indeed, we have developed handy philosophies that allow us to endure the
conflicts, to calm our conscience, and to continue our daily profitable business.
xiv Biomedical Magnetic Resonance: Proceedings of the International Workshop
Richard R Ernst
Nobel Laureate
Laboratorium für Physikalische Chemie
ETH Hönggerberg HCI
8093 Zürich, Switzerland
Preface
This volume consists of contributions from the invited speakers of the “International Workshop
on Biomedical MR” organized by the Department of NMR, All India Institute of Medical
Sciences, New Delhi from January 12-15, 2005 as a satellite event of the International
Conference on Magnetic Resonance in Biological Systems (ICMRBS). The workshop covered
exploding areas of the applications of magnetic resonance techniques in biomedicine.
Clinical imaging has shown faster advancement during the last two decades and especially
the field of MR continues to progress at an impressive speed that requires periodic up-
date. The organizers of the ICMRBS felt that it would be ideal to organize a workshop
on biomedical applications of MR in this part of the world where the use of MRI is still
at its infancy.
The workshop addressed various aspects of MRI and MRS starting from the fundamental
physics to the latest advances. Several eminent scientists including Prof. Richard R Ernst,
Nobel Laureate, delivered lectures in the workshop. Gathering of experts from various
countries like Australia, Canada, France, Germany, India, Switzerland, UK and USA enabled
the participants of the workshop to interact and exchange freely their scientific ideas. In
addition, we had participants from Bangladesh, Canada, Israel, Nepal, Russia, Singapore
and USA.
This volume contains twenty-nine chapters and is organized in such a fashion so as to
maintain continuity of thought process with several illustrations. The authors have tried
to make complex methodologies understandable and interesting to knowledgeable readers
who may not be specialists. In particular, it is hoped that this volume will prove valuable
to graduate students and young scientists who plan to enter the fascinating area of
biomedical magnetic resonance.
The organizers would like to thank the Director and Dean of the All India Institute of
Medical Sciences. The Department of Science and Technology and the Department of
Biotechnology of the Government of India, the Indian National Science Academy,
International Union of Pure and Applied Biophysics (IUPAB), International Society for
Magnetic Resonance in Medicine (ISMRM), National Magnetic Resonance Society of India
(NMRS), Indian Biophysical Society (IBS), Siemens Ltd., Bruker Spectro-Spin, and German
Remedies, Kodak India Ltd., Varian Inc., USA and Amersham Health are thanked for
financial support to the workshop. I acknowledge the help and assistance rendered by my
colleagues and staff, in particular Mr Virendra Kumar, Mr KA Danishad, and Dr Uma
Sharma in the preparation of this volume.
Above all, I thank all the speakers who contributed enthusiastically their lecture notes
in the form of an article. This volume has emerged only out of their enthusiasm and
cooperation. The efficiency of the publishers Jaypee Brothers Medical Publishers (P) Ltd.,
and their cooperation is greatly acknowledged. Last, but not least, I would like to thank
my parents and my family for their patience, understanding, and encouragement.
NR Jagannathan
Sponsors
1
Historical Perspectives of MRI
Edwin D Becker
*MRI is also widely employed in such areas as plant physiology, food technology, and materials research.
This presentation does not delve into these applications.
2 Biomedical Magnetic Resonance: Proceedings of the International Workshop
evacuated container could be deflected by moment of the lithium nucleus, and the RF.
an inhomogeneous magnetic field. 2 The Rabi received the Nobel Prize in 1944 for
beam split into two components, depending the discovery of NMR.
on whether the nuclear magnetic moments The NMR in molecular beams provided
of the hydrogen nuclei were oriented physicists with an elegant method to
parallel or antiparallel to the magnetic field. measure nuclear magnetic moments with
Stern’s molecular beam technique was able much higher precision than could be
to provide estimates of the magnitude of obtained previously. However, the practical
nuclear magnetic moments and spawned use of NMR really stems from its first
increasingly more elegant experiments, observation in bulk materials—solids, liquids
particularly in the laboratory of II Rabi at and even gases at normal pressures. The
Columbia University. In seeking ways to physicist CJ Gorter had made two
improve the precision of the measurement, unsuccessful attempts to observe NMR in
Rabi was led to the idea that in a magnetic bulk materials (1937 and 1943),4 and it was
field a nucleus with spin orientation parallel not clear that the weak NMR signals could
to the field would exist in a different energy be detected. The signal from a molecular
level from one with antiparallel orientation, beam arises from all nuclei in the beam; but
and that absorption of a quantum of energy in bulk materials, the theory of absorption
at precisely the correct frequency could and induced emission makes it clear that
cause nuclei to “flip” from one orientation the signal would come from only the
to the other. The energy v needed to satisfy difference in the populations in the two
the Bohr relation energy levels—about 1 in 100,000. However,
just after the World War II two groups
hν = E2 – E1 = (γ/2π)B0 ...(1)
succeeded in detecting NMR. On December
turns out to be in the radiofrequency (RF) 15, 1945, Purcell, Torrey and Pound (MIT/
range, generally 1 to 900 megahertz (MHz), Harvard) found the resonance of hydrogen
depending on the magnetic field strength. In nuclei in a large sample of paraffin wax in
equation 1, h is Planck’s constant, Ei are the a waveguide cavity.5 About a month later,
magnetic energy levels, γ is the nuclear Bloch, Hansen and Packard (Stanford) used
magnetic moment divided by the nuclear a different experimental approach to find
spin quantum number, and B0 is the strength the resonance in a small sample of liquid
of the applied magnetic field. In December water. 6 These experiments, carried out
1937, Rabi and co-workers subjected a beam independently and concurrently, were
of LiCl molecules to an RF of 3.518 MHz as published in January 1946 and earned Bloch
and Purcell the Nobel Prize in 1952. Purcell’s
the molecules passed through a static
address on this occasion presaged the
magnetic field. They varied the strength of
myriad applications that would be found
the magnetic field and found that at one for NMR:7
particular value the beam was deflected and “I remember in the winter of our first
its intensity on the detector abruptly experiments, just seven years ago, looking
dropped.3 This was the first observation of on snow with new eyes. There the snow
nuclear magnetic resonance (NMR), the term lay around my doorstep—great heaps of
“resonance” describing the very precise protons quietly precessing in the earth’s
relation in equation 1 that had to be fulfilled magnetic field. To see the world for a
among the magnetic field, the magnetic moment as something rich and strange is
Historical Perspectives of MRI 3
molecules are much lower than the values gave insights into physiological function.
observed for free water, probably, the result Surface coils, however, were limited to
of restricted and anisotropic motion of studies of tissue near the surface, not deep
water molecules in the vicinity of biological within the body. The need for better
macromolecules. localization methods for NMR research and
In 1971, Raymond Damadian made an the potential for using NMR information
important discovery that would have long- obtained in the living human body for
term consequences.8 With the background medical diagnosis provided the impetus for
of observations of cellular water, Damadian development of new techniques.
anticipated possible differences in NMR
relaxation times for water in normal cells INVENTION OF NMR IMAGING
and cancer cells. He measured T1 and T2 in
The breakthrough came in 1971, with Paul
pieces of tissue excised from normal rats
Lauterbur’s conception of the use of linear
and from fast-growing tumors that had been
magnetic field gradients to localize NMR
implanted into other rats, and found that
signal information along one dimension and
each of the eight tumor samples had longer
to construct a two- or three-dimensional
relaxation times than any of the normal
image from a collection of such one-dimen-
samples. However, a number of further
sional data sets.9 Lauterbur recognized that
studies in several laboratories showed that
the basic resonance equation (1) would be
with slow-growing mouse tumors and with
modified when a magnetic field gradient
excised human tissues the distinction
Gx is applied along direction x:
between normal and malignant tissue was
not so clear-cut. Although tumors tended hν = (γ/2π)B0(1+Gxx) ...(2)
to have longer relaxation times (partly, at The resonance frequency thus provides a
least, because of higher water content), there measure of the location of the signal along
was such a wide variability that relaxation the x direction for a non-uniform macro-
measurements could not be used as a scopic object such as an animal or a human.
definitive method to distinguish among Lauterbur first demonstrated the application
tissues that are normal, malignant, or of this idea a year later, using a commercial
pathological but not malignant. Never- NMR spectrometer (Varian Model A-60) and
theless, the attention of a number of NMR a sample consisting of two capillaries filled
investigators had been drawn to the with water inside a 5 mm diameter sample
possibility of using this method in medical tube filled with D2O. He used combinations
diagnosis. of the normal x and y shim coils to generate
Meanwhile, in a number of laboratories, magnetic field gradients in various direc-
attention was being given to NMR studies tions in the xy plane, then applied a “back
within a living animal—initially worms and projection” method to generate a 2D image
a rat tail, which fit into standard NMR tubes, from these several data sets. These results
but later to small animals that could be were published in a short paper in Nature
accommodated in a large bore magnet. Little early in 1973.10 His paper emphasized the
useful information could be obtained from a basic physics and the key role of the inter-
spectrum of the entire animal, but localization action of the static and RF fields. To reflect
by means of surface coils permitted spectra the importance of these two factors, he
to acquired, particularly with 31P NMR, that coined the name zeugmatography from the
Historical Perspectives of MRI 5
Greek word zeugma, “that which is used for Peter Mansfield.11 Mansfield and his co-
joining.” He showed that the experimental workers were exploring high resolution
image could be made dependent on relaxa- NMR in solids. In 1972, Mansfield devised
tion times, and he correctly forecast the a method using pulsed magnetic field
future wide applicability of the technique: gradients to create a diffraction pattern that
“The variations in water contents and could provide information on atomic sepa-
rations in crystals. The first demonstrations
proton relaxation times among biological
of the feasibility of the concept were given
tissues should permit the generation, with
in a paper in the Journal of Physics in 197312
field gradients, large compared to internal
and at a Colloque AMPERE in Krakow,
magnetic inhomogeneities, of useful zeugma-
Poland in September 1973. There Mansfield
tographic images from the rather sharp
learned of Lauterbur’s initial imaging paper
water resonances of organisms, selectively
and recognized that his method could also
picturing the various soft structures and
be used for imaging of general macroscopic
tissues. A possible application of consider-
objects, not just crystalline solids. Mans-
able interest at this time would be to the in
field’s recollection of his initial reaction to
vivo study of malignant tumours, which have
Lauterbur’s paper is interesting:11
been shown to give proton nuclear magnetic
“I was struck by the stark contrast
resonance signals with much longer water
between our two approaches to imaging. In
spin-lattice relaxation times than those in
my pulsed approach, I had formally pro-
corresponding normal tissues.”
posed the optical analogy of plane-wave
“The basic zeugmatographic principle
scattering through a mathematical frame-
may be employed in many different ways,
work where reciprocal lattice space or k-
using a scanning technique, as described
space was introduced and in which the k
above, or transient methods. Variations on
vector was proportional to the product of
the experiment, to be described later, permit time and gradient vector. The concept of k-
the generation of two- or three-dimensional space in MRI, of course, plays an important
images displaying chemical compositions, role these days in categorizing different
diffusion coefficients and other properties imaging sequences in terms of the k-space
of objects measurable by spectroscopic trajectories.”
techniques. Although applications employing Mansfield’s laboratory soon showed how
nuclear magnetic resonance in liquid or a magnetic field gradient and frequency-
liquid-like systems are simple and attractive selective pulses could be used to select
because of the ease with which field gradi- specific slices in a three-dimensional object
ents large enough to shift narrow resonances to permit 2D imaging of each slice; and later,
by many line widths may be generated, he developed the technique of echo-planar
NMR zeugmatography of solids, electron imaging, which provides images much more
spin resonance zeugmatography, and analo- rapidly than the conventional methods.
gous experiments in other regions of the Lauterbur and Mansfield shared the Nobel
spectrum should also be possible. Zeugmato- Prize in 2003 for their independent develop-
graphic techniques should find many useful ment of MRI.
applications in studies of the internal
structures, states, and compositions of micro-
OTHER EARLY APPROACHES TO
scopic objects.”
NMR IMAGING
A very different approach to MRI was
initiated concurrently and independently by Lauterbur’s initial paper did not attract wide
6 Biomedical Magnetic Resonance: Proceedings of the International Workshop
attention in the NMR community, but his 1974, at the meeting of the International
presentations at two NMR conferences had Society of Magnetic Resonance (ISMAR) in
dramatic effects. Bombay (now Mumbai). Two members
of the audience from the University of
2D FT Method Nottingham—Waldo Hinshaw and William
At the Experimental NMR Conference Moore—were inspired to begin a new app-
(ENC) in Raleigh, North Carolina, in April, roach as soon as they returned home. They
1974, Richard Ernst heard Lauterbur’s talk developed a simple method that was easy
on zeugmatographic imaging and imme- to implement with equipment then avai-
diately recognized that the two-dimensional lable, the sensitive point method. They
NMR method he was beginning to develop applied oscillating magnetic field gradients
could be used with switched magnetic field in the x, y and z directions concurrently
gradients to obtain images. In Mansfield’s across the sample, so that only the sample
terminology, Ernst’s method scanned k- in a very small volume remains in resonance,
space in a different and more efficient hence contributes to the imaging signal. By
manner than Lauterbur’s initial back projec- altering the electrical parameters of the coils
tion method. Shortly thereafter, Kumar,
used to create the gradients, the sensitive
Welti and Ernst13 obtained an image, again
point could be moved through the sample
of two tubes of water, with repetitive
experiments using an x gradient that is and eventually produce a 3D image. 15
stepped through successive time periods Within a few months, this simple method
after the exciting RF pulse is applied (so- had been developed to produce images of
called phase encoding), while the y gradient small objects such as fruits and vegetables.
is applied during data acquisition (frequency The problem is that a signal is generated
encoding). As this 2D imaging method began from only one point at a time, a very
to be used in more sophisticated practical inefficient procedure that requires a lengthy
applications, it became clear that distortions period for data acquisition for a large
could be introduced with the variable time sample. Improvements were made to
periods for the phase encoding step. This
generate a sensitive line, rather than a point,
artifact was overcome by Edelstein, Hutchi-
and this method was used in early human
son, Johnson and Red path14 by using a
constant time but variable strength field imaging, as described later, but this techni-
gradient for phase encoding. With this so- que was never developed extensively in
called spin warp refinement, the basic commercial application.
Lauterbur concept, implemented in Ernst’s
2D manner, has become the standard FONAR
method for MRI diagnostic imaging. A somewhat similar method that relied on
obtaining signal from only one small portion
Sensitive Point Method
of sample at a time was developed by
Other approaches to NMR imaging, deve- Raymond Damadian. Following his obser-
loped in the mid-1970s, were initially useful; vation that relaxation times in excised tissue
but ultimately, proved to be inefficient for from some tumors were longer than those
most practical imaging applications. One in normal tissue, Damadian began to think
arose from a talk by Lauterbur in January about obtaining NMR information from
Historical Perspectives of MRI 7
within a human body. In a patent application to scan a macroscopic volume, and the
in 1972 that covered his work on excised magnet must be large enough to accommo-
tissue, he also speculated on a large in vivo date this movement.
NMR system in which the RF could some- In spite of these disadvantages, Dama-
how be focused into a narrow beam:16 dian and co-workers were able to construct
“In Figure 2 (not shown here) an electro- a magnet for animal studies and obtain an
magnet shown in cross-section is designed image of the thoracic cavity of a mouse in
to have sufficiently large dimensions to hold 1976, with an estimated resolution of 3 mm.17
They also showed the image of a mouse in
a mammal or human being to be examined.
which a tumor had been implanted in the
A transmitter probe is provided with a beam
upper thorax. By 1977, they had constructed
focusing mechanism for focusing the radiated a 0.05 T magnet of 53 inch diameter, with
magnetic energy from the radiofrequency an NMR frequency of 2 MHz, and were
generator into a beam having a narrow cross- able to demonstrate an image of a human
section. This probe is slidably mounted on a chest.18 With the very low field and point-
helical track and positioned so that the by-point method, the signal/noise was poor
radiated beam is orthogonal to the direction and it took several hours to complete a scan.
of the field provided by the electromagnet. The resolution was poor; but major features,
The transmitter probe is moved on the track such as the heart and lungs, could be
by means not shown so that the probe may identified. Damadian pursued this method
scan the entire body to be examined. Also with the establishment of the FONAR
mounted on the track is a receiver probe of Corporation, which produced a few instru-
the same design as the transmitter probe ments based on the FONAR method.
However, by 1981, the FONAR Corporation
which detects a beam having the same cross-
joined other companies in adopting the
sectional width as the beam radiated by the
Lauterbur field gradient principle as
probe...” embodied in the Ernst 2D method with spin
Focusing RF of several meter wavelength warp modification.
into a narrow beam flies in the face of well-
known optical principles, Damadian turned DEVELOPMENT OF HUMAN MRI
his attention to the design and construction
of a large, low field magnet where the static The first live human NMR image was
field B0 could be shaped with suitable shim published by Mansfield and Maudsley in
coils to obtain a small homogeneous volume, 1977—a line scan of the internal anatomy of
a finger, an appendage small enough to be
with the homogeneity rapidly falling off
inserted into a standard NMR magnet.19
outside this volume. Damadian called the
Contrast between bone, bone marrow,
method “field focusing NMR” and coined nerve, artery and other tissues was demon-
an acronym FONAR. FONAR is similar to strated. Also, in 1977, Andrew et al20 used
the sensitive point method in obtaining data the multiple sensitive point method and a
inefficiently, from only one small volume at larger gap electromagnet to image a hand,
a time. Moreover, FONAR has the addi- while Hinshaw, Bottomley and Holland
tional disadvantage that the location of the examined Bottomley’s wrist and discussed
homogeneous volume is fixed by the design potential medical uses.21
of the magnet, so the subject must be physi- Serious application to most anatomical
cally moved in three orthogonal directions studies required a magnet capable of holding
8 Biomedical Magnetic Resonance: Proceedings of the International Workshop
a human being. The necessary scale-up from initially at 0.5 T; but by the early 1980s, a
traditional NMR sample tube sizes of 5 to field of 1.5 T (about 60 MHz for proton
20 mm diameter to nearly a one meter bore NMR) became the industry standard. By
for a whole human body was a daunting October, 1987, sufficient clinical data had
task, which could be accomplished only with been obtained for the National Institutes of
a corresponding reduction in the magnitude Health in the United States to convene a
of the magnetic field. Most early efforts Consensus Conference, in which a “jury” of
were directed toward electromagnets with radiologists and other clinical experts could
an air core, designed for a field of about assess the state of the art and make
0.1 T (4 MHz for 1 H NMR), except for recommendations on optimum uses of MRI.
Damadian’s 0.05 T superconducting magnet. The following excerpts from the Conference
In 1978, Mansfield et al observed the first Statement highlight the prevailing views:26
image of the human abdomen,22 and Clow “Even in the short period of its use, it
and Young reported the first NMR image has proved to be unusually rewarding in
of a section through a human head. 23 the detection, localization, and assessment
Features such as the eyeballs and ventricles of extent and character of disease in the
in the brain were clearly visible. By 1980, central nervous, musculoskeletal, and
Moore and his colleagues at Nottingham cardiovascular systems. In the brain, for
were able to demonstrate one advantage of example, it has a proven capacity to define
NMR imaging over X-ray computed tomo- some tumors and the plaques of multiple
graphy (CT) in showing the first sagittal sclerosis provided by no other technique. It
and coronal sections of a human head.24 In is a competing imaging method in the
another paper, the same year they showed evaluation of many other organs. Additional
that NMR images could reveal a wide range prospective studies comparing MRI with
of pathology in the head and commented other diagnostic methods are essential in
on the “dramatic ... new advance in neuro- those areas where the method has shown
radiological capability with its potential promise but where its precise role has not
impact on clinical management.”25 Their yet been defined. This consensus develop-
clinical results provided examples that ment conference does not purport to include
included tumors, aneurysms, circulatory all of the applications of MRI to the pediatric
malformations and chronic sinus infection. patient, a subject that will require separate
As these successful applications became consideration... The full potential of MRI
more widely known, interest accelerated has not been reached, and continuing
among a number of companies in developing refinement of equipment, contrast agents,
MRI instruments commercially for a poten- and software may be anticipated...”
tially very large and lucrative market in The final sentence quoted was prophetic.
medical diagnostic imaging. There is a MRI has become widely available and is
fascinating story—too lengthy to be presen- regarded as an indispensable diagnostic
ted here—of the concerted efforts in several modality. New generations of magnets have
companies in the USA and Europe to rapidly been developed at both higher and lower
convert laboratory prototypes to clinically magnetic fields for specific purposes. Faster
useful instruments. Superconducting mag- scanning methods have become available,
nets with a 1 meter bore were developed and the quality of images has steadily
Historical Perspectives of MRI 9
lines of equal intensity, it was not too effective pulse sequence,30 later improved
difficult to make the mental transform and by Meiboom and Gill (the CPMG sequ-
picture the cw spectrum. However, for ence),31 that formed the basis for making
systems with unequal line separations and reliable measurements of T 2 . Carr and
unequal intensities, the process was more Purcell also introduced the 180°-τ-90° pulse
difficult. A “hot” topic at the time was the sequence to measure T1. These sequences
recently discovered 1H chemical shift in have had enormous impact in MRI, as T1-
ethanol (CH3CH2OH), which displayed a and T2-weighted images play a critical role
readily interpretable cw spectrum—three in diagnosis.
lines of intensities 3:2:1. The corresponding The pulse sequences for diffusion-
FID, however, was a complicated beat weighted images can also be traced back to
pattern. To convince visitors of the equi- the early 1950s when the CPMG sequence
valence of the two responses, Carr made was used to measure diffusion in liquids
what in current NMR imaging would be under the influence of a steady magnetic
called a “phantom”, three pieces of rubber field gradient. A much more effective
of volumes 3:2:1, which were placed in a method was introduced in 1963 when
magnetic field gradient that simulated the Stejskal and Tanner32 showed that a pulsed
chemical shifts in ethanol. The FID obtained field gradient offered independent control
from this phantom agreed with that from of the magnitude and length of the gradient
liquid ethanol itself, thus verifying Carr’s without interfering with the measurement
interpretation. of the spin echo. In fact, the pulsed field
gradient had been introduced originally by
Echoes Hahn and co-workers33 in 1955 to avoid
spurious echoes, an application that survives
Whatever pulse sequence is used to obtain
in imaging methods as crushers and spoiler
an NMR image, a spin-echo is invariably
gradients.
involved in some way. The discovery of Other applications of echoes abound in
the spin echo by Erwin Hahn in 1949 ranks imaging. The stimulated echo from a 90o-τ-
as a milestone in NMR,28 since it introduced 90o-τ-90o pulse sequence was also discovered
a principle of reversibility that might at first by Hahn and now appears in imaging
appear to be physically impossible. The spin- methods. The gradient-recalled echo (GRE) was
echo has proved to be widely applicable in first reported by Herman Carr in his PhD
all sorts of NMR experiments. The physics thesis in 1952.27 Rather than reversing the
of the spin-echo will be covered elsewhere; direction of the gradient to create the echo,
here I comment only on its accidental dis- he rotated the sample in the gradient (the
covery. Hahn was investigating the use of first example of sample spinning). However,
short RF pulses when he observed what he there was little application of the GRE until
calls “a weird signal” that appeared when it was introduced in the form of a pulsed
no pulse was being applied.29 He initially gradient as a key element of imaging
considered it an artifact but persisted in methods.
exploring the phenomenon and soon recogni-
zed that he had observed a refocusing of
CONCLUSION
magnetization that had apparently decayed
to zero. Carr and Purcell investigated the This short chronology of some early deve-
spin echo in detail and developed a more lopments in NMR only scratches the surface
Historical Perspectives of MRI 11
in describing the conceptual advances, the 14. Edelstein WA, Hutchison JMS, Johnson G,
innovative techniques that have been intro- Redpath T. Phys Med Biol 1980; 25: 751.
duced, and the extremely wide range of 15. Hinshaw WS. Phys Lett 1974; 48A: 87.
applications for NMR that have come about 16. Damadian RV. US Patent 3 789 832 (Filed March
in the 60 years since the discovery of NMR 17, 1972, Issued February 5, 1974).
17. Damadian R, Minkoff L, Goldsmith M, Stanford
in bulk materials. Modern MRI and its close
M, Koutcher J. Science 1976; 194: 1430.
relative, localized NMR spectroscopy, are 18. Damadian R, Goldsmith M, Minkoff L. Physiol
feasible only because this whole array of Chem Phys 1977; 9: 97.
technology has now come together to create 19. Mansfield P, Maudsley AA. Br J Radiol 1977;
the powerful methods we now employ. 50: 188.
There is no sign of any abatement in further 20. Andrew ER, Bottomley PA, Hinshaw WS,
rapid advances in this field. Holland GN, Moore WS, Simaroj C. Phys Med
Biol 1977; 22: 971.
21. Hinshaw WS, Bottomley PA, Holland GN.
REFERENCES Nature (London) 1977; 270: 722.
1. Becker ED (Ed) Historical Perspectives, Vol. 1 22. Mansfield P, Pykett IL, Morris PG, Coupland
of Grant DM, Harris RK (Eds) Encyclopedia of RE Br J Radiol 1978; 51: 921.
Nuclear Magnetic Resonance Chichester, 23. Clow H, Young IR. New Scientist 1978; 588.
England: John Wiley and Sons, 1996. 24. Holland GN, Hawkes RC, Moore WS J Comput
2. Estermann I, Stern O. Z Phys 1933; 85:17; Frisch Assist Tomogr 1980; 4: 429.
R, Stern O Z Phys 1933; 85:4. 25. Hawkes RC, Holland GN, Moore WS,
3. Rabi II, Zacharias JR, Millman S, Kusch P. Phys Worthington BS. J Comput Assist Tomogr 1980;
Rev 1938; 53:318. 4: 577.
4. Gorter CJ. Physica (The Hague) 1936; 3: 995; 26. Magnetic Resonance Imaging: National Insti-
Gorter CJ, Broer LJF. Physica (The Hague) 1942; tutes of Health Consensus Development
9: 591. Statement, Office of Medical Applications of
5. Purcell EM, Torrey HC, Pound RV Phys Rev Research, NIH, Bethesda, MD, 1987.
1946; 69: 37. 27. Carr HY PhD Thesis, Harvard University,
6. Bloch F, Hansen WW, Packard M Phys Rev Cambridge, MA, 1952. See also “Early Years of
1946; 70: 474. Free Precession Revisited,” Reference 1, 253-
7. Purcell EM, Les Prix Nobel 1952. 60.
8. Damadian R. Science 1971; 171: 1151.
28. Hahn EL. Phys Rev 1950; 77: 297.
9. Lauterbur PC. “One path out of many—how
29. Hahn EL. “Pulsed NMR—A personal history,”
MRI actually began”. Ref. 1, 445-49.
Reference 1, 373-78.
10. Lauterbur PC. Nature (London) 1973; 242: 190.
30. Carr HY, Purcell EM. Phys Rev 1954; 94: 630.
11. Mansfield P. “A personal view of my involve-
ment in the development of NMR and the 31. Meiboom S, Gill D. Rev Sci Instrum 1958; 29:
conception and development of MRI”. Ref. 1, 688.
478-81. 32. Stejskal EO, Tanner JE. J Chem Phys 1965; 42:
12. Mansfield P, Grannell PK. J Phys C 1973; 6: 288.
L422. 33. Anderson AG, Garwin RL, Hahn EL, Horton
13. Kumar A, Welti D, Ernst RR. J Magn Reson JW, Tucker GL, Walker RM. J Appl Phys 1955;
1975; 18: 69. 26: 1324.
12 Biomedical Magnetic Resonance: Proceedings of the International Workshop
2
Introduction to
Biological NMR Spectroscopy
Girjesh Govil
Soon after its discovery, NMR became an there has been major progress in decoding
indispensable tool in chemical and physical of gene sequences of various organisms,
sciences. For several reasons, its applications sequencing and structure determination of
to life sciences had to wait another three macromolecules such as proteins, carbo-
decades. New techniques in excitation and hydrates and nucleic acids, cellular metabo-
detection, better probe designs, break- lism and understanding of how organs
function. In this brief presentation, I shall
through in electronics, advancement in
show how NMR has made impact on
magnet technology and development in
modern areas of life sciences, focusing on
computer software and hardware, led to topics not covered by other speakers in this
new frontiers in NMR, and this in turn workshop.
opened its applications in biological sciences.
Today, NMR is an indispensable tool in CLASSICAL VIEW OF NMR
structural determination of large biological SPECTROSCOPY
molecules, understanding biochemistry of
cellular systems, imaging and spectroscopy We note that NMR is observed with nuclei
of whole organs (MRI and MRS), clinical having spin (I). In the presence of magnetic
medicine, pharmacology and several other field (B0), the angular momentum of such
areas of life sciences. nuclei causes a precessional motion of their
The foundations of modern biology were magnetic moment (μ) around the z-axis, with
laid during the period 1940-1970. For an angular velocity ω0 = -γB0 (radians/sec).
example, it was established that nucleic acids Under thermal equilibrium, more nuclei are
(DNA and RNA) are carrier of genetic aligned parallel to B0 and there is a net
information. The information coded in the magnetization (Mz) in the z-direction. If we
form of sequence of DNA bases is translated apply an oscillating radiofrequency (RF) field
into specific proteins, which are responsible (B1) in the x-direction, with a frequency equal
for functions of cells. Cells, in turn, are to ω0, then the net magnetization will tend
responsible for the chemistry and functions to flip from the z-direction to the xy plane
of whole organs. During the last 40 years, depending on the strength and duration of
Introduction to Biological NMR Spectroscopy 13
the RF pulse. The flip angle is given by θ = 1000 MHz. One generally uses 1H resonance
γB1t (radians), where B1 is the strength of frequency rather the magnitude of the field
the RF field and t is the time duration of to indicate the magnetic field of the
the applied field. Modern NMR techniques spectrometer. The sample volume used is
use RF pulses, which are characterized by around 5 mL, which is placed in a sample
the flip angles that they produce. Thus, a tube of 5 mm diameter. The pole gap is
π/2 (90°) pulse flips the net magnetization relatively narrow. When working with
from the z-axis to xy plane and produces a animals and humans, lower magnetic fields
transverse magnetization (Mxy). A π (180°) are used for safety consideration (usually
pulse inverts the net magnetization to the less than 4 T), but the pole gap is much
negative z-direction. Pulses with small flip larger to accommodate the animal. In
angles rotate only a portion of magnetization addition to the main magnetic field, electro-
vector (M) in the transverse plane. Most nic coils are used to achieve high level of
experiments are conducted using more than
field homogeneity. Field gradients are used
one pulse applied at specified periods of
to change the field to suit the requirement
time. The motion of M may appear complex
of the experiment.
as we have the Larmor precession and the
The second component of NMR equip-
spin flip superimposed. It is conceptually
ment is an arrangement for generating RF
simpler to describe the motion of the NMR
powers at desired frequencies. Modern
nucleus in a rotating frame of reference.
One can imagine the viewer standing at the NMR experiments are usually performed,
origin of the reference frame and rotating using strong RF fields in the range 0.01 to
with an angular frequency equal to the 0.4 T or several hundred kilowatts. These
Larmor precession. Such a description RF pulses are applied for short durations,
greatly helps in understanding the behavior with a very precise and specific time frame.
of net magnetization. A pulse programmer helps to attain such an
objective. RF pulses may cause heating and
Observation of NMR Spectrum: There are proper arrangements for maintaining the
three basic elements of an NMR spectro- temperature are made.
meter. A magnet with a very homogeneous
The signal is detected by a receiver coil
magnetic field is used to lift the degeneracy
and then processed by a computer. Several
between nuclear energy levels. Higher the
types of editing procedures are built in the
field, larger is the gap between the ground
computer software, all of which are part of
and the excited states, and higher is the
the spectrometer console. For optimum
population difference between the two
signal-to-noise (S/N) ratio, there has to be
states. Thus, higher magnetic fields help in
a very precise coupling between the
better sensitivity. We will see later that it
excitation and detection hardware.
also helps in a better dispersion of NMR
lines. Thus, in solution studies on bio- A Simple Pulse Sequence for NMR Excita-
molecules, one tries to use the highest tion and Detection: The simplest pulse
possible magnetic field. Spectrometers using sequence used in NMR is the free induction
B0 as high as 23.5 T are available, which decay (FID) following an excitation pulse as
correspond to a 1H resonance frequency of illustrated in Figure 2.1. A 90° pulse is used
14 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 2.1: A simple pulse sequence used in NMR. The FID signal is detected as a function of time. Instead of a
90° pulse, one can use a softer pulse (flip angle less than 90°), which allows system to return to equilibrium
at a faster rate
to excite the sample, which tips the orienta- in the single-pulse FID experiment (as well
tion of the net magnetization from the z- as in other pulse experiments) is in the time
axis to the xy plane. This pulse establishes domain. The Fourier transform (FT) of S(t)
“phase coherence” as immediately after the gives the spectrum as a function of frequency
pulse, all nuclei rotate together (coherently) S(ν).
around the z-axis, in the transverse plane.
A signal is induced in the detector coil, NMR NUCLEI IN LIFE SCIENCES
which is called the FID. This signal decays All living systems have five major elements,
with a particular time constant (T2) and C, H, N, O and P. Of these, the naturally
signal is detected as a function of time. Since abundant isotopes of C (12C) and O (16O)
the transmitter pulse is several order of do not have magnetic moments. The more
magnitude stronger than the detected signal, abundant isotope of N (14N) has spin 1. For
the collection of the signal is delayed by a O, 17O has magnetic moment, but its spin is
few milliseconds for the receiver coil to 5/2. Nuclei with spin > ½ have quadrupole
recover and respond to the FID. The moments. Such nuclei give relatively broad
excitation and detection can be repeated signals. Fortunately, we have four spin ½
several times to get a good time-averaging nuclei, 1H, 13C, 15N and 31P, which serve as
and improve S/N. The signal S(t) received reporter groups and provide us with a
Introduction to Biological NMR Spectroscopy 15
wealth of information about biological stable isotopes and are available at reason-
systems. Their NMR properties are listed in able costs. Gene cloning methods can be
Table 2.1. used to prepare reasonable quantity of
It is this rich arsenal of information compounds enriched in these isotopes for
carriers, which makes NMR such a powerful NMR studies. 13C or 15N labeled substrates
method. For example, P is present in can be used in studies of metabolism.
relatively smaller number of biological In addition to above, use has been made
molecules. However, some of these, such as of certain other nuclei for specialized
adenosine triphosphate (ATP) are involved applications. 2H (deuterium) has been used
in providing energy for essential processes in studies on biological membranes. 19F has
of life. 31P is the nucleus of choice for been used for studying drug metabolism.
studying bioenergetics in cells and organs. Nuclear polarized 3He and 129Xe have been
In muscle and brain, another energy-rich used in imaging of lungs. Use of 7Li and
23
compound is phosphocreatine (PCr). When Na has also been reported.
sufficient oxygen and fuels are available to
facilitate ATP synthesis, abundant amount NMR PARAMETERS
of PCr is produced. During energy crisis It may appear from the above discussion
(hypoxia, ischemia or cell damage), PCr that each atomic nucleus having a magnetic
serves to maintain ATP levels so that cellular moment shall give a single line spectrum.
work can continue. However, if ATP is not This is true if we look at the 1H NMR in
synthesized and PCr stores are depleted, water. However, what makes NMR useful
some of the cellular functions can no longer is the fact that nuclei are not isolated and
occur. electronic clouds in their environment
Both H and C are elements present in all modify their behavior. Let us discuss some
biological molecules. 1 H and 13C NMR of these properties.
provide information on all classes of
biological systems. These spectra are Chemical Shift (δ): Figure 2.2 shows 1 H
relatively more complex, but are rich in NMR spectrum of a small DNA duplex
information. Even 1H resonance in a simple containing 12 base pairs. It is obvious that
molecule like water (which constitutes almost the spectrum has several hundred lines. The
31 P spectrum like wise has several lines.
75% of living systems) provides us with a
wealth of information on biological organs These arise because different 1H or 31P are
as will be discussed extensively in this in different chemical environments. The
workshop. The natural abundance of both electrons modify the magnetic field seen by
13C and 15N are low. Both 13C and 15N are the nucleus, and the field (Bn) at the site of
16 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 2.2: 1H and 31P NMR spectrum of a self- complementary DNA duplex dissolved in water and having the
sequence GGTAGIACTACC. Positions of different 1H resonances are marked with reference to the shift of
sodium 2,2- tetradeutero-3-trimethyl-silylpropionate used as standard. Standard chemical nomenclature has
been used for labeling different atoms in the oligomer.
the nucleus is slightly different from the coil. The difference can then be correlated
one (B0) applied. with structural changes, and the associated
Bn = B0 (1 – σ) changes in the magnetic fields arising from
conformational differences. Instead of
σ is called the shielding or the screening
constant. The differential screening of nuclei absolute value of σ, one uses the difference
due to electrons and other local fields in from a standard compound to express
the vicinity of the nuclei under study is chemical shifts (δ = σref – σ, or (ν–νref)/ν).
called chemical shift. One has to apply higher The values of δ are of the order of 10–6 and
magnetic field (at fixed frequency) to get are, therefore, expressed in parts per million
the resonance. (ppm). In this form, δ is a dimensionless
Several short- and long-range factors quantity. However, chemical shifts are also
influence σ and a theoretical calculation is expressed as differences in frequencies in
difficult. However, it is possible to break- Hz (Δν = B0Δσ). In this form, the shift is
down contributions from various factors and proportional to the applied field. For
estimate changes in values of σ, rather than example, on a 100 MHz spectrometer, 1 ppm
their absolute values. For example, one may corresponds to 100 Hz; while on a 1000 MHz
compare the chemical shift values of the instrument, it is equal to 1000 Hz.
same protons in an ordered state of a Let us look at the spectrum in Figure
macromolecule and the values in a random 2.2, more carefully. One finds that different
Introduction to Biological NMR Spectroscopy 17
1H have different shifts and these are number of intervening bonds is more than
characteristic of the chemical groups. The three. In the case of cytosine, the interaction
thymine CH3 protons come at around 1.7 occurs via the H5-C5-C6-H6 fragment and
ppm and are at the highest field. The signals is about 7 Hz. On the other hand, coupling
in the 12-15 ppm range are due to hydrogen between T(CH3) and the T(H6) protons is a
bonded imino protons. What is more, even four bond interaction and its magnitude is
protons with similar chemical groupings small.
such as the two thymine methyls have Dipolar Couplings (Dij): Nuclei also interact
different chemical shifts. directly due to their proximity in space, as
Peak Intensities: The next thing we notice each nuclear magnet exerts a magnetic field
is that the CH3 proton lines have much on other spins in its close proximity. This is
larger amplitude than the cytosine H5, H6 called direct magnetic dipole-dipole inter-
protons. The intensities in NMR spectra are action or dipole coupling and results in the
directly proportion to the number of protons splitting of the resonance of each interacting
giving rise to the signal and provide relative 1
H into two, with a separation:
number of 1 H in the chemical groups.
However, amplitudes may be different if ΔB = 3μ (3cos2 θij –1) (μ0/4π)/rij3
resonance lines have different line widths. Here, μ0 is the permeability of the medium,
NMR spectrometers have a built-in subro- μ is the nuclear magnetic moment, rij is the
utine to provide integrated areas of various distance between nuclei i and j and θij is the
lines and thus to provide relative intensities. angle between the internuclear vector (rij)
Scalar Coupling (Jij): A careful look at the and the applied field, B0 . Thus, dipolar
cytosine H5, H6 proton signals reveals that interaction contains geometrical information
each of them are doublets separated by about the molecule. In solutions, because of
about 7 Hz. This splitting arises because the the rapid motion of the molecules, the angle
H5 and H6 protons interact with each other θ varies with time. The time average of the
through an interaction called scalar coupling. term (3cos2 θij–1) is zero and, therefore,
Such an interaction (Jij) between nuclei i and dipole-dipole interaction is not observed
j, occurs through the electronic cloud joining directly. However, if a molecule is dissolved
the two atoms. It depends on the number in an anisotropic liquid crystalline solvent,
and the nature of chemical bonds joining then the value of this term may not average
atoms i and j, hybridization of the bonding to zero, and the geometrical information
orbitals, and other stereochemical factors.
can be retrieved. This interaction is also a
J values between directly bonded nuclei are
dominant contributor to relaxation rate,
large. For example, J value for directly
which is manifested in the form of nuclear
bonded 13C and 1H is around 125 Hz for
Overhauser effect (NOE).
saturated carbon atoms (sp3 hybridized C),
150 Hz for ethylenic C, and 220 Hz for Relaxation Rates and Line-widths: Imme-
acetylenic C. Three bond coupling J (A-B- diately after the application of B0 field, the
C-D) usually follow a cos2ϕ dependence on population of the two nuclear spin energy
the dihedral angle (ϕ) around the B-C bond. levels is equal, the net magnetization Mz
J values are relatively small when the = 0, and the system is in a non-equilibrium
18 Biomedical Magnetic Resonance: Proceedings of the International Workshop
state. The build up of equilibrium magneti- The relaxation times depend on two factors:
zation obeys a first order rate equation: the strength of the interaction responsible
for energy transfer and the time-scale of
dMz/dt = (M0–Mz)/T1 the motion causing the fluctuations in the
T1 is called the spin-lattice or the longi- local magnetic fields. For non-viscous
liquids, such as pure water, T1 and T2 are
tudinal relaxation time. Immediately after
almost equal. However, in general, the
excitation by a 90° pulse, the net magneti-
transverse magnetization decays faster than
zation in the z-direction is zero, and Mz
the longitudinal magnetization.
returns to its equilibrium value with the The line-width of the resonance signal
same time-constant. at half-height is related to T2 and is, in fact,
The built up or decay of the z-magneti- equal to 2/T2 . The decay of transverse
zation requires exchange of energy between magnetization can also be caused by in-
nuclear spins and the surrounding (lattice). homogeneities in the magnetic field (ΔB0).
In liquids, the dominant process for such an One therefore defines another time
energy exchange for spin ½ nuclei is the relaxation time T2*
magnetic dipole-dipole interaction. Rota-
1/T2* = 1/T2 + γΔB0/2
tional and translational motions of a
molecule in a liquid cause a fluctuating The line-width is given by 2/T2*.
magnetic field. The components in the trans- We will learn later that relaxation
verse plane with component at the Larmor parameters T1, T2 and T2*, even in a simple
frequency can stimulate transitions between molecule such as water, are influenced by
the two states of the nuclear spin system. factors such as its interaction with other
The magnetic energy received by the molecules in the tissue, flow and diffusion,
environment is transformed into thermal paramagnetic molecules and other factors.
energy. Both intra- and inter-molecular These properties form the basis of medical
interactions can contribute to relaxation. For imaging by NMR.
13C, the relaxation is dominated by intra-
Quadrupolar Interactions: Nuclei with spin
molecular 1H-13C dipole-dipole interaction. quantum number > ½ possess a charge
For 1H, the relaxation mechanism involves distribution, which is not spherically
1H-1H dipole-dipole interactions, both inter-
symmetric. These nuclei, therefore, have a
molecular (particularly with solvents) and quadrupole moment Q. This can interact
intramolecular. with the electric field gradient (q) at the
The magnetization in the xy plane (Mxy) nucleus and contribute to relaxation. In case
decays with a different time-constant T2, of deuterium ( 2 H), the value of Q is
which is called spin-spin or transverse relatively small (Q = 2.77 × 10–3) and, there-
relaxation time. An important mechanism fore, NMR signals are relatively sharp. In
for T2 relaxation is energy transfer within fact, the quadrupolar coupling constants
the nuclear spin system. Transition in the (e2 qQ) can be used for obtaining useful
spin state of a nucleus changes the magnetic information in certain biological problems.
field at the nearby nuclei and contributes to On the other hand, 14N and 17O resonances
shortening of the life-time of the spin state. are fairly broad.
Introduction to Biological NMR Spectroscopy 19
Nuclear Overhauser Effect (NOE): The NOE With the availability of cryoprobes and other
is defined as the fractional change in the technical advances in NMR, the concen-
intensity of the signal of the nucleus i, when tration limit may go down to nM ranges.
the transition due to another spin j is Resolution: We have seen, that even a small
perturbed. NOE arise from dipolar-dipolar oligomer gives a very large number of NMR
interaction between the two spins (i and j) signals. The 1H signals lie in a narrow range
and also depend on molecular motion. When
of 15 ppm. Thus, it is difficult to resolve all
intramolecular dipole-dipole interactions
resonances, particularly for nuclei, which are
dominate the relaxation, NOEs are inversely
in very similar chemical environment. In the
proportional to the sixth power of the inter-
above example, one notices the crowding
nuclear distances. In a molecule, if one
of signal in the sugar 1H regions. Therefore,
distance is known (e.g. H5-H6 distance in
cytosine), then the other distances can be it is preferable to use higher fields for
estimated. This is one of the key information biomolecular studies and for in vivo
in determining macromolecular structures. spectroscopy.
Chemical Exchange: Another factor, which Assignments: To make meaningful conclu-
influences NMR signals, is exchange between sions, one needs to know which signal is
different chemical entities. For slow associated with which nucleus. In the
exchange (on the NMR time-scale), we see example above, there are two thymines and
signals from the two species involved in indeed one gets two 1H signals in the CH3
chemical exchange. When the exchange rate region. But how do we find as to which
is fast, one gets a spectrum, which is the signal belongs to which thymine? Assign-
time-average of spectra for the two ment of signals is one of the first steps in
exchanging species. This information can be applications on biological systems.
used to obtain thermodynamic and kinetic Water Signal: In most chemical applications,
information for the exchange reaction. NMR studies are conducted using
deuterated solvents such as D2O or CDCl3.
DIFFICULTIES IN STUDYING However, for biological applications, studies
BIOLOGICAL SYSTEMS BY NMR have to be performed in H2O, which is the
Why it took 30 years for NMR to move milieu of living systems. D2O is toxic. Even
from chemical to biological systems. There in studies on macromolecules, D2O is avoi-
are several problems which one encounters ded, to prevent deuterium exchange with
when dealing with biological systems. the labile NH and OH protons, which
Sensitivity: By its very nature, NMR is a provide valuable information on hydrogen
relatively insensitive technique. In earlier bond networks. With two protons, the
days, one required solutions of 100 mM molar concentration of water protons in
concentrations to obtain a decent NMR aqueous solutions is almost 110 M, against
spectrum. With the advent of FT technology, which signals from mM concentrations of
advances in probe designs, magnet and the molecules of interest have to be picked.
computer technology, the limit has come Fortunately, FT NMR has given us power
down to mM concentrations. The techno- to suppress undesirable resonances. Two
logies are improving every year leading to such molecules from our view-point are
new advancements in biological research. water and fat.
20 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Broad Lines: The line-widths in most organic nature of mixing during the period t1. Some
compounds are around 0.3 Hz. The lines in critical experiments in biomolecular studies
biological macromolecules are broader are J-Correlated Spectroscopy (COSY) (and
because of shorter T 2 due to their high several advanced versions of COSY such as
molecule weight, which leads to lower HMQC, TOCSY), Nuclear Overhauser
mobility. The line-width increases with the Spectroscopy (NOESY) and ROESY. The
molecular weight and, therefore, studies on basic pulse sequences for such experiments
large proteins are not only complicated by
are shown in Figures 2.3A to D. Spin-echo
problems of resolution and assignments but
and inversion recovery experiments, which
also by the higher widths of NMR signals.
are extensively used in MRI are also multi-
Some solutions to such problems have been
suggested recently. The line-widths in cells pulse experiments. Recently, such experi-
and organs are even more. ments have also found applications in MRS.
New pulse sequences are being developed
2D NMR SPECTROSCOPY every year and it is difficult to keep count
of the ever-increasing acronyms in the
Several problems discussed in the previous literature of pulse NMR.
section have been resolved through the use
The following advantages of 2D
of two-dimensional (2D) and multi-
spectroscopy are obvious:
dimensional NMR spectroscopy. Special
techniques of pulse NMR allow spectra to 1. Since we can spread the peaks in two
be recorded with two independent dimensions, cross peaks have a better
frequency dimensions F1 and F2. The signals dispersion of signals. In heteronuclear
in such spectra are characterized by two experiments, the larger chemical shift of
frequencies (f1, f2). Depending on the pulse the second nucleus helps in resolving
sequences used, the two frequencies may signals from protons with very similar
correspond to the same nuclei (homonuclear chemical shifts.
2D) or different nuclei (heteronuclear 2D). 2. Homonuclear correlation parameters
The diagonal peaks in a homonuclear 2D between nuclear spins, such as J and
NMR (f1 = f2) correspond to the 1D NMR. NOE can be obtained, which in turn, can
The off-diagonal peaks (commonly called be used for structural determination.
cross peaks) have important information 3. Correlations between various NMR
about the relationships between different nuclei (1 H, 13C, 15N and 31 P) can be
spins. Different interactions between the established. This helps in assignments.
spins can be picked up depending on the 4. Information on chemical exchange can be
pulse sequence. obtained using ROESY experiments.
A general scheme of 2D NMR is shown 5. Pulse sequences have been developed
in Figure 2.3. A second time domain is which suppress signals from water or
created by performing 1D experiments with other undesirable species.
various incremental times during the
NMR AT DIFFERENT LEVELS OF
evolution period t1, during which the nuclear
BIOLOGICAL SYSTEMS
spins interact with each other through
various type of interactions. The various 2D Information transfer in biological systems,
NMR experiments differ essentially in the flows from 1D information present in DNA
Introduction to Biological NMR Spectroscopy 21
Figs 2.3A to D: Use of pulses to generate 2D spectrum. (A) General scheme of 2-dimensional NMR; it usually
consists of (i) a period during which the spin system of interest is prepared; for example, by generation of
transverse magnetization through a 90° pulse, (ii) an evolution period t1 during which it develops under
factors such as Larmor precession or J/D couplings, (iii) the detection time t2. (B) By a systematic variation of
evolution time a second time axis (t1) is created. (C) Pulse sequence for a simple COSY spectrum consists of
two 90° pulses separated by the evolution time t1. (D) Pulse sequence for a NOESY spectrum
22 Biomedical Magnetic Resonance: Proceedings of the International Workshop
sequence, through m-RNA, t-RNA, and proteins or even protein folds involved in
ribosomal RNA to amino acid sequences in biological functions of living systems. The
proteins. The 3D structures of proteins are human genome project has witnessed a
responsible for activities of cells. Though major breakthrough, raising the hope that
all cells are derived from the same genomic biological processes will be understood at
material (paternal gene from spermatozoa molecular level. At this stage, we do not
and maternal gene from the ovum), the know the exact gene sequences which are
initial fertilized egg cell retains its properties expressed. However, several proteins with
only during the first few rounds of division. similar functions have similar 3D structures
They later differentiate into cells for (e.g. kinases, dehydrogenases) and, there-
different organs, which have different fore, knowledge of the structure of a parti-
properties and functions. This is achieved cular class of protein from one organism,
by differential expression of the genes and may help us to understand a particular
thereby, changes in the cellular levels of reaction in the cell or organ of another.
different proteins. Further, cells from the NMR and X-ray crystallography are the
same organ may behave differently, if major techniques used to unravel 3D
subjected to stresses such as disease, changes structures of biological molecules. Several
in diet, chemical and environmental factors, NMR parameters provide structural infor-
etc. For example, if one ingests a toxin or a mation. These include distance information
drug, the protein levels may not change, from NOE, orientation constraints from
but it may inhibit a critical enzyme and dipolar couplings (using liquid crystalline
reduce its activity. solvents), backbone and side chain dihedral
Thus, a biologist is interested in knowing angles from scalar couplings, secondary
sequence, the 3D structure and function of structural elements from chemical shift
biological molecules, organization and changes, location of hydrogen bonds from
functions of macromolecular assemblies, chemical shifts or exchange studies, etc. The
cellular functions as well as chemistry of structural parameters are normally fed into
whole organs. NMR has found applications structural algorithms, which provide struc-
at all these diverse levels of biological ture(s) consistent with the structural para-
processes. meters. Unlike crystallography, NMR does
not provide the structure directly. At this
MACROMOLECULAR STRUCTURES stage, only medium size macromolecules and
AND ORGANIZATIONS molecular assemblies have been studied
One of the most rewarding areas of NMR using NMR. One advantage of NMR
is study of the three-dimensional structures methods is that the structures are obtained
of biological macromolecules (proteins, in water solutions and are not dictated by
nucleic acids and sugars), their dynamic crystal forces. It has been realized that
behavior and their structure-function rela- biological macromolecules are not rigid and
tionship. The structures of a large number have internal motions, which in fact, plays
of such molecules have been reported, but a dominant role in their functions. NMR
our knowledge is far from complete. As has proved advantageous in exploring
yet, we do not know the total number of motions of biomolecules.
Introduction to Biological NMR Spectroscopy 23
The next level at which NMR has helped or which are difficult to assay by standard
is in understanding macromolecular chemical methods. The only problem is
organization. In recent years, studies have related to the low sensitivity of the method,
been made on complex macromolecular limited at this stage to mM ranges.
assemblies, such as protein-nucleic acid Cell metabolism is a highly intricate net-
complexes, biological membranes, glyco- work of coupled reactions. These include
proteins and glycolipids. The use of recently biosynthetic (which usually require energy)
developed high resolution solid-state NMR and oxidative pathways (from which living
has made a major impact on such studies. systems obtain energy). Energy stored in
the form of fatty acids, or sugars is released
STUDIES OF BODY FLUIDS, during their oxidation and stored in the
TISSUES AND CELLS form of ATP or PCr. There are three major
stages in energy-producing metabolism,
Cell function is the result of an intricate
glycolyis, Krebs cycle and oxidative phos-
network of functioning proteins, small phorylation. During glycolysis, glucose is
molecules and ions and external factors, metabolized to pyruvate through a series
which may activate or control enzyme of reactions. One molecule of glucose is
functions. Though enzyme functions have oxidized to two molecules of pyruvate, two
been studied in vitro, one understands in molecules of NAD are reduced to NADH
vivo biological functions only by working at and there is a net production of two
cellular level. NMR has proved invaluable molecules of ATP. Certain organisms
to understand cell metabolism and factors, survive entirely on glycolysis as energy
which control and modify such actions. NMR source. In these cases, pyruvate may be
of cells, tissues, and body fluids has, fermented to alcohol or some other molecule
therefore, become an important area of and simultaneously, NADH is oxidized back
research in biological and medical sciences. to NAD. During anaerobic conditions in
Metabonomics is a recent term coined for brain or in spermatozoa, the final product
this field, which is aimed to understand at is lactic acid. In presence of oxygen, pyru-
a molecular level, the behavior of cellular vate is converted to acetate, which is then
systems and aberrations in their function as further metabolized in the Krebs cycle. The
a result of diet, stresses and changes during two carbons are oxidized to CO 2 and
cell development and differentiation. The coenzymes FAD and NAD are reduced. In
non-invasive nature of NMR measurements, the final stage of oxidative phosphorylation,
offer several advantages over conventional which occurs in mitochondrial membrane,
techniques. While most analytical techniques the reduced coenzymes are oxidized and
are capable of detecting compounds for energy released is stored in the form of
which the technique has been designed, ATP. The energy pathways are controlled
NMR is not biased towards a particular at several stages. For example, if there is
compound. It does not require precise excess of ATP, then glucose metabolism
selection of analytical conditions to obtain stops and it is stored in the form of gluco-
quantitative information from endogenous gen. This simple picture may be modified
metabolites. It can simultaneously detect in different organs or organisms or under
molecules which are expected, unexpected stresses such as disease.
24 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Study of cell metabolism under aerobic is the male reproductive organ. It acts as a
conditions (presence of oxygen) require highly efficient storage system and enables
special modifications in the probe and in spermatozoa to mature and preserves/
sample handling. However, glycolysis, enhances their motility and fertilizing
inactive cells, cell free extracts, body fluids capacity. There are three main parts of
and tissues can be analysed using a normal epididymis: caput is the birthplace of
probe in a high resolution NMR spectro- spermatozoa, corpus where they are in semi-
meter. I shall spend some time on cellular matured stage and cauda where they are
NMR since this subject is extremely impor- fully matured. During the passage through
tant in following the area of magnetic epididymis, spermatozoa undergo major
resonance spectroscopy (MRS) of whole morphological and biochemical changes. The
organs, and is not covered by other next stages in the cycle of spermatozoa are
speakers.
capacitation, a process where spermatozoa
are activated, and acrosomal reaction where
STUDIES ON SPERMATOZOA
the cap region delivers hydrolytic enzymes
As an illustrative example, we discuss so that it can fuse (fertilize) with the egg.
studies on spermatozoa, the paternal cells These two processes take place in the female
in reproductive biology of mammals. reproductive tract. Changes in the bio-
Importance of such studies in fertility chemistry of spermatozoa in all the five
regulation, artificial insemination, and in steps have been followed using NMR.
understanding cellular and molecular Identification of Small Molecular Weight
biology of spermatozoa is obvious. From Compounds: 1H and 13C NMR allows direct
the view point of NMR, spermatozoa are detection of the molecules present in the
extremely friendly, as they can survive for cells (Figs 2.4A and B). It may be noted that
a long time under anaerobic conditions and macromolecules do not contribute to such
do not settle because of their motility. spectra as their lines are relatively broad
General features of sperm morphology, and concentrations are low. Typically, 105
and its diversity in different animals has cells suspended in Dulbecco medium have
been the subject of extensive investigations been used for obtaining 2D homonuclear
using microscopic techniques. Its overall DQF-COSY and 1H-13C HSQC spectra. The
structure can be divided into three parts; wealth of information available in such
(a) head, which contains the paternal spectra is obvious. The chemical composition
chromatin and has a cap-like cover called and relative concentrations of molecules in
acrosome, which participates in egg penetra- cells changes dramatically, not only from
tion and fusion, (b) mid-piece containing species to species but also within samples
mitochondria and which contributes to most from the same group of animals. The signals
of the NMR signals, and (c) tail which is and levels of amino acids such as Ala, Arg,
responsible for the movement in the seminal Asp, Gly, Glu, Ile, Leu, Lys, Met, Pro, Thr,
fluid (motility). Motility and fertilizing Val, and molecules such as lactate (Lac), m-
ability are the major markers of sperma- inositol, glycerophosphorylcholine (GPC),
tozoan activity, which can be beneficially or hypotaurine and uridinediphosphoglucose
detrimentally influenced by a variety of (UDP) could be identified and their
physical and chemical factors. Epididymis, concentrations measured. The relative
Introduction to Biological NMR Spectroscopy 25
Figs 2.4A and B: 2D 1H-13C (J-correlated) HMQC spectra, from sperm cells derived from two different regions
of goat’s reproductive tract. As the cells mature, there are marked changes in their chemical composition,
which is reflected in these spectra
Fig. 2.6: 31P NMR spectrum of active cells provides vital information on bioenergetics, including levels of
ATP, PCr and intracellular pH. A similar methodology is followed in MRS
Introduction to Biological NMR Spectroscopy 27
in TIFR and supported by the DST, DBT and CSIR, J Cavanagh, WJ Fairbrother, AG Palmer, NJ
is gratefully acknowledged. Skelton. Protein NMR Spectroscopy: Principles
and Practice. San Diego Academic Press Inc.,
1996.
FURTHER READING K Wuthrich. NMR of Proteins and Nucleic Acids.
New York; John Wiley and Sons, 1986.
1. Early development of NMR spectroscopy in G Govil and RV Hosur. Conformation of Biological
chemical sciences is elegantly covered in: Molecules: New Results from NMR, Berlin;
JA Pople, HG Schneider, HJ Bernstein. High Springer-Verlag 1982.
Resolution Nuclear Magnetic Resonance, McGraw 5. For cells, body fluids and metabonomics: No
Hill Publications 1959. good books, but some recommended articles
For more theoretical treatment see: are:
A Abragam. Principles of Nuclear Magnetism. Sudha Srivastava, Girjesh Govil. Applications of
Oxford: Clarendon Press 1961. NMR to the study of cells and body fluids. Current
2. The basic principles of NMR spectroscopy have Organic Chemistry 2001; 5: 1039-57.
been described in several text books. For a Cath O’Driscoll. Metabonomics on the map.
somewhat simplified version see: Chemistry in Britain 2002; 25-28.
H Gunter NMR Spectroscopy: Basic principles, JP Shockcor, Elaine Holmes. Metabonomic
concepts, and applications in chemistry, 2nd edn. Applications in Toxicity Screening and Disease
Chichester John Wiley and Sons 1995. Diagnosis. Current Topics in Medicinal
3. An excellent book for theory and experimental Chemistry 2002; 2: 35-51.
pulse FT NMR spectroscopy is: JK Nicholson, J Connelly, JC Lyndn, E Holmes.
G Bodenhausen, H Kogler, RR Ernst. Principles Metabonomics: a platform for studying drug toxicity
of Nuclear Magnetic Resonance in One and Two and gene function, Nature Reviews 2002; 1: 153-
Dimensions, New York; Oxford University Press 61.
1985. RG Shulman, F Hyder, DL Rothman. Biophysical
4. For protein and macromolecular NMR one may basis of brain activity: Implication for neuroimaging.
consult: Q Rev Biophysics 2002; 35: 287-25.
Fundamentals of Magnetic Resonance Image Production 29
3
Fundamentals of Magnetic
Resonance Image Production
P Raghunathan
gradient pulse of a duration Δt along the by pulsing more than one gradient coil; a
second axis (the y-axis) will accumulate a 45° oblique axis would use equal amplitudes
phase along that axis: of two orthogonal gradients.
To accomplish MR imaging, the three
φy = γGyyΔt ...(3)
gradient coils must be activated suitably for
Phase changes are cyclic, and anything that (i) slice selection, (ii) phase encoding, and
undergoes cyclic changes has a frequency. (iii) frequency encoding or ‘readout’. We
Therefore, by bringing about changes in the shall discuss these modular operations in
spin phase using phase encoding gradients this order, which is the usual sequential
(e.g., G y in Eq. 3), we are essentially order of their appearance in standard MRI
achieving another dimension in frequency- pulse sequences.
encoding.
Slice Selection
Physical Gradients of the MRI Scanner The first step in MR image production is
Three sets of independent gradient coils are the selection of an anatomical slice. To do
used in a scanner with their axes oriented this, it becomes necessary to confined the
along the x-, y- and z- directions. Direct RF excitation selectively to the ‘region of
current, pulsed for several milliseconds interest’ (ROI) of the tissue under diagnostic
through any of these coils, produces the examination. This may be achieved by
gradient pulse Gx , Gy or Gz. In the case of applying a bandwidth-limited RF pulse in
conjunction with the magnetic field gradient
a superconducting magnet, which is used in
Gsl; for example, Gsl could be Gz, applied to
most mid-field and all high-field (>1.5 T)
resonate a perpendicular plane in the ROI.
MRI scanners, the z-direction is conven-
The frequency selective RF pulse is essen-
tionally defined along its long axis. The
tially a pulse tuned to have a small band-
three gradient coils may be activated to width Δω, which is typically 1-2 kHz around
produce a slice selection gradient (Gsl), a
frequency encoding gradient (Gfr), and a
phase encoding gradient Gph. Each of these
may be chosen to dertermine a certain tomo-
graphic plane of the human anatomy and
will be assigned, through the operating
software, to one of these three ‘logical’
operations. For example, in Figure 3.1, we
show the orientation of the logical frequency
encoding and phase encoding gradients for
the axial, sagittal and coronal tomographic
planes when the logical ‘slice selection’ Fig. 3.1: (Top) The orthogonal x,y and z gradient field
gradient is taken perpendicular to the plane. directions inside the MRI magnet.
(Bottom) The logical frequency encoding and phase
Because of the availability of the three
encoding gradients for the axial, sagittal and coronal
gradient coils, controlled alterations in the planes, with the logical slice selection direction taken
effective magnetic field can be generated along z.
along any orthogonal axis. Furthermore, (A to P: Anterior to Posterior, S to I: Superior to Inferior,
oblique imaging axes can also be defined L to R: Left to Right)
32 Biomedical Magnetic Resonance: Proceedings of the International Workshop
progressively stepped up and played out encoding that slice to produce a (ω, φ)
for each x position in the matrix, e.g., 256 ‘spatial frequency – spatial phase’ data
times for a 256 × 256 picture data array. matrix. This matrix constitutes an array of
Gph, applied perpendicular to both Gsl and pixels. Image contrast contributed by each
G fr, in the only gradient that changes pixel will be later seen to be related to the
amplitude in a series of programmed steps characteristics of tissue in the corresponding
during the data acquisition loop of a stan- voxel (volume element). Transverse magneti-
dard 2-D imaging sequence. zation within each pixel contributes a specific
signal waveform, i.e., specific frequency,
Frequency Encoding phase and amplitude, to the composite RF
Following the slice selection and phase signal. Each composite RF signal, or echo, is
encoding modules we apply the Gfr gradient the sum of RF components from all the
for producing a space-encoded dispersion voxels in the image plane.
of the MR signal which is collected as an
Dephasing FID, and Its ‘Rephasing’ to
‘echo’. (The description of an echo, and the
Create an ‘Echo’
need for producing it, are discussed in the
next section). As the echo signal is detected This section is an interlude to examine what
by the RF receiver coil – the ‘probe’ of the an ‘echo’ signal is, and why we need to
MRI scanner – it is simultaneously digitized create it. Admittedly, the MR signal used
by an analog-to-digital converter (ADC) for for imaging is present immediately after the
later Fourier transformation. We note that RF pulse and is, in fact, the FID. This signal,
Gfr is switched on perpendicular to the Gsl however, decays or ‘dephases’ with a time
direction, and must be applied for each value constant T2 or T2* depending on the chemical,
of Gph. physical and magnetic environment of the
In Figure 3.2 we illustrate the processes tissue 1H nuclei. In order to give ourselves
of ‘selecting’ an anatomical slice, and then sufficient time for spatially encoding the MRI
signal, the FID must be delayed from the
RF pulse and recovered by rephasing it a
little later. The is done by creating an echo.
Two types of rephased echoes are in use in
MR imaging: the spin-echo (SE), and the
‘gradient-recalled’ echo or, simply, the
gradient-echo (GE).
Spin-Echo
Magnetization in the xy plane starts to
dephase immediately following the initial
90° RF excitation. Dephasing is the disper-
Fig. 3.2: A slice of thickness Δωsl, selected in the
transverse orientation (top), and the xy imaging plane sion of the individual spin moments that
(bottom). For clarity, the spatial frequencies (wavy comprise the macroscopic magnetization
lines) and phases (arrows) are shown only in alternate vector in the xy plane, and it occurs both
elements of the pixel array due to the intrinsic spin-spin interactions
34 Biomedical Magnetic Resonance: Proceedings of the International Workshop
amongst the precessing spins and due to TR. When longitudinal recovery of the spin
local magnetic field in homogeneities. moments towards equilibrium is occurring
The way we compensate for dephasing in one excited slice, a spin-echo can be
is called rephasing. This may be achieved aquired from another slice.
by the reversal of the phase of the spin Conventional SE imaging is associated
moments by a refocusing 180° RF pulse. As with long image acquisition times since the
a result, the dispersing spin phases now flip pulse sequence repetition time, TR, must be
over in the transverse (xy) plane and pro- sufficiently long to allow recovery of the
gressively rephase to form a spin echo (SE) magnetization to thermal equilibrium prior
signal. The time between the initial 90° RF to the next 90° RF pulse. Reducing TR
pulse and the maximally rephased echo is should, in principle, lead to a saving in time
labeled TE (‘time-to-echo’). In the SE signal, in a repetitive image scan. Such a time –
any inhomogeneities present in the static B0 saving is possible through use of the
field and any magnetic susceptibility gradient echo, as we shall see below.
contributions to the tissue ROI from the
surrounding structures are corrected (i.e., Gradient-Echo
get refocused), and the signal intensity To understand the gradient-echo (GE), we
decays exponentially in time due to the recall that an initial, small flip-angle (<90°)
intrinsic spin-spin interactions only, as RF pulse (α-pulse) may be used to flip the
exp (–TE/T2). precessing tissue magnetization into the (xy)
We note, however, that a sequence of plane, producing the FID. Instead of using
the 90° and 180° RF pulses alone do not an inverting RF pulse to rephase the
constitute the imaging sequence. For doing decaying spin moments, we now employ a
this, both phase encoding and frequency gradient reversal strategy by using a
encoding gradients must be applied as well, ‘dephase-rephase’ readout gradient pair.
inevitably increasing the minimum achiev- That is, a negative gradient (-G x) is first
able TE. Additionally, the slice selection used, which is immediately followed by a
gradient must also be applied to define the positive gradient of the same amplitude
image plane. These features of a basic SE
imaging sequence are illustrated in Figure
3.3, where the important parameters TE and
TR (which is the ‘time-to-repeat’ the imaging
sequence in a repetitive scan) are also shown.
To acquire multi-slice images, the 90° and
180° RF pulses are repeated with different
RF center frequencies to excite similar slices
centered at different positions along the
direction of the slices selection gradient (see
Eq. 5). A series of different slices can be
excited and detected in an ‘interleaved’
manner. That is, more than one slice is
excited (and the echo digitized) in one TR
period. One Gph is played out per slice per Fig. 3.3: The basic spin-echo imaging sequence
Fundamentals of Magnetic Resonance Image Production 35
(+Gx). This gradient reversal rephases the phase encoding gradient Gph is also switched
spin moments and forms an echo the on so that spin information in the ortho-
gradient-echo (GE) – for signal detection. gonal direction gets space-encoded.
The GE is particularly advantageous for
short TR, small RF pulse angle (α < 90°) Use of the Parameters TR and
imaging protocols because of the shorter TE in Producing Image Contrast
time necessary for gradient reversals, In the previous sub-section two important
compared with 180° RF pulse flips of the timing parameters, TE and TR, have been
magnetization. However, unlike in the SE introduced. The parameters can be varied
sequence, magnetic gradient reversal does to achieve contrast sensitivity in MR images.
not rephase the so-called ‘T2* - dephasing’ The MR image intensity represents the tissue
due to magnetic field and magnetic characteristics of a specific slice through the
susceptibility inhomogeneities. If the echo patient’s body. Primarily, the intensity
delay TE from the initial α-RF pulse is depends on two factors, one of which
varied, an exponential decrease in signal represents an exponential signal decay due
intensity, exp (-TE/T2*) with a time constant to the T2 relaxation process, and the other
T2* will be observed, with T2* < T2. an exponential signal re-growth due to the
The gradient-echo imaging sequence is T1 relaxation process. Taking the case of
shown in Figure 3.4. Its application shows the most frequently used SE imaging
how the signal is moved away from the RF sequence, the quantitative dependence of
pulse and how the data is allowed to range the MR echo intensity, S, on T1 and T2 may
over both negative and positive values of be written as 9,10
spatial frequencies in a signal data acquisi-
tion. At the same time that this dephasing TR TE ...(6)
S = AM0 1 – exp – exp –
frequency encoding gradient is applied, the T1 T2
T1 TE
S sin
0
exp –
TR T2 * ...(7) as those depicted in Figure 3.3 show that a
1 – cos exp –
T1 number of individual events occur concur-
rently such as the application of RF pulses
It may be seen from Eq. 7 that in GE- to the patient’s body, tuning magnetic
based imaging sequences in general, the gradient pulses on and off, and digitizing
contribution of α and TR determines the and storing the RF signals emitted by the
amount of T1-weighting, while TE deter- tissue 1H nuclei. Most of these events are
mines the amount of T2*-weighting. Also associated with, and determine, the spatial
use of low flip angles leads to more rapid characteristics of the image. Each imaging
scans, if somewhat weaker signals compared cycle produces one composite time-domain
with SE sequences. sinusoidal signal, which has a component
Typical 1H nuclear T1 and T2 values for coming from each individual voxel.
various human tissues at B0 values of 1.0T Although the individual sinusoids are phase
and frequency encoded, it is not possible to
and 1.5 T are set forth in Table 3.1. Whereas
unscramble them from one composite signal
the T2’s are relatively independent of B0,
waveform. It is necessary to acquire a series
T1’s are found to be generally shorter at
of composite signals (or ‘views’) by repeat-
the lower B0 value. By choosing TE and TR ing the data acquisition cycle. Let us,
appropriately, these T1 and T2 values can therefore, briefly chronicle the series of
be exploited for selective imaging of a tissue events leading to the filling up of the entire
type and, therefore, for delineating a 2D data matrix.
pathology (e.g., tumor). It is this unique After the slice has been selected by the
flexibility provided by MRI in image simultaneous application of the initial RF
acquisition, and the ability to introduce pulse and the Gsl gradient, the raw data
dynamic levels of soft tissue contrast for acquisition cycle starts. During each cycle,
probing tissue proton density as well as lasting for TR, 1H nuclear spins in individual
tissue distribution, that renders it such an voxels become phase and frequency encoded
important modality in diagnostic radiology. as per the following sequence of
programmed steps. Phase encoding is
Table 3.1: Typical 1H nuclear T1 and T2 values of produced by Gph that is pulsed on briefly
living human tissue at 1.0 T and 1.5 T for a time Δt and then turned off. The
Tissue T1 (ms) T2 (ms) frequency encoding G fr gradient is then
1.0 T 1.5 T 1.0 T 1.5 T switched on, and stays on with a constant
amplitude during the buildup, peaking and
Grey matter 810 920 100 100
decay of the signal echo. The ADC window
White matter 680 780 90 90
is also left open during this period so that
Fat 240 260 85 80
Muscle 730 860 45 50
the echo is ‘read out’, i.e., sampled and
Cerebrospinal fluid 2500 3000 1400 1420 digitized. During each cycle, the sequence
Blood 525 600 260 260 of gradient activity and data acquisition is
so adjusted that the echo peak occurs at
Fundamentals of Magnetic Resonance Image Production 37
time TE, and at the instant of its formation of distinct pixels, with each pixel in turn
the spin voxels is each row are in phase. being associated with the proton density
Because the digital signal comprises the sum and relaxation time characteristics of the
of all the precession induced time domain tissue in that voxel.
signals of the slice-selected 1H nuclei, it is
Fourier transformed to yield a spatial Image Reconstruction and the
frequency distribution and thus a one- ‘Display’ Matrix
dimensional proton spatial distribution.
The image data matrix (or display matrix)
With each repetition of the acquisition
is obtained via the 2D Fourier transfor-
cycle, the amplitude of Gph is incremented
mation of the raw data matrix, and this is
stepwise, to impart a unique phase change
performed by the array processor of the
along the phase encoding direction, whereas MRI scanner. The array of spatial frequencies
Gfr has the same amplitude every time it is thus becomes converted into final image
applied during readout. As the rate of phase amplitudes, and this is depicted in Figure
change for each spin moment along the 3.5. The image matrix is a frequency map of
phase encoding axis has a unique frequency the 1H nuclear signal intensity from a volume
distribution, a second Fourier transfor- element weighted by the T1 and T2 values
mation is used to yield projections along of the tissues contained within the volume.
this axis. The immense diagnostic utility of suitably
The data matrix is filled out in this weighted MR images is extensively
fashion, and the rows become distinctly documented in the literature9-11, and just a
coded by their linear variation in the phase couple of prototypical illustrations are given
angle (Eq. 3) while the columns become involving pathologies of the brain (Figs 3.6A
coded by their linear variation in preces- and B) and spine (Figs 3.7A and B).
sional frequency (Eq. 2). The entire 2D data As implemented in most clinical MRI
matrix, thus becomes parsed into an array scanners, image reconstruction creates two
Fig. 3.5: Fourier transform relationship between the ‘raw data’ spatial frequency matrix and the final image
matrix. The latter is a 256 × 256 matrix; the pixel intensities show T1 – and T2 – weighting according to the TR,
TE pulse sequence parameters used
38 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 3.6A and B: Mid-brain sagittal MRI: (A) T1– weighted (TR = 600 msec; TE = 25 msec), demonstrating only
cortical atrophy; (B) T2– weighted (TR = 2500 msec; TE = 90 msec), showing multiple ‘bright’ foci both in the
white matter and in the corpus callosum, due to Multiple Sclerosis
Figs 3.7A and B: Sagittal spine MRI: (A) T2– weighted and (B) T1 – weighted images. The extramedullary
soft tissue mass shows up better (black arrow) in the T1– weighted image
Fundamentals of Magnetic Resonance Image Production 39
important image types called magnitude and zation (due to the tissue 1H nuclei) in space.
phase images. They arise basically due to Each digital data sample is one Fourier
the fact that the transverse magnetization coefficient, or one spatial frequency
component that is responsible for producing component. The variable we use to denote
the MR signal is a mathematically complex spatial frequency is ‘k’. Therefore, the 2-
quantity. The phase image becomes dimensional space in which the FT operation
significant whenever extraneous factors is defined is called ‘k-space’, with its
contribute to some phase errors across the components being labelled kx and ky. If x
imaging plane. The most important of these and y represent the two space variables
factors is patient motion, especially if it defining an object, kx and ky represent the
occurs in the presence of an applied gradient. two spatial frequency variables. If the units
The usual method for separately recove- of x and y are, say, cm, then the units of kx
ring magnitude and phase images is to and ky would be cycles cm–1 or, simply,
measure the in phase or real (R) and cm–1. Spatial frequency is the rate of change
quadrature or imaginary (I) components of
of frequency as a function of distance. A
the complex magnetization by comparing the
more appropriate term than ‘spatial
echo signal to a ‘clock’ reference RF
frequency’ would be ‘spatial phase’ since kx
waveform and demodulating. Each
and ky actually express the phase ‘advance’
component, R and I, may then be utilized in
or ‘retardation’ the spin moments experience
the usual 2D reconstruction procedure to
per unit tissue length in the x- and y-
create either the absolute magnitude or
directions.
‘modulus’ (M) image according to M =
The symbol ‘k’ is a carry-over from the
(R 2 + I 2 ) 1/2 , or the ‘phase’ (P) image
earlier ‘wave vector’ concept used in optics
according to P = arctan (1/R). The modulus
reconstruction sacrifices phase information and crystallography, and there exists a
and, in the process, eliminates phase errors. transparent analogy with the familiar
It represents the actual magnetization levels reciprocal space concept in crystal physics. Just
within each voxel and is therefore used as in the diffraction limit in optics, it is the
wavelength λ = 2π/k that decides the spatial
regularly for displaying SE and GE diagnos-
resolution achievable in MRI, and not the
tic images. The phase image occasionally
wavelength of the RF at the Larmor fre-
comes in handy, e.g., for depicting blood
quency. The axes kx and Ky constitute the
flow, or for high-lighting magnetic suscepti-
k-space grid of ‘raw data’ which is the
bility or other phase-dependent information.
Fourier inverse, or the diffraction pattern, of
‘k-space’: Raw Data the MR image. We can examine this
diffraction pattern with appropriate ‘low
Matrix by Another Name
frequency’ and ‘high frequency’ filters and
The concept of k-space is important for a convince ourselves of the following
proper understanding of the subtleties properties which are of crucial importance
involved in Fourier imaging.12,13 The raw in clinical MR imaging: (i) low spatial
data that are acquired in MR imaging are frequencies become encoded in the central
digital samples of the Fourier transform of area of k-space (around k=0), and these
the ‘object’, which in our case is defined as determine gross image features and image
the arrangement of the transverse magneti- contrast; (ii) the high spatial frequencies,
40 Biomedical Magnetic Resonance: Proceedings of the International Workshop
which are present at the peripheries of k- The EPI may be implemented in the GE
space, contain edge information and control or SE version. If a 180° RF pulse is inserted
spatial resolution. between the initial excitation pulse and the
All MRI imaging sequences strive to fill start of signal sampling, the sequence
k-space with optimum trade-offs in scan becomes the SE EPI.
time, signal-to-noise ratio, and resolution The EPI is somewhat limited by the fact
with minimal artefacts. Just exactly how that signal-to-noise ratio is relatively poor.
magnetic gradient trajectories fill k-space is Each line must be digitized within a very
what differentiates the wide variety of pulse short time, since all lines have to be scanned
sequences in use today. As a particularly before the T2 decay of the spin moments
important example of fast k-space filling, following excitation. Short acquisition time
we shall briefly discuss echo planar imaging for each line requires a large RF receiver
(EPI) in the next section. bandwidth and, consequently, noise is
higher. Spatial resolution is also usually
Echo-Planar Imaging (EPI) limited.
The EPI is a ‘snapshot’ strategy to map the The above limitations notwithstanding,
multiple phase-encoded lines of k-space after today’s rapid and spectacular developments
only one RF excitation. In other words, it is in the applications of MRI to real-time
a technique wherein the complete data set functioning of the brain and other organs
required to reconstruct an image is acquired have become possible mainly due to ultra-
from the MR signal produced by a single quick and ingenious strategies of mani-
RF excitation of the selected slice, provided pulating k-space.
that the magnetic gradients creating the
sampling trajectory could be switched CONCLUSION
rapidly enough and with sufficient magni- We have discussed the basic principles of
tude. The original concept, due to Mans- space-encoding the frequencies and phases
field,14 employed a continuous scan path of time domain in vivo MR signals to
zigzagging rectilinearly through the entire produce clinically useful MR images. Linear
k-space plane. This requires an ingenious magnetic field gradient pulses, when
manipulation of the encoding gradients imposed on the main magnetic field in a
whereby Gx is inverted after each line-scan controlled manner, achieve (i) selective
while Gy is applied very briefly in ‘blips’ activation of tomographic tissue slices, and
after completion of each line. Inversion of a (ii) the encoding of spin-echo or gradient
gradient reverses the scan direction in k- echo MR signals from an activated slice into
space. Gx is preferably strong and rapidly spatially resolved frequency and phase
switched to obtain very fast echoes (1 per dimensions. The 2D raw data acquired after
millisecond), while Gy is blipped to produce scanning a slice plane are shown to comprise
pulses coinciding with the zero crossings of spatial frequencies which are then Fourier-
the G x gradient. Programmed to act in transformed into a 2D MR image. The ‘k-
synchrony, these gradients generate the fast space’ concept is introduced for developing
zigzagging route through the k-plane. The a comprehensive understanding of the 2D
MR signal is continuously sampled during raw data array. Fast MRI pulse sequences
this k-space traverse, and the method is are distinguishable, in principle, by how
therefore, ultrafast and efficient. rapidly magnetic gradient trajectories fill
Fundamentals of Magnetic Resonance Image Production 41
Fig. 4.1: Sagittal MR image that is T1 weighted and Fig. 4.2: T2-weighted MR scan of the brain showing
in which contrast is mainly dependent on relaxation a large brain tumor and associated edema
times. The corpus callosum, for example, is bright,
whereas the cerebrospinal fluid is dark mainly
because of their different relaxation times compared includes the remainder of the host (water)
to the surrounding brain molecule as well as other solute and solvent
molecules. Spin-lattice relaxation occurs
such images are sensitive to a variety of because of magnetic interactions between
clinical disorders and may exhibit very high the nuclear spin dipoles and the local
contrast. For example, Figure 4.2 shows a randomly fluctuating magnetic fields that
brain tumor in an image that depends exist on an atomic scale inside any medium.
strongly on T2. Here the relaxation time of These originate mainly from neighboring
the cancerous tissue is more than 100 percent magnetic nuclei such as other hydrogen
increased compared to the normal brain. protons (e.g. within a water molecule, each
To understand the nature of the infor- hydrogen affects the neighbor) and are
mation portrayed by such images, it is modulated by the motion of other surroun-
worthwhile discussing what factors may ding dipoles in the lattice which have
affect the relaxation times of water within components fluctuating with the same
tissues. For proton NMR, after being distur- frequency as the resonance frequency. The
bed by an RF pulse, the nuclear magneti- recovery is very efficient when there is a
zation does not spontaneously recover very local fluctuating field that can provide a
fast, but relies almost entirely on interactions magnetic perturbation at the Larmor fre-
between the water molecules and the quency. For example, each proton in a water
surrounding medium to re-equilibrate. R1 is molecule has a neighboring proton that is
called the spin-lattice relaxation rate. The also a magnetic dipole which generates a
“lattice” denotes the molecular environment magnetic field at the neighboring proton of
surrounding a hydrogen nucleus, and about 5 gauss (0.5 milli Tesla). This field is
Contrast Mechanisms in MRI 45
constantly changing in amplitude and is different from that at lower fields. The
direction as the water molecule rotates effect of the molecular motion is usually
rapidly and moves about in the liquid. It expressed by a correlation time, τ c ,
also changes as a result of intermolecular characteristic of the time of rotation of a
collision, translation, or chemical dissociation molecule or of the time of its translation
and exchange. The magnetic field experi- into a neighboring position. Relaxation rates
enced by any nucleus will fluctuate with a in simple liquids are affected, for example,
frequency spectrum that is dependent on by viscosity, temperature, and the presence
the molecular tumbling due to the random of dissolved ions and molecules, which alter
thermal motion of the host and surrounding the correlation times of molecular motion
molecules. The mean strength of the local or the amplitudes of the dipolar inter-
field is determined by the strength of the actions.
magnetic dipoles in the medium and how Whereas T1 is sensitive to radiofrequency
close they approach to the hydrogen nuclei. components of the local field, T 2 is also
The component of the frequency spectrum sensitive to low frequency components.
which is equal to the resonant frequency, When an ensemble of nuclei is excited with
(or, for reasons beyond our discussion, twice RF a transverse component of magnetization,
as high) is effective in stimulating an energy orthogonal to the applied field direction,
exchange to induce recovery back towards may develop, and it is this component that
equilibrium, i.e., T1 relaxation. In liquids the rotates with the Larmor frequency and
characteristic frequencies of thermal motion induces the MRI signal in a receiver coil. T2
are of the order of 1011 Hz or higher, much reflects the time it takes for the ensemble
greater than NMR frequencies of 107–108 to become disorganized and for the trans-
Hz. Consequently, the component of the verse component to decay. Since any growth
frequency spectrum from molecular motion of magnetization back toward equilibrium
that can induce T1 relaxation is small and must correspond to a loss of transverse
the process is slow. As the molecular motion magnetization, all contributions to T1 relaxa-
becomes slower, either due to lower tion affect T2 at least as much. In addition,
temperature, or increased molecular size, components of the local dipolar fields that
the intensity of the fluctuations of the oscillate slowly, at low frequency, may be
magnetic field at the resonance frequency directed along the main field ( Bo ) direction
increases, reaches a maximum, and then and thus, can modulate the precessional
decreases again as the energy of the motion frequency of a neighboring nucleus. Such
becomes increasingly concentrated in frequency perturbations within an ensemble
frequencies lower than the NMR range. of nuclei result in rapid dephasing of the
Thus, R1 passes through a maximum value transverse magnetization and accelerated
when the molecular tumbling rate matches spin-spin relaxation. Since the low frequency
the Larmor frequency as the molecular content of the local dipolar field increases
motion becomes slower. It may be noted monotonically as molecular motion pro-
from this that relaxation times depend on gressively slows, although T1 passes through
the frequency of the NMR measurement, a minimum value, T2 continues to decrease
and thus are usually shorter at low field and then levels off, so that T1 and T2 then
strengths. Thus the contrast at higher fields take on quite different values.
46 Biomedical Magnetic Resonance: Proceedings of the International Workshop
In the picture developed above, relaxa- water relaxation times are shorter than in
tion results from the action of fluctuating pure water because of the presence of large
local magnetic fields experienced by protons, macromolecules and chemical species that
which stimulate the return to equilibrium promote relaxation. Proteins and other
of an excited population of spins. In pure macromolecular structures produce, via
water the dominant source of such effects protons on their surfaces, dipolar fields that
is the dipole-dipole interaction between fluctuate relatively slowly and which do not
neighboring protons, mainly between average out on the NMR timescale, and
hydrogen nuclei in the same water molecule. these are efficient sites for relaxation. Water
The tumbling of each water molecule then molecules or single protons may exchange
causes the weak magnetic field produced between these sites and the rest of tissue
by each proton to fluctuate randomly, and water, thereby spreading the effect. Proteins
at the site of a neighboring proton these are often considered to contain one or more
random alterations in the net field produce layers of hydration, which is often loosely
relaxation. The time scale characteristic of termed the “bound water” fraction, and this
the dipolar interaction reflects molecular may play an important role in mediating
motion and clearly is expected to influence interactions between the bulk aqueous
the efficacy of relaxation. Qualitatively, medium and the macromolecular surface.
when there is a concentration of kinetic Some specific chemical side groups, notably
motion in the appropriate frequency range, hydroxyl and amide moieties, are known
relaxation will be efficient. We can envisage to be efficient conduits for relaxation, so
other types of motion which will be too when these occur in high abundance the
rapid or too slow to be effective. The key relaxation will be fast, but in general the
important descriptor is the correlation time, large array of environments and chemical
τc which measures the time over which the species within tissue usually make it
local fluctuating field appears continuous impossible to identify specific interactions
and deterministic. It represents the time it that dominate. Figure 4.3 shows MR images
takes on average for the field to change that are sensitive to relaxation times of two
significantly. eggs – one boiled and one fresh. In the
In simple liquids such as water the boiled egg the proteins are cross-linked and
molecular motion is rapid and on average heat denatured, and this induces gross
is isotropic. The motions are so fast that changes in the water relaxation times
relaxation is not very efficient—the dipolar compared to the fresh egg, even though the
fields fluctuate too rapidly to be very effi- elemental composition has not altered.
Solutions of macromolecules and biologi-
cient, and the motion averages out any net
cal tissues are chemically heterogeneous and
effects of the local fields (so-called motional
thus, water in such media may experience a
averaging). In pure water, T 1 is several wide variety of different environments and
seconds long, whereas in tissues water chemical species with which to interact. Even
relaxation times are often substantially in simple protein solutions, there may be
under 1 second. In pure water most of the different ranges and distributions of
relaxation is accounted for by the effects of correlation time, coupling strengths and
intra-molecular dipolar effects from protons molecular dynamics, that affect the local
within the same H2O molecule. In tissue, dipolar fields experienced by water protons.
Contrast Mechanisms in MRI 47
In general, larger proteins produce more water proton relaxation will depend on how
efficient relaxation per unit mass than effectively, and at what rate, these effects
smaller molecules. An even greater variety are spread through the rest of the water
of different scales and types of constituents population. Such exchange processes have
occur within cells and whole tissues. Tissues profound effects on the observable NMR
contain diverse, freely tumbling solute ions relaxation phenomena. In a time of 50 msec,
and molecules such as small proteins and water molecules diffuse distances of the
lipids as well as relatively immobilized or order of 20 μm, so that they sample many
even rigid macromolecular assemblies such different environments on the cellular level
as membranes and mitochondria. Tissues are
within the timescale of relaxation.
also spatially inhomogeneous, containing
In many (though by no means all)
many different types of cell or structure,
and there may exist multiple compartments situations, very rapid exchange occurs
that are not connected or in which water between bulk water and bound and inter-
transport is restricted. Nonetheless, facial water in biological systems. Consider
although tissues are markedly heterogeneous the situation in which there are two such
at the cellular level, NMR relaxation will populations in rapid exchange; one is a bulk
still reflect the average character of local solvent phase with relaxation time Ta and
dipolar fields experienced by water protons. the other is a small hydration layer fraction
whose relaxation time Tb is much shorter
Multi-compartment Systems and the because it is in an efficiently relaxed environ-
Effects of Exchange ment associated with the “dry” solute
material. If one gram of the dry weight
If at any time only a small proportion of biological material directly hydrates and
water protons are in close juxtaposition to decreases the relaxation times of C grams
efficient relaxation sites, then the average of water, and this then exchanges rapidly
Fig. 4.3: T2 -weighted MR images of two eggs. The right egg is fresh and has a long T2, whereas the left egg
has been boiled. The heat denatures and cross-links the macromolecules in the egg which drastically reduces
the relaxation times of the egg white
48 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 4.4: T2 sensitive echo planar images through a human leg. The image on the right was obtained after
2 minutes dorsiflexion exercise after which the relaxation time of the anterior tibialis muscle increases
5
RF Pulse Sequences Including
Rapid Imaging Sequences
Rao P Gullapalli, Jiachen Zhuo
the extreme ends of the k-space are subjected The high frequency components, which are
to a more severe phase dispersion. Since at the periphery of k-space on the other
the starting phases of the views, closer to hand provide resolution to the images. One
the center of k-space receive the least amount should remember that k-space is band
of phase dispersion, the echo signal is the limited in terms of frequencies in each of
highest for these views. This also suggests the directions. Of course, in order to obtain
that these views will have preserved in them a high resolution image one could sample
the low frequency content. Signals that have longer and longer. It should be remembered
low frequency information also contribute that the gain in resolution comes at a cost
towards the signal to noise of the image. in terms of scan time and a loss of signal to
Conversely echoes that are encoded with noise. Hence, an optimum balance between
the largest phase encode gradient, such as sampling time and signal to noise should be
the 128th view will have faced a severe de- maintained when it comes to resolution.
phasing of the echo signal. The large amount All images as seen in MR are a result of
of de-phasing provided by the phase encode a two-dimensional Fourier transform of the
gradient essentially removes all the low raw data that is obtained from the RF
frequency components within the signal and receiver. The realization of the source of
the read gradient captures only the high contrast and resolution and the improve-
frequency components of the excited slice. ment in gradient technology has allowed
Essentially low frequency components get many an investigator to develop novel pulse
encoded in the center of k-space and higher sequences that provide snapshot images.
and higher frequency components are
encoded as one travels to the periphery of
the k-space. This concept is crucial and forms
the basis for the design of novel pulse
sequences.2,3
Thus as the number of k samples increase,
the spatial resolution of the image is
increased. In order to construct a complete
image from k x and k y all possible
combinations of k must be sampled along
each axis for the desired resolution. In order
to gain an understanding of pulse sequence
design it is necessary for one to understand
the source of contrast and resolution within
an image. The configuration of a typical
k-space is seen in Figure 5.1 along with the
location of the different frequency
components and their contribution to the
Fig. 5.1: Configuration of k-space showing the
image. The center of k-space, which mainly
distribution of different frequency components and
contains low frequency components the main sources that contribute to contrast and
provides the contrast in the resulting image. resolution
54 Biomedical Magnetic Resonance: Proceedings of the International Workshop
You may recall that a standard spin-echo contrast is T2-weighting with an echo time
sequence takes 8 minutes and 32 seconds of 100 ms then one has to wait 50 ms to
to complete the acquisition of a T2-weighted apply the refocusing pulse followed by
image at a TR of 2 sec and 256 × 256 another 50 ms to capture the echo. During
resolution. This sequence is shown in Figures both the wait periods either the signal is
5.2A and B along with its associated scheme decaying or is being build-up to form the
of filling k-space. Only one line in k-space echo. Rather than waiting for this decay
is filled for every TR. Recall that higher and build-up during the imaging sequence
spatial frequencies are encoded at the it is possible with the new high-speed and
periphery since maximum de-phasing occurs self-shielded gradient technology to
through the use of a large phase encode generate multiple echoes during that time.
gradient. Lower spatial frequencies are That is, echoes may be generated every few
encoded during the time that the phase milliseconds, say every 10 ms, for as long
encode gradient is small or absent. as the signal might last. While one could
The limitations of such a sequence is that use this sequence to obtain images at TE’s
only one line in k-space gets encoded per in multiples of 10 ms, one could also use
TR and hence the duration of the scan is these echoes cleverly to cover k-space with
TR × desired resolution. If the desired the goal of minimizing scan time. For
A B
Figs 5.2A and B: (A) Pulse diagram for a spin-echo sequence, and (B) its corresponding k-space filling
scheme. The slice-selection process leaves one at the center of k-space. The x- and y-gradients then moves
one across or up and down the k-space depending on the polarity of the individual gradients. In this example,
although the x- and y- gradients are applied simultaneously we have shown for simplicity, the vector moving
along the x-direction first and then moving up to the maximum point indicating the application of maximum
phase-encode gradient. The 180o slice-selective refocusing pulse moves this vector through the origin to the
other side of k-space. At this point a positive read-gradient is applied and simultaneously the analog to digital
converter is turned on which reads out the first line in k-space. This process is continued until each of the lines
is read-out by appropriate scaling of the phase-gradient during each TR
RF Pulse Sequences Including Rapid Imaging Sequences 55
example, one could construct a multi-echo number of refocusing pulses used is typically
sequence using 16 refocusing rf-pulses with called the echo train length (ETL) or the
an echo spacing of 10 ms. Such a sequence TURBO-factor. The ETL or the TURBO-
could potentially generate 16 different k- factor gives an indication of the speed of
spaces that provided 16 different images such a sequence when compared to a tradi-
with different T2-weightings. Alternatively, tional spin-echo sequence.4,5 For example a
one could construct a single k-space from 16 ETL sequence would be 16 times faster
the different echoes by forcing the desired or have a TURBO-factor of 16 compared to
echo (or T2-weighting) to occupy the center a conventional spin-echo sequence. Such class
of k-space and those echoes farthest from of sequences is called Fast Spin-Echo (FSE)
this desired weighting to occupy the or TURBO spin-echo sequences. The example
periphery of k-space. The T2-weighting of shown in Figures 5.3A and B has a TURBO
the echo that is at the center of k-space factor of 4. Please note that while in this
would dictate the contrast in the image example the fourth echo was chosen to be
whereas the rest of the echoes would the center of k-space any of the other echoes
provide resolution to the image. Which of could have been used to encode the center
the 16 echoes occupies the center of k-space just by changing the phase encode gradients
is determined by the T2-weighting desired to obtain the desired contrast.
in the image. An example of a four-echo The inherent advantage of FSE is its
scheme is shown in Figures 5.3A and B speed compared to a conventional spin-echo
where the fourth echo is used to encode sequence. This advantage has to be taken in
the center of k-space, the first echo encodes context with other issues that arise when
the periphery of k-space and the other two using FSE. Since the number of RF-pulses
echo make up the intermediate space. The required to produce an FSE image is much
A B
Figs 5.3A and B: (A) Pulse diagram of a four-echo-fast spin-echo sequence and (B) the k-space associated
with this sequence. It should be noted in this sequence that after every echo is read the phase encode
gradient is rewound prior to the application of the next refocusing pulse. Please follow the k-space traversal
diagram at the center of k-space but after the first refocusing pulse. For the second half of k-space the
polarities of the phase gradient change
56 Biomedical Magnetic Resonance: Proceedings of the International Workshop
greater than a conventional spin echo on the resolution required. It is also used
sequence, heat deposition (measured as to image the abdomen as it allows one to
specific absorption rate (SAR)) to the patient minimize on respiratory artifacts by
becomes a factor and increases linearly with obtaining images within a breath-hold.
the number of refocusing pulses used per Currently, single shot FSE sequences are
unit TR. This becomes even more of a used routinely for cholangiopancreatography
limiting factor at field strengths greater than (MRCP) where they have proven to be an
1.5 Tesla since SAR increases as square of excellent alternative for ERCP and also in
the field strength. At higher field strengths diffusion-weighted imaging where the goal
either longer inter-echo spacing needs to be is to create diffusion anisotropy maps or
used or refocusing RF pulses with less power perform fiber tractography.9-11
need to be used. Another issue is the The above sequence type shows how
contrast of the images themselves. When refocused spin echoes can be used to make
comparing an FSE image with a standard up the k-space required for image formation
spin-echo sequence, it is obvious that the with a given contrast. One could also do
FSE images have a smooth look to them. the same using refocused gradient echoes
This is because there is some amount of T2 where the echo is reversed several times
smoothing that occurs since several echoes using the read-out gradient. Such an
were responsible for the creation of the FSE acquisition is commonly called the echo-
image. Hence when relating an FSE image planar imaging (EPI) technique. 12 Each
to a standard spin-echo image the echo time reversal of the gradient creates another
is referred to as the effective echo time or echo, which can be phase encoded to a
Teff.5 One would also notice that the FSE different line in k-space. Figures 5.4A and B
images have a somewhat reduced contrast show a typical single-shot EPI sequence
(such as between gray and white matter) where all the k-space data is acquired after
since the images are subject to magnetization a single RF-excitation. Notice that every
transfer effects due to the application of a alternate line in k-space is read in the
series of 180o pulses.6 Fat is bright on T2- opposite direction. This is due to the nature
weighted images using FSE. This is due to of the acquisition where every other echo is
the fact that the multiple train of closely obtained with reversed polarity of the read
spaced refocusing pulses essentially gradient. In such an acquisition, one has to
eliminates the J-coupling modulation reverse these lines in k-space prior to 2D-
enabling the fat to be bright.7 fourier transform. Any off-resonance signal
In clinical practice, FSE is routinely used (such as fat) will encounter a position shift
for T2-weighted imaging. A traditional fluid due to the long duration of data sampling
attenuation inversion recovery (FLAIR) (50-100 ms). Fat that is 225 Hz away from
sequence would take unreasonably long water frequency will be shifted by about 12
period of time to complete the scan due to pixels for a read-out time of 50 ms and will
its requirement of long inversion time and appear as an artefact. Hence it is necessary
echo time even at low resolution. 8 FSE to minimize these off-resonance artefacts by
makes such imaging possible as it cuts down good shimming over the region of interest
the scan time to within 4 minutes depending and also by suppressing these off-resonance
RF Pulse Sequences Including Rapid Imaging Sequences 57
A B
Figs 5.4A and B: (A) Pulse diagram for a single-shot spin-echo EPI sequence and (B) its traversal in k-space.
Following a single excitation, the data is read-out by reversing the read gradient while at the same time
‘blipping’ the phase gradient. Every alternate line then needs to be reversed, and phase corrected prior to
Fourier transform
multi-shot EPI is that the requirements for these sequences have also been explored and
the gradients are not as stringent as for single- show excellent promise for the future.26
shot EPI.19 In such situations multi-shot EPI A relatively new sequence that incorpo-
provides a better alternative to standard rates both spin echoes and gradient echoes
gradient-echo sequences when temporal is called the GRASE. Figures 5.6A and B
resolution is required. One should remember shows a typical GRASE acquisition and its
that the potential for artifacts increases with associated k-space traversal. GRASE can be
the number of potential discontinuities in k- thought of as a combination of FSE and
space. To accurately correct for these artifacts EPI.27,28 For each spin echo formed one could
it may be necessary to obtain an additional generate several gradient echoes. Such an
navigator echo that can be used to minimize acquisition has several advantages. Firstly,
these artifacts.20 the received signal is kept alive for a longer
All the above sequences described above duration due to refocusing; secondly,
including the EPI sequence obtain data in a susceptibility artifacts are much reduced
rectilinear fashion and the corresponding compared to EPI; thirdly, the acquisition
k-space essentially follows a raster scanning will have generally a better signal to noise
system. Several variations from this raster compared to EPI; and finally, the acquisition
scanning have been successfully implemented is less prone to run into SAR limitations
by various investigators. For example it is compared to a similar FSE acquisition since
possible to sample k-space radially by the 180o pulses are spaced further apart.
turning on the x- and y-gradients simul- While the use of GRASE has been shown
taneously with varying proportions. The for several applications, its use might
reconstruction of such a data would then increase as higher and higher field strength
use projection reconstruction techniques as magnets become part of the main stream.29
done in CT.21 Perhaps the most useful and In general, GRASE might be thought of as
promising acquisitions among the variants a class of sequences that encompasses both
of EPI is the spiral scan where the coverage FSE and EPI. In FSE for each refocusing
of k-space is accomplished in a spiral fashion pulse there is a gradient-echo, whereas in
starting from the center of the k-space.22,23 spin-echo EPI for each refocusing pulse there
Figure 5.5 shows a typical k-space trajectory are multiple gradient echoes. So one could
used in spiral imaging. Both the x- and think of FSE as a GRASE factor of 1 whereas
y-gradients spiral out starting at the center in the example shown in Figures 5.6A and
of k-space. This technique makes efficient B the GRASE factor is 3 where one obtains
use of the available gradients as the three echoes per refocusing pulse. Similarly,
gradients are smoothly varying and is the GRASE factor in a single-shot EPI
inherently less sensitive to motion.24 Since sequences would be the number of gradient
the acquisition is not Cartesian in nature, echoes obtained to cover k-space.
the raw data obtained needs to be
regridded to a Cartesian co-ordinate system STEADY-STATE IMAGING PHYSICS
prior to Fourier transform. Other variants
on spiral imaging such as the Rossette has Gradient-echo sequences can also be used
also been proposed for efficient sampling with similar scanning parameters as the spin
of k-space.25 Multi-shot and 3D versions of echo sequences but the image quality is
RF Pulse Sequences Including Rapid Imaging Sequences 59
rather poor due to susceptibility effects at the object being imaged. As the TR is
longer echo times and artifacts due to flow. shortened such that it gets comparable to
However, the biggest advantage of gradient the T2 of the tissue, the MR signal persists
echo type scans is the ability to perform from one excitation to the next. So as the
fast imaging or steady-state imaging. Steady excitation process continues from TR to TR,
state imaging involves the use of excitation the signal strength and contrast of the
pulses usually less than 90o at a very rapid resultant image are governed by the history
rate usually at the rate where TR<<T2 of of the spins over several TR’s.
Fig. 5.5: k-space filling scheme for a spiral EPI scan. In this case both the x- and y-gradients spiral out from the
center of k-space. The sequence is less sensitive to flow artifacts and makes efficient use of the gradients.
60 Biomedical Magnetic Resonance: Proceedings of the International Workshop
When using conventional sequences, the completely decay. During these first few
TR is generally much longer than the T2 of TR’s or RF excitations, the magnetization
the tissues being imaged. In other words, will be found in a different direction each
the transverse relaxation process is complete time resulting in different signals during
prior to application of the next RF pulse to each TR interval (for a mathematical
encode the next phase-encode view. Hence treatment of steady-state spin physics,
the chance of any spins having a history of please see the appendix). This creates
the previous excitation is negligible. stimulated echoes whose amplitude is half
However, in steady state imaging where that of a regular spin-echo.30,31 Depending
TR approaches or becomes less than the T2 on the spacing of the pulses and the
of the imaging region the history of the relaxation properties of the tissues under
spins persists for several TR’s since the consideration this stimulated echo could
magnetization has not had a chance to interfere with the desired echo and cause
interference. Figures 5.7A and B show the TR is reduced and manifest as bright bands
effect of closely spaced RF pulses when they (commonly called FLASH bands) parallel to
are unequally spaced (a) and when they are the frequency encode direction. The
equally spaced (b). Typically n RF pulses brightest of these bands occur around the
applied to a spin system can generate up to physical center of the phase encode gradient
(3n-1 - 1) echoes following the last RF pulse. since the spins in this region receive the
The stimulated echoes produced are typically least amount of dephasing during the
T 2 -weighted and their origin is from application of the phase encode gradient.
previous RF pulses. It is necessary then to As a result these spins preserve the history
de-phase this unwanted signal prior to the of previous excitations and introduce some
application of the next RF pulse. T2-weighting into the images. These bands
As seen in Figure 5.8, after several also occur in places where the phase
excitations the RF and the magnetization encoding pulse is able to produce a complete
come to a steady state wherein the 2 π rotation of magnetization. The strength
magnetization due to each RF pulse is exactly of these bands is a function of T1, T2, TR,
compensated by changes due to precession and flip angle. To appreciate the contrast
and relaxation during the short TR interval. mechanisms in steady state imaging, it is
However artifacts begin to develop as the essential to understand that the image
B
Figs 5.7A and B: (A) Three RF pulses spaced unequally between them will produced five echoes one of
which is the stimulated echo, (B) A train of equally spaced RF pulses produce several echoes
62 Biomedical Magnetic Resonance: Proceedings of the International Workshop
relaxation properties. Contrast manipulation and read gradients are reversed. While the
is done through the appropriate choice of first a pulse will pull down some magneti-
these sequence parameters. TR determines zation onto the transverse plane, the next a
the optimum flip angle to be used in order pulse will form the spin-echo and the
to obtain the maximum signal to noise ratio subsequent a pulses will form a combination
as given by the Ernst angle. Long TE’s of spin and stimulated echoes. The
provide images with T2*-weighting whereas stimulated echo will be coincident with the
shorter TE’s produce images with high spin-echo since the RF pulses are spaced
signal-to-noise ratio (SNR) with minimal equidistantly in time. The effective TE of
motion artifacts. The choice of an such a sequences is (TR+TE) since the echo’s
appropriate flip angle will provide images origin is truly from a previous RF excitation.
ranging from proton density weighted (low Subsequent echoes will have contributions
flip angles) to highly T1-weighted images from even earlier RF excitations. One
(higher flip angles). As a general rule for should expect an image which is more
T1-weighting in the images one could use a heavily T 2 -weighted compared to that
short TR (50 ms), short TE (4 ms) and a obtained from GRASS or FAST imaging
high flip angle (50 degrees). Of course one sequence types albeit with much lower signal
could use even a shorter TR and a higher to noise ratio. Such rapid imaging sequences
flip angle for even higher T1-weighting but that produce T 2 -weighted images are
the reader should be cautioned that the SNR commonly called Contrast Enhanced-FAST
will be greatly diminished. To obtain proton (CE-FAST), PSIF (reverse of FISP) and
density weighting one could use a long TR, Steady State Free Precession (SSFP, GE).31,32
short TE and a small flip angle. For T2*- For more details on the different acronyms
weighting a long TR, long TE and a small used for different flavors of gradient echo
flip angle is preferred. The small flip angle sequences please see reference 38.
and the long TR ensures minimization of
any T1 effects in the image.32-34 TrueFISP
In the case of GRASS, FISP, and FAST, Improvement in gradient coil technology has
the free induction decay of the signal coming produced coils with higher and higher peak
from the RF is sampled, while the spin-echo gradients while at the same time reducing
and the stimulated echo components the rise times. These gradients are also
emanating from the string of RF pulses are capable of performing at high duty cycles
kept away from the sampling window. Since with excellent eddy current compensation.
the RF pulses are fired at constant intervals Because of this, techniques such as FISP
the spin-echo and the stimulated echo which were first described in 1986 have
overlap. One can then ignore the FID part become practical in the clinical setting as
of the signal and move the sampling this technique demands high duty cycle.34
window so as to capture the spin-echo The TrueFISP (Siemens) sequence, also
signal, which has more T 2 -weighting known as Fast Imaging Employing Steady-
associated with it. The sequence diagram state Acquisition (FIESTA, GE) and Balanced
for such a pulse sequence resembles that of Fast Field Echo (Balanced FFE, Philips), uses
the sequence for GRASS except that the slice fully balanced gradients in all directions
64 Biomedical Magnetic Resonance: Proceedings of the International Workshop
(slice, read, phase encoding directions) to since phase errors accumulate with longer
refocus all the transverse magnetization as TR’s. Steady state magnetization is critical
shown in Figure 5.9.39 This allows for the for TrueFISP which also minimizes the off-
steady state of both the longitudinal and resonance effect. However, it takes several
transverse magnetization to be maintained. TR’s (equivalent to a few T1’s of the tissue
Therefore TrueFISP can be used with very being imaged) to reach this steady state.
large flip angle without spin saturation Techniques are available through the use of
problem. Due to the intrinsic high SNR, it is steady-state preparation pulses to bring the
then possible to use the highest available magnetization to a steady state in a shorter
receiver bandwidth to achieve even shorter time.42
TRs.
Since the signal in TrueFISP is a mixture DESS (Dual Echo in the Steady State)
of echoes refocused from all the previous Another sequence called the DESS is a
RF excitations with different amplitudes, the variation of the fast gradient echo sequences
signal behavior is a complicated function of such as FLASH or GRASS which uses long
the flip angle, TR, T1, T2. Nevertheless, the readout gradient to focus a second gradient
image contrast of TrueFISP is generally echo as shown in Figure 5.10 to introduce
believed as T2-T1-weighted.40 So TrueFISP T2-weighting to the image.43 The first echo
has found its most applications in cine heart has mixed T1/T2-weighting with high SNR
imaging or coronary arteries imaging due and the second echo is strongly T2-weighted
to its high contrast between blood and but with low SNR due to T2 decay. Signals
myocardium41. Further, the ultra-fast speed from both echoes are then averaged to
also makes it very suitable for breath hold achieve a reasonable T2-weighting and high
imaging in order for higher resolution, SNR. One main problem with DESS is its
shorter imaging time and hence less motion sensitivity to motion due to the prolonged
artifacts. TR. 3D-DESS has been routinely used in
Due to the fact that TrueFISP signal cartilage imaging for a high contrast noise
depends on coherences set up from several ration between the thin sheet of cartilage (low
RF pulses prior, it is very sensitive to off- T2) and joint fluid (high T2).
resonance effects. Hence the use of long By adjusting the echo times for the two
TR’s could lead to image quality problems echoes, one could also vary the relative
phase of fat and water, thereby making the magnetization is then imaged with a rapid
sequence sensitive to chemical shift differen- gradient echo sequence that captures the T2
ces. This sequence has been used to identify contrast. For more detailed theoretical
adenomas of the adrenal glands by descriptions of the above techniques the
simultaneous acquire in-phase/opposed- reader will find references 47, 49 and 50
phase images.44 helpful.
Other forms of steady state imaging that Burst imaging is another novel technique
make clever use of steady state sequences derived from the behavior of steady state
for a desired contrast are also available. pulse sequences. In this technique a series
One could prepare the longitudinal magneti- of RF pulses are applied in the presence
zation such that the magnetization has a T1 of a small x-gradient. 51,52 This is followed
or a T2 history prior to rapidly imaging the by a 180 o pulse that refocuses all the
magnetization. 45-47 Such sequences are echoes that are then read-out using the same
generally termed magnetization prepared x-gradient to sample all the echoes as shown
rapid acquisition techniques. One such in Figure 5.11. A y-gradient is also applied
sequence uses an inversion pulse followed simultaneously to move the echoes to
by a delay prior to capturing all the views various parts of k-space.53,54 The inherent
required to make a single image. The delay signal-to-noise of this scan is low compared
after the inversion pulse is to let the to an EPI sequence since the full echo is
magnetization recover to a point where the never sampled and the echo-intensity is
contrast between any two tissues is the distributed through k-space.51 Theoretically,
greatest and follow this up with a rapid the SNR of such a scan is reduced by a
gradient-echo imaging scheme. Such a factor of √N where N is the number k-
sequence commonly called magnetization space lines sampled in the burst acquisition.
prepared rapid acquisition gradient-echo While the utility of burst imaging is limited
(MP-RAGE), provides a highly T1-weighted due to its poor SNR it can be useful in special
scan with the T1 contrast being far superior applications of fMRI where the acoustic
to the one obtained with a regular T 1 - noise of the scanner needs to be low. Burst
weighted sequence. Similarly one could also acquisitions tend to have low acoustic noise
obtain a very highly T2-weighted image by compared to EPI acquisitions since there is
preparing the longitudinal magnetization no rapid switching of gradients.
such that it has a T2 history. Such a sequence
is called driven equilibrium fourier trans-
form or DEFT acquisition.48 The preparation
part of this sequence consists of a normal
spin echo(90x-180y-90-x) pulses. The phase
of the second 90o pulse would be adjusted
such that the transverse magnetization,
which is T 2 -weighted is now in the Fig. 5.11: Pulse sequence diagram for BURST
longitudinal direction. This longitudinal sequence
66 Biomedical Magnetic Resonance: Proceedings of the International Workshop
A B C
Figs 5.12A to C: Free precession of the steady state signal. (A) At time t=0 the magnetization is rotated by a
RF pulse with an angle α. (B) This magnetization precesses about the main field determined by the local
inhomogeneities at an angle ϕ between time t=0 and t=TR and is a function of the relaxation properties of the
signal. (C) The magnetization returns to its steady state
68 Biomedical Magnetic Resonance: Proceedings of the International Workshop
23. Noll DC, Cohen JD, Meyer CH, Schneider W. 38. Elster AD. Gradient-echo MR imaging: Techni-
Spiral k-space MR imaging of cortical activation, ques and acronyms. Radiology 1993;186:1.
J Magn Reson Imag 1995;5:49. 39. Duerk JL, Lewin JS, Wendt M, Petersilge C.
24. Meyer CH, Hu BS, Nishimura DG, et al. Fast Remember true FISP? A high SNR, near 1-
spiral coronary artery imaging. Magn Reson second imaging method for T2-like contrast in
Med 1992;28:202. interventional MRI at .2 T. J Magn Reson
25. Noll DC, Peltier SJ, Boada FE. Simultaneous Imaging 1998;8:203-8.
multislice acquisition using rosette trajectories 40. Zur Y, Stokar S, Bendel P. An analysis of fast
(SMART): A new imaging method for functional imaging sequences with steady-state transverse
MRI. Magn Reson Med 1998;39:709. magnetization refocusing. Magn Reson Med
26. Yen YF, Han K, Heiss S, Birdwell RL, Herfkens 1988;6:175-93.
RJ, Sawyer-Glover AM. Glover GH. Dynamic 41. Fuchs F, Laub G, Othomo K. TrueFISP—
breast MRI with spiral trajectories: 3D versus technical considerations and cardiovascular
2D. J Magn Reson Imaging 2000;11:351. applications. Eur J Radiol 2003;46:28-32.
27. Oshio K, Feinberg DA. GRASE (gradient- and 42. Busse RF, Riederer S. Steady-state preparation
spin-echo) imaging: A novel fast MRI technique. for spoiled gradient echo imaging. Magn Reson
Magn Reson Med 1991;20:344. Med 2001;45:653-61.
28. Feinberg DA, Oshio K. Gradient-echo and spin 43. Hardy PA, Recht MP, Piraino D, Thomasson D.
echo) MR imaging: A new fast clinical imaging Optimization of a dual echo in the steady state
technique. Radiology 1991;181:597. (DESS) free-precession sequence for the
29. Feinberg DA, Kiefer B, Litt AW. High resolution imaging cartilage. J Magn Reson Imag
GRASE MRI of the brain and spine: 512 and 1996;6:329-35.
1024 matrix imaging. J Comput Assis Tomogr 44. Namimoto T, Yamashita Y, Mitsuzaki K,
1995;19:1. Nakayama Y, Makita O, Kadota M, Takahashi
30. Frahm J, Merbold K-D, Hanicke W, et al. M. Adrenal masses: Quantification of fat content
Stimulated echo imaging. J Magn Reson 1985;64: with double-echo chemical shift im-phase and
81. opposed-phase FLASH MR images for
31. Haacke EM, Wielopolski PA, Tkach JA. A differentiation of adrenal adenomas. Radiology
comprehensive technical review of short TR, 2001;218:642-46.
fast, magnetic resonance imaging. Review of 45. De Lange EE, Mugler JP III, Bosworth JE, et al.
Magnetic Resonance in Medicine 1991;3:53. MR imaging of the liver: Breath-hold T1-
32. Gyngell ML. The application of steady-state weighted MP-GRE compared with conventional
free precession in rapid 2DFT NMR imaging: T2-weighted SE imaging - Lesion detection,
FAST and CE-FAST sequences. Magn Reson localization, and characterization. Radiology
Imaging 1988;6:415. 1994;190:727.
33. Gyngell ML. The steady-state signals in short- 46. Lee H, Price RR. Diffusion imaging with the
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34. Chien D, Edelman RR. Ultrafast imaging using 47. Mugler JP III, Brookemen JR. Three-dimensional
gradient echoes. Magn Reson Quarterly magnetization-prepared rapid gradient-echo
1991;7:31. imaging (3D MP RAGE). Magn Reson Med
35. Oppelt A, Graumann R, Barfuss H, et al. FISP 1990;15:152.
- a new fast MRI sequence. Electromedica 48. Becker ED, Ferretti JA, Farrar TC. Driven
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36. Haase A, Frahm J, Matthaei D, et al. FLASH new method for nuclear magnetic resonance
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Overview of an MRI System 71
6
Overview of an MRI System
RS Saini
MR SYSTEM OVERVIEW
Fig. 6.1: Figure gives a general overview of the major components of the
MR system and their interconnections
Overview of an MRI System 73
and further processing. This procedure in assurance programs. They contain material,
new systems is performed by software while which can produce MR signal. Water is the
in earlier systems dedicated hardware was most commonly used material in the MR
used for this purpose. phantom.
7
In vivo MR Spectroscopy
Stefan Röll
experiments. Larger macromolecules use a small surface coil to excite and detect
exhibiting low T2 values become visible in signals from the superficial region of
spectra as baseline modulations. interest. However, for reaching region of
An expensive way of lowering the interests within the body in brain, prostate
detection limit of MRS is increasing field or breast MRS, additional localization by
strength. For clinical studies, field strengths slice selection or phase encoding is required.
ranging from 0.2 up to 7 T have been used With single “voxel” spectroscopy (SVS), a
today. Within this range, noise increases single spectrum from a defined location is
approximately linearly with B0, while signal acquired; chemical shift imaging (CSI) or
amplitudes increase quadratically. However, spectroscopic imaging (SI) refers to the
the expected linear net-increase in SNR can simultaneous acquisition of multiple voxels.
be only observed if the same field The location of the spectra is conveniently
homogeneity is reached. As susceptibility planned on anatomic MR reference images,
based distortions increase with increasing acquired beforehand. While since the
B0, many areas of the human body show a introduction of MRS many localization
less homogeneous B0 at high B0 compared techniques have been invented and tested,
to low B0 despite shimming. This is the major only the few most robust ones have
source of signal loss at high fields. As T1 prevailed in clinical practice. Those are
increases and T2 decreases with increasing described below.
B0, there is also the tendency of relaxation
counter balancing field-dependent SNR gain; Single Voxel Spectroscopy
however, the variation of in vivo relaxation The single voxel spectroscopy sequence
properties is minor for many metabolites of STEAM (stimulated echo acquisition mode)
interest for 1H MRS.l,2 Apart from SNR, the detects the “stimulated echo” generated by
other user-relevant quantity positively three consecutively applied RF-pulses of 90o
affected by increased field-strength is flip-angle each (Fig. 7.1).3,4 Each of the RF-
spectral resolution. The chemical shift pulses is slice-selective. Since their
dispersion Δf of a spectral line with respect underlying gradient pulses are applied along
to a reference line scales linearly with B0. mutually orthogonal directions, these pulses
To allow for comparison of spectra obtained define a cuboidal subvolume, i.e. the voxel:
from different B0 scanners, the ordinate of The first pulse selects a slice within the
spectra is usually scaled by the relative sample, the second orthogonal pulse selects
dispersion with respect to B0, in parts per a pencil-shaped cuboid from within the slice,
million: Δf 106 / f0. So while the spectral and the third pulse cuts the voxel out of the
positions of two identical singlet lines pencil shape. However, the three pulses will
remain identical at B0 and 2B0, they appear not only generate the desired stimulated
half as wide or twice as good separated at echo, but seven other “coherence pathways”
2B0. will be populated: FIDs generated by each
of the three pulses, and spin-echoes
LOCALIZATION TECHNIQUES
generated by any two of the pulses need to
For studying, e.g. calf muscle or tumors close be eliminated since magnetization of those
to the body surface, it may be sufficient to signals has not experienced complete
In vivo MR Spectroscopy 79
the sequence of choice compared to STEAM. are incremented with each acquisition, while
A minor additional advantage of the spin- the readout period is again reserved solely
echo sequence is a lower sensitivity against for resolving the spectroscopic dimension.
diffusion, due to shorter intervals between An extreme example is the 3D-CSI sequence
spoiling gradient pulses. An advantage of shown in Figure 7.3, achieving spatial
the STEAM sequence is shorter minimum resolution along all spatial dimensions by
echo times during which magnetization phase-encoding. The spatially resolved
decays by T2; during the second and the spectra are obtained from the k-space data
third RF-pulse of the STEAM sequence, i.e. by 4-D Fourier transform. Unlike the
the “mixing period”, the magnetization of position of a slice selection profile, the
the stimulated echo is stored as longitudinal position of a CSI-grid realized by phase-
magnetization decaying only by T 1 . encoding can be modified retrospectively
Furthermore, some coupled resonances by fractions of the voxel size. In a 2D-CSI
show higher signals at intermediate echo- sequence, one spatial dimension is selected
times when detected by STEAM.5 by a slice-selective RF-pulse, while only the
When used for detection proton signals, other two dimensions are phase-encoded.
these SVS techniques are used in To minimize the above mentioned field
combination with a water suppression dependent T2* signal loss, it is necessary to
sequence module. In the brain the water minimize the delay TD between the center
signal is 105 times more intense than typical of the pulse (when the echo maximum is
metabolite signals. Given that the dynamic formed) and the start of the acquisition.
range of the receiver is sufficient, a smooth Such 2D- or 3D-CSI sequences of only a
water signal could be removed from the short acquisition delay are especially useful
spectrum in a post-processing step.
However, tiny modulations of the water
signal can appear as severe distortions with
respect to the metabolite signal scale. Hence,
the water signal is preferably suppressed
experimentally, usually by frequency-
selective excitation in combination with
dephasing. Among often used sequence
modules applied before localization are
CHESS and WET, and among those applied
during the sequence are MEGA and DRY-
STEAM.1
to avoid heavy T2-weighting in non proton coils. As a consequence of the effect, the
applications where often T2-values are low. VOIs superimposed over the anatomical
Another strategy for minimizing T2* loss images as shown in the graphical user
in CSI is generating an echo. Adding phase interface for positioning are only precisely
encoding to the SVS sequences during a correct for one frequency offset or one
period when magnetization is transverse, spectroscopic signal. It should be left to the
one arrives at hybrid 2D- or 3D-CSI user of a spectroscopy sequence to decide
sequences. Those are successfully used to which signal the precise positioning of
especially in brain 1H MRS. The volume VOIs refers to; e.g. instead of using the
selected by the slice selective pulses covers system resonance frequency which is usually
a region of interest of the inner brain which the one of the water signal, the user may
is further resolved by phase encoding, while choose an spectroscopic intermediate signal
the selection of intense subcutaneous or bone as e.g. the choline signal, to reach the
lipid signals can be avoided. Another smallest average deviation for all signals of
advantage of hybrid CSI sequences is that interest. As a consequence of the chemical
the FOV defined by phase-encoding can be selection artefact in hybrid CSI, the voxels
made small without the risk of signal at the borderline of the VOI are not
folding, as long as the selection profile is completely excited, and may not be
sufficient to effectively suppress any outside quantitatively evaluated without further
signals. measures (note however that the effect only
An important limitation of the usage of occurs with slice selection but not with
slice selection pulses in SVS or hybrid-CSI phase encoding, so that the CSI-grid is not
sequences is the chemical shift selection shifted!). One way of handling the effect is
artefact. As the spectroscopic signals precess numerical correction. Another possibility is
with different Larmor frequencies, thus exciting a larger VOI than planned, and
superimposing another frequency offset to avoiding the excitation of outer volumes by
the one caused by the slice selection saturation pulses which can be designed for
gradient, they are excited at different higher bandwidth than selection pulses. A
positions. Quantitatively, the position shift third possibility is the usage of spectral-
of the slice center Δz for 2 signals of a spatial selection pulses.
frequency difference Δf is given by The minimal measurement time of a
Δz = 2π Δf/γ Gslice conventional CSI experiment discussed so
= 2π Δf tpulse slice_thickness/BWTP. far is given by the number of phase encoding
The frequency difference Δf depends steps along each direction, times TR, i.e.
linearly on the field strength B0. To minimize TA = Nx Ny Nz TR.
chemical shift displacement, one seeks to By reduced phase encoding covering
maximize the slice selection gradient Gslice, only an spherically or elliptically shaped
by designing pulses of a high band-width- inner portion of k-space, the CSI
time product BWTP, and by realizing the measurement time is significantly reduced.
pulses with minimal pulse duration tpulse This strategy is advantageous when e.g. a
using maximum transmitter voltage. The spherically symmetric Hamming k-space
latter can be achieved by using local transmit filter is applied for reducing voxel-bleeding
82 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Matrix Spectroscopy
Using multiple element receive “matrix”
coils, the sensitivity of clinical MRS can be
substantially increased. The development of
multi-channel hardware is on-going for 1H
detection, and it may be transferred for
detecting other nuclei. The problem of com-
bining the data from the single receive
Figs 7.4A to D: The effect of voxel bleeding: In the
case of k-space filtering by a spherical Hamming filter,
elements is more involved for complex MRS
only citrate and choline signals from within the data than for magnitude imaging data, but
phantom container are visible (A), the point-spread- was described already by Roemer et al. 7
function shows only minor sidelobes (C), in the One arrives from the complex signals Si of
unfiltered case; lipid signals from another outside the coil elements i at a combined signal Scomb
container appear (B), the point-spread-function
by
shows significant contributions originating from
outside areas Scomb = λ(r) Σi Wi(r) Si(r)
In vivo MR Spectroscopy 83
The purpose of the complex weighting can be normalized to the reception profile
factors Wi is two-fold. They contain the of the body coil.
appropriate phase correction terms for Figures 7.5A and B show an example
ensuring coherent complex signal addition where the sensitivity of an MR exam of
and avoiding signal cancellations. cortical and white matter voxels was tripled
Furthermore, they ensure the maximum SNR by using a 23 channel coil, as compared to
of the combined data by an additional the usage of a single element receive coils.
weighting of the signals Si with their signal The potential of clinical matrix 1H spectro-
intensities. This self-weighting property of scopy appears high when considering its
the combination algorithm leads to the possible realization at very high field
additional advantage that contributions of strengths such as 3T or 7T.
low SNR data are virtually negligible, thus
facilitating the selection of coil elements. Fast-CSI
After computing the sum term, a
normalization of signal amplitudes over all Especially in the case of 3D CSI, the
locations is to be carried out in the case of conventional CSI experiment described
CSI data. Various choices of λ(r) have been above may takes too long. The goal of fast
suggested. Data-inherent procedures well- CSI techniques is to reduce the minimum
known from imaging such as sum-of-squares measurement time. With respect to sequence
normalization can be applied also to sensitivity, i.e. the SNR reached per unit
spectroscopy. Even better homogeneity is measurement time, the conventional CSI
achieved when in a pre-scan imaging experiment serves as a benchmark. As
experiment, the sensitivity profiles of the usually spectroscopic experiments are
selected coil elements are compared to the limited by the need of reaching sufficient
profile of the large and homogeneous body SNR, fast techniques are hardly of practical
coil. Using this pre-scan data the CSI data importance when sensitivity is sacrificed.6
A B
Figs 7.5A and B: Upon coherent, appropriately weighted combination of each channel’s data, the multiple
array brain coil increases signal to noise especially of cortical voxels (A) as compared to a single-array head
coil (B)
84 Biomedical Magnetic Resonance: Proceedings of the International Workshop
On the other side, since e.g. matrix of phase encoding steps along one direction,
spectroscopy enhances the sensitivity, fast i.e. a typical factor of 16.
CSI matrix sequences not compromising with
sensitivity are useful, e.g. to buy spatial POST-PROCESSING
resolution without prolonging acquisition
times. The task of MRS post-processing is to
Among the fast sequences8,9 which almost evaluate at least relative metabolite
reach the sensitivity of the conventional concentrations and to display results. It
sequence are those which use an echo-planar appears hardly possible to device a general
readout module, designed to simultaneously purpose algorithm for absolute metabolite
encode spectral and spatial dimensions quantitation, due to a number of parameters
during signal acquisition (Fig. 7.6). Thus, usually not determined during a clinical
minimal measurement time of such an EPI- MRS exam (such as T1 and T2 values of
CSI experiment is reduced compared to a metabolite signal in pathological tissue), and
conventional CSI experiment by the number due to the fact that a ground truth for
verifying in vivo results is hardly available.
Hence, for each metabolite signal an
intensity value in arbitrary units is usually
obtained by a prior-knowledge based fitting
procedure, which can be compared to other
intensities of the same spectrum.
Quantitation of short TE 1H clinical spectra,
showing a large number of partially poorly
resolved signals and possibly artefactual
lipid signals, is one of the most ambitious 7. P Roemer, W Edelstein, C Hayes, S Souza, O
problems of MRS post-processing (Fig. 7.7). Mueller. The NMR phased array. Magn Reson
Med 1990;16:192-225.
REFERENCES 8. J Scheidler, H Hricak, D Vigneron, K Yu, D
Sokolov, L Huang, C Zaloudek, S Nelson, P
1. R de Graaf. In vivo NMR spectroscopy Wiley, Carroll, J Kurhanewicz. Prostate cancer:
Chichister, 1998. Localization with three-dimensional proton MR
2. T Ethofer, I Mader, U Seeger, A Ludolph, W spectroscopic imaging—clinicopathologic study.
Grodd, U Klose. Comparison of metabolite T1 Radiology 1999;213:473-80.
relaxation times in different brain regions at 9. T Scheenen, D Klomp, S Röll, J Fütterer, J
1.5 and 3 Tesla. Proc. ISMRM, 2003;434.
Barentsz, A Heerschap. Fast acquisition-
3. EL Hahn. Spin echoes. Phys Rev. 1950;80(4):
weighted three-dimensional proton MR
580-94.
spectroscopic imaging of the human prostate.
4. J Frahm, KD Merboldt, W Hänike. Localized
Magn Reson Med 2004;52:80-88.
proton spectroscopy using stimulated echoes.
J Magn Reson 1987;72:502-508. 10. V Govindaraju, K Young, A Maudsley. Proton
5. J Hennig. Coupling effects in volume selective NMR chemical shifts and coupling constants
1H spectroscopy of major brain metabolites. for brain metabolites NMR Biomed 2000;
Magn Reson Med 1991;21:82-96. 13:129-53.
6. R Pohmann, MV Kienlin, A Haase. Theoretical 11. S Neubauer. High-energy phosphate meta-
evaluation and comparison of fast chemical bolism in normal, hypertrophied and failing
shift imaging methods. J Magn Reson human myocardium Heart Failure Rev 1999;
1997;129:145-60. 4:269-80.
86 Biomedical Magnetic Resonance: Proceedings of the International Workshop
8
Multi-dimensional NMR
Spectroscopy and Editing in vivo
M Albert Thomas, Nader Binesh, Kenneth Yue, Hyun-kyung Chung,
Steven Han, N DeBruhi, A Aude, Anand Kumar
spectrum plotting intensity versus two axes, localization. 49 A human brain 2D COSY
the dimensions of which depend on the spectrum using a 2 T MRI scanner was
specific 2D NMR technique.22 recorded in a gross occipital volume of 5 ×
There have been several attempts during 6 × 8 cm3 which required in a total sampling
the last fifteen years in implementation and duration of 1 hour and 42 minutes. Homo-
evaluation of 2D NMR spectroscopy on the nuclear decoupled in vivo 1H MR spectra
MRI scanners and NMR spectrometers. using constant time chemical shift encoding
Sotak et al made use of 2D-zero-quantum were presented by Leibfritz and co-
(ZQ) spectra on a 2 T scanner to visualize workers.50 Non-localized versions of COSY
the lactic acid resonances in the presence of spectra have also been recorded in rat brain
interfering lipid resonances in phantom and rabbit kidney by other researchers using
models and mouse tumors.23 Crozier et al the high field NMR spectrometers.51-52
used a multiple quantum edited 2D sequence A major goal of this presentation is to
to visualize the excitatory neurotransmitters give an overview of recent progress with
glutamine and glutamate in phantom and the implementation of several localized 2D
rat brain, although this work was carried MRS sequences on the whole body 1.5 T
out on a 4.7 T spectrometer.24 Other high- and 3 T MRI scanners.
resolution spectrometer work has been
focused toward spatially localized 2D SPATIALLY RESOLVED MULTI-DIMEN-
studies and in vivo studies.25-32 Desmoulin SIONAL 1H MR SPECTROSCOPY AND
and Seelig showed a localized SECSY study EDITING
of a rat.25 van Zijl et al recently utilized 2D
{ 1 H- 13C}- spectroscopy to visualize 13C- Single-and Multi-voxel-based One-
labeled glucose, glutamine, glutamate, and dimensional 1H MR Spectroscopy
lactate in the cat brain in vivo.26 Other recent High concentration of water in biological
studies have attempted 2D DQ spectroscopy tissue makes MR imaging quite sensitive.
in human tissues.33-34 Different versions of However, this leads to a major problem in
the localized COSY sequence have also been MR spectroscopy since the metabolites are
implemented previously.35-45 McKinnon and tens of thousands of times lower in
Bosiger proposed a conventional COSY concentration than water. Thus, in order to
sequence with hard RF pulses (90°-t1-90°) obtain an acceptable spectral quality from
followed by three volume selective 180° RF the metabolites, MRS techniques must
pulses. 35 Haase and co-workers incorporate ways of minimizing or
implemented a COSY combined with an eliminating the water signals. Other basic
outer volume suppressing sequence, namely challenges in clinical 1D MR spectroscopy
LOCUS. 36 Many previous attempts to are lipid suppression and optimal spatial
develop localized 2D COSY spectra yielded localization of the volume of interest (VOI)
only the phantom results35-38 or, rat brain including single versus multiple volume
spectra using a surface coil without a built- elements (voxel).
in localization sequence.46-48 A new gradient First, water suppression can be extended
enhanced COSY in combination with VOSY from changes in hardware for data
used the STEAM sequence for volume acquisition (digitizers), pulse sequences, to
88 Biomedical Magnetic Resonance: Proceedings of the International Workshop
echo from the desired volume. This echo is from the column chosen by the two RF
different from the “race-track” echo analogy pulses at a time TE1/2 after the 180o pulse.
of the spin echo. This stimulated echo This echo is echoed again by another 180oss
corresponds to the analogy of “pancake” pulse in the remaining orthogonal plane and
echo.57 the second echo is actually from the desired
Point Resolved Spectroscopy or PRESS volume which is acquired and processed.57
(90oss-180oss-180oss) involves the selection Hence, PRESS is nothing but a double-spine
of a slice from a given volume with the use echo based MRS sequence. Figure 8.1
of a frequency selective 90o RF pulse in summarizes different protocols including
conjunction with a magnetic field gradient both single- and multi-voxel localized MRS
(90oss). However, the use of the gradient including global CHESS water suppression
dephases the spins from the chosen slice and OVS. The single-voxel based STEAM
which are now in the transverse plane.2,57 and PRESS are denoted in Figure 8.1 as
Hence, a negative lobe is added immediately STEAMSV and PRESSSV.
after the RF pulse to rephrase the phase- Spectroscopic imaging (SI), also called
scrambled magnetization during this chemical shift imaging (CSI), enables
excitation. The FID produced from this slice distilling both chemical shift as well as spatial
is ignored since the objective is to elicit information from the acquired signal.58-59
signals from a small voxel within the slice. Because of the latter challenge, it has several
After a duration of TE 1/2, a frequency similarities in mode of data acquisition with
selective 180o RF pulse with a magnetic field MR imaging like the use of phase encoding
gradient (180oss) in a plane orthogonal to gradients. However, the data processing in
that of 90oss is used. This creates an echo this case requires both spatial and time
Fig. 8.1: 1D MRS protocol including water and fat suppression and signal acquisition
90 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fourier transforms to decode the chemical shift expresses the local electronic
information. The PRESSCSI and STEAMCSI environment and is predominantly an intra-
sequences in Figure 8.1 can be used where molecular interaction, Molecular motion also
the basic PRESS and STEAM sequences influences this shift significantly. Thus, a
remain the same; however, the field primary manifestation and measure of the
gradients are modified for spatial encoding. chemical bond between the nuclear spins,
The first slice selection gradient along with i.e., the nuclear-nuclear coupling via the
the RF pulse is the same but spatial bonding electrons, is the exclusively intra-
information along the two dimensions of molecular J-coupling, which is independent
the chosen slice are encoded via phase of the applied magnetic field. Exploitation
encoding gradients. Phase encoding of this interaction is the crucial link to the
gradients do not change the frequency of molecular chemistry of the compound under
the spins in the received signal since they study. A map correlating the peaks that
are not on during signal acquisition. Instead, belong together in the same spin system is
they serve to momentarily change the phase quite important and a 2D spectrum provides
of the spins. The spins return to the same such a map. Two-dimensional NMR
frequency under the influence of the external spectroscopy enables converting a crowded,
magnetic field after the gradients are overlapping 1D MR spectrum to a better-
switched off but retain the phase memory resolved 2D spectrum through the addition
difference relative to the spatial dimension. of a spectral dimension.22 The problems due
These phase encoding gradients are repeated to the overlap in the 1D spectrum
several times between the remainder of the introduced by the multiplicity of various
RF pulses each time repeating the entire peaks due to J-coupling could be removed
sequence but with the phase encoding by separating the interactions due to
gradient changing slightly. This successive chemical shift and indirect spin-spin coupling
change is done in a stepwise fashion with (J) along the two dimensions in 2D MR
the gradient amplitude changing thus; spectroscopy. Instead of a standard 1D
several samples of a frequency are spectrum plotting intensity versus a
produced. The data can then be Fourier singleaxis (i.e., chemical shift), 2D NMR
transformed to allocate signal intensities of spectroscopy techniques produce a 2D
different frequencies to appropriate voxels. spectrum plotting intensity versus two axes,
the dimensions of which depend on the
Chemical Shift Correlated specific 2D NMR technique.
MR Spectroscopy Before applying these multi-dimensional
For MR spectroscopy of a particular nucleus, MR techniques in human tissues, each
namely hydrogen (1H), one counts on the sequence needs to be converted first into
differences in internal spin interactions which localizing a volume of interest (VOI) using
are due to differing inter and intramolecular three orthogonal slice-localizing RF pulses.
environments. These differences in the A second task is to minimize the number of
molecular environment are of several types RF pulses essential for creation of Hahn’s
and are hence, responsible for different spin-echo or coherence transfer echoes.
features of a spectrum or an image. The Shown in Figure 8.2 are different modules
Multi-dimensional NMR Spectroscopy and Editing in vivo 91
Fig. 8.2: 2D MRS protocol including volume localization, 2D spectral sampling, etc.
92 Biomedical Magnetic Resonance: Proceedings of the International Workshop
σ1 ∝ Iy ...(2)
After an evolution during the interval of
2Δ, the spins evolve under the influence of
J coupling, but the chemical shift will be
refocused resulting in an echo characterized
by
σ2 ∝ Iy cos(2πJΔ) – 2IxSzsin (2πJΔ) ...(3)
The spin-state (σ 2) after the evolution
due to both chemical shift (ω1(I)) and J-
coupling with its coupled partner during
the t 1 evolution can be calculated as
discussed earlier.60
Fig. 8.3: 2D L-COSY sequence A second pair of Bo gradient crusher
pulses was transmitted around the last slice-
selective 90o RF pulse. The spin state after
(t1) for the second dimension was inserted
an evolution during the last set of B o
immediately after the formation of the Hahn
gradient crushers,
spin-echo. In addition to the slice-selective
σ5 ∝ -0.5 cos(2πJΔ) [Iycos (ω1(I)t1) cos(πJt1) +
180o RF pulse, there were refocusing B o
Ixsin (ω1(I)t1) cos (πJt1)+ 2IzSxcos (ω1(I)t1) sin
gradient crusher pulses before and after the
(πJt1)-2IzSy sin (ω1(I)t1) sin(πJt1)] + 0.5 sin
last 90o RF pulses. Each time point of the
(2πJΔ) [Iycos (ω1(I)t1) sin(πJt1) + Ix sin (ω1(I)t1)
pulse sequence is marked as shown in Figure
sin (πJt1) - 2IzSxcos (ω1(I)t1) cos (πJt1)+2IzSy
8.1 to evaluate the time course of
sin (ω1(I)t1) cos(πJt1)] ...(4)
magnetization that will take into account
Eqn. (4) was calculated under the
the rotation after each RF pulse and
assumption that the gradient crushers after
evolution during the Bo gradient pulses and
the last sliceselective 90o RF pulse. It is also
the incremental period.
evident from Eqn. [4] that the coherence
A theoretical analysis to calculate the
transfer from spin I to S is characterized by
amplitudes and phases of L-COSY diagonal
2IzSx and 2IzSy. The 2D signal acquired after
and cross peaks in a weakly coupled two
the last crusher gradients is given by,
spin (I=1/2, S=1/2) system is discussed here.
s(t1, t2) = Tr {(Ix) σ5} exp(-iω2 (I)t2) exp(-t1/
A product operator formalism was used to
T2) exp (-t2/T2) [1-exp (-TR/T1)] ...(5)
evaluate the time course of the magneti-
A similar equation can also be calculated
zation. 22,60 The amplitude and phase
for S spin resulting in a coherence transfer
characteristics at different time points of an
to I spin. A double Fourier transformation
L-COSY spectrum are derived here. Prior
along both t1 and t2 axes will result in a
to the first 90o RF pulse, the spin-state is
two-dimensional MR spectrum as a function
explained by
of two frequency variables (F1, F2) described
σo ∝ Iz ...(1)
by,
where I is the spin under consideration and
S is its J-coupled partner. After the rotation S(F1,F2) = s(t1,t2) dt1dt2 ...(6)
by the first 90o RF pulse, the spin-state is Fourier transformation of the standard
explained by acquisition dimension converts each FID to
Multi-dimensional NMR Spectroscopy and Editing in vivo 93
a frequency spectrum. The 2D data set matter brain phantom using a Siemens 3 T
S(t1,t2) becomes S(t1,F2). A second Fourier MRI scanner. The brain phantom had several
transform applied to the data along the metabolites at physiological concentrations
incremented echo time dimension t1 converts as shown below: 8.9 mM NAA, 0.7 mM
the oscillating phases of each of the coupled GABA, 2.1 mM Asp, 0.9 mM Ch, 7 mM Cr,
metabolite peaks to their respective 1 mM Glc, 12.5 mM Glu, 2.5 mM Gln, 2 mM
frequency components. The 2D data set GSH, 4.4 mM mI, 0.4 mM Lac, 3.6 mM PCr,
S(t1,F2) then becomes S(F1, F2). The final 2D 0.6 mM PCh, 1.8 mM Tau, 0.3 mM Thr,
spectral matrix has the information of both 1 mM PE and 1 mM DSS in a phosphate
chemical shift and J-coupling along the F2 buffer solution to maintain pH of 7.2. 2D L-
dimension and only J-coupling along the F1 COSY spectra were recorded using the
dimension. Ernst and co-workers reported following parameters: 27 ml voxel, TE = 20-
that the diagonal peaks were dispersive 30 ms, TR = 2s, total number of scans = 768
where as the cross peaks were absorptive (96 Δt1 increments and 8NEX/Δt1). The total
when two hard 90o RF pulses were used to duration for each 2D scan with water
acquire the basic 2-pulse COSY spectrum.62 suppression was approximately 24 minutes.
One drawback of including the gradient Before zero-filling to (2048 × 256), the 2D
pulses is due to the mixed line shapes in the raw data was apodized using skewed
2D NMR spectra.60 Eqn. [4] clearly indicates squared sine-bell filter along t2 and squared
that both the diagonal and cross peaks of sine-bell filter along t1. The resultant 2D
an L-COSY spectrum have mixed phases spectrum was displayed in the magnitude
along the F 1 axis. In contrast to the mode.
amplitude modulation in conventional COSY,
the phase modulation in L-COSY is caused L-SECSY
by the evolution during the Bo gradient Three-dimensional (3D) localized spin-echo
pulse before the last 90o RF pulse.60 Pure correlated spectroscopic (L-SECSY) was
phase L-COSY spectrum can be recorded derived from the basic SECSY originally
using a quadrature detection method reported by Nagayama et al.64 Modification
along the F1 axis described by Doddrell and of L-COSY by inserting equal incremental
co-workers41 that will require two separate periods before and after the last 90o slice-
P- and N- type spectral acquisition and selective RF pulse in Figure 8.3 results in L-
recombination of the two data sets. SECSY. The diagonal peaks of L-SECSY lie
Relaxation during the gradient pulses before on a line at F1 = 0 and the cross peaks are
the last 90o RF pulse will cause further losses symmetrically disposed on both sides of the
in signal intensities. diagonal. Similar to L-COSY, the singlet
The L-COSY sequence has been resonances appear on the diagonal. The cross
implemented on the GE (General Electric peaks can be identified on a line cutting
Healthcare Technologies, Waukesha, WI) through the diagonal with an angle of 45o.
and Siemens (Siemens Medical Systems, Shown in Figure 8.5 (Plate 1) is a 2D L-
Erlangen, Germany) 1.5 T and 3 T MRI SECSY spectrum of a gray matter brain
scanners. Shown in Figure 8.4 (Plate 1) is a phantom, same as the one used for L-COSY.
2D L-COSY spectrum recorded in a grey A Siemens 3 T MRI scanner and the
94 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the dominant diagonal peaks (F2 = F1): (i) the total creatine pools, and a second peak,
oil phantom: the olefinic protons at 5.4 ppm, possibly between the olefinic and
the poly-methylene protons (CH2)n at 1.4 magnetically equivalent polymethylene
ppm and methyl protons at 1.0 ppm. (ii) the protons of unsaturated lipids. These two
muscle phantom: (i) the residual water at peaks are marked as 1 and 2 in Figure 8.9.
4.8 ppm, the methyl and methylene protons In the J-coupled metabolites and lipids,
of Cr/PCr at 3.0 ppm and 3.9 ppm, tri- coherent magnetization transfer leads to
methly protons of Ch/PCh) at 3.2 ppm and COSY-type cross peaks. At TM = 300 ms,
imidazole protons of Car at 7.0 ppm and 8 these cross peaks were absent. As explained
ppm, respectively. Two pairs of cross peaks two decades ago, dipolar interactions
were identified at the following locations: between neighboring protons of different
(F2 = 5.4 ppm, F1 = 2.9 ppm), (F2 = 2.9 ppm, molecular species modulated by random
F1 = 5.4 ppm), (F2 = 5.4 ppm, F1 = 2.2 ppm), tumbling leads to cross relaxation and
and (F2 = 2.2 ppm, F1 = 5.4 ppm). The first magnetization transfer between the protons
two cross peaks are due to the indirect spin- of different species.22 A non-equilibrium
spin (J) coupling between the methylene state between the magnetically coupled
protons connecting olefinic groups and water and metabolites is created
immediately after the second 90oss pulse in
olefinic protons in polyunsaturated fatty acyl
the 2D L-EXSY sequence. A 2D L-EXSY
chains. 74 The last two cross peaks were
spectrum records transfer of magnetization
possibly due to J-coupling between CH2 next
within protons of one molecule or between
to an olefinic group and the olefinic
two different molecular species effected by
protons.75 In agreement with the assignments
the intra- and inter-molecular magnetization
reported by Szczepaniak et al,76 the diagonal
exchange mechanisms. It was also reported
peaks of glycerol backbone protons were
earlier that magnetization may be exchanged
also identified at (F2 = F1 = 4.3 ppm), (F2 =
among molecular species by coherent and
F1 = 5.3 ppm), and the cross peaks (F2 = 5.3 incoherent transfer processes.22 The build
ppm, F1 = 4.3 ppm), (F2 = 4.5 ppm, F1 = 4.0 up rates of 2D L-EXSY peaks have been
ppm), (F2 = 4.3 ppm, F1 = 5.3 ppm) and (F2 further investigated in human muscle after
= 4.0 ppm, F1 = 4.5 ppm). The diagonal and recording several 2D spectra with TMs
cross peaks of the methylene and methyl varying between 100 ms and 2 s.
protons of saturated fatty acids overlapped
with unsaturated fatty acids in the aliphatic CLINICAL APPLICATIONS OF 2D L-COSY
region (0.9 - 4 ppm). In the 2D spectra of In the following section, our recent findings
the muscle phantom, there were also weak on three clinical applications of 2D L-COSY
cross peaks between the imidazole and in i) a liver disorder and a neurological
aliphatic protons of carnosine. disorder, namely late-life major depression,
A 2D L-EXSY spectrum recorded in the and breast cancer in vivo will be discussed.77-
soleus muscle of a healthy volunteer is 79 Two-dimensional L-COSY spectra of these
shown in Figure 8.9 (Plate 2) using TM of three patient cohorts were recorded using
300 ms. Two different exchange cross peaks a GE 1.5 T MRI scanner (GE Healthcare
were recorded in human calf muscle: first Technologies, Waukesha, WI) equipped with
peak, between the mobile tissue water and echo-speed-gradients operating at a slew
Multi-dimensional NMR Spectroscopy and Editing in vivo 97
rate of 120 T/m/s. A body RF coil was glutamine/ glutamate to creatine ratio (Glx/
employed for RF transmission, and a single Cr) in the parietal lobe.92-93 These changes
3” surface coil in the first two groups and have also been reproduced in the studies of
a phased-array breast coil for reception in minimal HE patients with volumes localized
the third group involving breast cancer in other regions of the brain. 94-96 One-
patients. dimensional MRS, used in these studies,
suffers from the spectral overlap of various
Hepatic Encephalopathy (HE)
metabolites resonating within a limited
Clinical features of HE range from subtle spectral range, which may lead to inaccurate
changes in sleep patterns, anxiety, and quantification. In addition, some metabolites
personality changes to profound cognitive at physiological low concentration are also
impairment, confusion, disorientation, and not visible. Hence, there is a need to address
finally coma. They are graded from 1-4, these limitations with 1D MRS. 2D L-COSY
depending on the clinical severity.80 Over was investigated to record the changes in
the past few decades, the focus has shifted cerebral metabolite levels in this group of
to a sub-group of patients with cirrhosis patients compared to healthy controls. A
who show no overt clinical signs and total of 18 patients (10 females and 8 males)
symptoms of encephalopathy, but who
with a mean age of 53.3 ± 10.6 years
perform poorly on psychometric tests when
undergoing evaluation for liver transplan-
compared to other patients with cirrhosis
tation and 21 healthy controls (13 females
and healthy controls.81-86 These patients are
and 8 males) with a mean age of 52.7 ± 12.6
categorized as having “subclinical” HE
years have been investigated. The diagnoses
(SHE). 87-90 This group of patients is
of the patients were alcoholic cirrhosis (n =
identified conventionally on the basis of a
3), primary biliary cirrhosis (n = 2), chronic
battery of neurocognitive tests and/or
hepatitis B cirrhosis (n = 1), autoimmune
electroencephalographic abnormalities.
hepatitis (n = 2), cryptogenic cirrhosis (n =
Significant findings in this group of patients
with SHE are impairment in tests of 1), and chronic hepatitis C cirrhosis (n = 9)
psychomotor speed, concentration, visual including a history of concomitant alcoholic
attention, tracking, visuo-spatial and fine liver disease in 2 patients.
motor skills while verbal memory skills are A 3 × 3 × 3 cm3 voxel was placed on the
relatively well preserved.86-91 However, it anterior cingulate region, since the
is not always possible to detect SHE by neuropsychological tests have indicated an
psychometric tests alone.82 Thus, a true alteration predominantly in the frontal tasks
single gold standard for the detection of that require attention and mental flexibility.
SHE is still not defined. A CHESS sequence was used for water
Ross et al first reported a triad of suppression. The following parameters were
signature changes observed in HE with 1H used: minimal TE = 30 ms, TR = 2 s, total
MRS of brain white matter: a significant number of scans = 768 (96 Δt1 increments
reduction in the myoinositol to creatine ratio and 8 NEX/Δt1), spectral width, F1 = 625
(mI/Cr), a decline in the choline to creatine Hz and F2 = 2500 Hz. The total duration for
ratio (Ch/Cr), and an increase in the each 2D scan was approximately 25 minutes.
98 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figures 8.10A and B (Plate 2) show the reduction in patients compared to healthy
2D L-COSY spectra of (A) a 44-year-old controls. The ratios of the cross peaks of
patient and (B) a 45-year-old healthy human NAA, Cr, Asp, PE, GABA, PCh, and ThrLac
subject, recorded in the anterior cingulate with respect to Cr_d did not show
using a voxel size of 27 ml. The 2D cross significant differences, but Tau/Cr_d ratio
peaks due to NAA, Cr, Glx, mI, Asp, PE, showed an increase (p=0.053) in the patients
PCh, Tau, ThrLac and GABA are marked. when compared to the healthy group. A
The mean ratios (± SD) of the metabolites stepwise discriminant analysis was
with respect to Cr_d in SHE patients and performed on the MRS ratios. The first ratio
healthy controls are summarized in Figure was found to be mICh/Cr_d, which on its
8.11. Statistically significant differences (p < own could correctly classify the patients and
0.05) were seen in the mean values of ratios healthy controls at 69 percent. Addition of
calculated from the cross peaks: Glx/Cr_d, more ratios improved the predictability with
mICh/Cr_d, mI/Cr_d. In addition, the four ratios in the order of mICh/Cr_d, Tau/
mean ratio of the diagonal peak of choline Cr_d, Ch/Cr_d and NAA/Cr_d. The total
(Ch_d/Cr_d) showed statistically significant correct classification from these four
variables was 87 percent (Table 8.1).
Addition of a fifth ratio did not improve
the classification any further.
prefrontal lobe, the hippocampus, and the were statistically significant. The volumes
head of the caudate nucleus, when compared of white matter, gray matter, and CSF in
with nondepressed control subjects.101 MRS the MRS voxels used for 2D L-COSY were
studies demonstrate an increase in the ratios calculated in 16 healthy women and 12
of Ch and mI with respect to Cr in the women patients using the 3D SPGR MRI
white matter regions with NAA levels data, and the mean values were 63.7 percent,
comparable to those seen in agematched 30.7 percent and 5.6 in healthy women, and
controls.100 However, “first generation” MRS 60 percent, 33.1 percent and 6.9 percent in
studies used single voxel, onedimensional elderly patients. Our preliminary study
(1D) 1H MR spectroscopy. While NAA, Cr, demonstrates the feasibility of applying the
Ch, and mI can be measured using 1D MRS, 2D L-COSY method in elderly subjects with
overlap of the resonances of these and without a clinical brain disorder.
metabolites hinders their precise
measurements. Further, this limitation results Human Breast Cancer
in poor reliability of detecting other Magnetic resonance spectroscopy is a
metabolites that are at lower concentrations. potentially useful and minimally invasive
On the other hand, two-dimensional (2D) technique for breast tissue classification. A
spectroscopy can overcome these problems number of prior investigators have applied
by the improved resolution enabled by the MRS to characterize breast tissue, with
second spectral dimension, 60-61 thus promising results. MRS of human breast
providing a more reliable method for tissues shows the resonances due to water,
measuring metabolites. 101 Our recent choline, nucleotides, saturated and
findings on comparing relative metabolite unsaturated fatty acids.103-110 Mackinnon
levels in patients diagnosed with major et al used one-dimensional (1D) MRS to
depressive disorder (MDD) to healthy distinguish 102/106 fine-needle biopsy
elderly subjects are presented in this section. samples of benign or normal tissue from
Our study samples comprised 16 healthy
female controls (mean age of 72 ± 7 years)
and 12 female patients diagnosed with major
depression (mean age of 72 ± 8 years). All
depressed patients met DSM-IV criteria for
major depressive disorder. Figure 8.12 (Plate
3) shows a 2D L-COSY spectrum recorded
in the frontal white matter region of a 61yo
patient diagnosed with major depression.
The 2D metabolite ratios of 12 patients and
16 controls are summarized in Figure 8.13.
Even though increase of metabolite ratios
such as mI/Cr_d, Glx/Cr_d, GABA/Cr_d
and PE/Cr_d, and decrease of NAA_d/
Cr_d were observed in patients compared Fig. 8.13: The metabolite ratios of elderly depressed
to elderly controls, none of these changes female patients and female elderly controls
100 Biomedical Magnetic Resonance: Proceedings of the International Workshop
data acquisition (20-30 minutes) necessary rated in rat brain using a 4.7 T MRI scanner
for the spectral averaging combination with and the cross peak images of mI and Tau
the second spectral encoding steps. When were recorded only. The 2D spectra derived
CSI or SI is added to 2D MRS, there will be from EPSI are complicated by artifacts
further increase of time due to additional contributed by strong coupling of various
stepping of the phase-encoding metabolite protons unavoidable at both 1.5 T
gradients.58,59 The metabolite images can be and 3 T MRI scanners. The artifacts in these
reconstructed from the 2D spatial and 2D multi-voxel based 2D MRS sequences need
spectral data projecting the cross peak to be simulated and further work is
volumes into the corresponding spatial necessary in the future to demonstrate the
images, a methodology termed as cross peak clinical potential of these multidimensional
imaging (CPI). 113,114 A combination of MR spectroscopic imaging methods.
16 x 16 spatial, 64 spectroscopic encoding
steps and repetition time (TR) of 1 s will CONCLUSION
result in a total duration of 4.6 hours.114 Broadband excitation and detection of the
Recent implementation of circular sampling J-coupled metabolites in human tissues
combined with 2D J-resolved spectroscopy noninvasively have been demonstrated using
was demonstrated by Renshaw and
different 2D MRS techniques. 2D cross peaks
coworkers using a 4 T whole body MRI
of more than ten J-coupled metabolites have
scanner resulting in a total duration of 2
been observed using L-COSY recorded in
hours.115
the frontal and occipital regions of human
Two different multi-dimensional MRS
brain.60,61,77-78 Other researchers have also
sequences have been proposed recently with
reported the implementation of different
a drastic reduction of total duration. First,
versions of 2D COSY.118-121 Simultaneous
Spielman and coworker introduced the first
acquisition of COSY from multiple volumes
application of time-varying gradients during
of interest was proposed by Delmas and
the read-out time to acquire multi-voxel
co-workers118 and two-voxel localized COSY
based 2D J-resolved spectral data in phantom
spectra of rat brain were recorded in a total
solutions containing lactate and ethanol
duration of 42 minutes using a Bruker 4.7 T
using a 1.5 T MRI scanner.116 Spiral-based
k-space trajectories were used in conjunction MRI scanner. A different pulse-sequence
with a 2D J-resolved sequence. By preceding (ISIS-COSY) was also proposed by Welch
the spiral read-out with a 2D preparation et al,121 where the volume was localized by
scheme, 128 spectral encoding with TR of outer-volume suppressed ISI (OSIRIS). Two
2 s resulted in a total duration of 17 major drawbacks of ISIS-COSY compared
minutes. Second, an asymmetric variant of to L-COSY were: (i) eight shots are
echo-planar spectroscopic imaging (EPSI) necessary to achieve the VOI and (ii) five
consisting of a train of trapezoidal read-out RF pulses are necessary to record the single
gradients, each followed by short refocusing voxel localized COSY leading to more
pulse was implemented by Meyer et al.117 dependence on the B1-field inhomogeneity.
A constant-time based COSY was used and In contrast to a recently implemented ISIS-
the total acquisition was 34 minutes. COSY sequence, 121 the 2D techniques
Application of this technique was demonst- discussed in this chapter are one-shot based.
Multi-dimensional NMR Spectroscopy and Editing in vivo 103
It has been shown recently that one- sequences need to be further evaluated.
dimensional MR spectra processed using the Further work is necessary to demonstrate
LCModel algorithm and due to severe the clinical potential of both single and
overlap of several spectral resonances in 1D multi-voxel based multidimensional MR
MRS in vivo, LC Model uses the individual spectroscopic imaging methods.
phantom spectra to come up with the
absolute concentrations of high and low- ACKNOWLEDGMENTS
concentrated metabolites. 19,21 In the
frequency domain, 2D L-COSY shows the This work was supported by the grants from the
National Institute of Health (MH065695) and the US
unambiguous presence of 2D cross peaks
Army Breast and prostate cancer research programs.
originating from several J-coupled Scientific support of the following people is gratefully
metabolites. However, further work is acknowledged: Dr. Lawrence Ryner and Dr. T.N
necessary in order to ascertain the Venkatraman in the implementation of several 2D
superiority of 2D L-COSY over the MRS sequences, Dr. Shida Banakar and Tai Dou and
quantitation of 1D MR spectra using LC- Ashwin Thomas during the preparation of this
chapter, and Mr. Art Ambrosio during the
Model. Additionally, different 2D MRS preparation of brain phantoms.
techniques discussed in this chapter need
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ABBREVIATIONS
11.
111. Mountford CE, Somorjai RL, Malycha P, Gluch 1. MRI- Magnetic Resonance Imaging
L, Lean C, Russell P, Barraclough B, Gillett D, 2. MRS- Magnetic Resonance Spectroscopy
Himmelreich U, Dolenko B, Nikulin AE, Smith 3. VOI- Volume of Interest
ICP. Diagnosis and prognosis of breast cancer 4. CHESS- Chemical Shift Selective Suppression
by magnetic resonance spectroscopy of fine- 5. RF - Radio Frequency
needle aspirates analysed using a statistical 6. OVS- Outer Volume Suppression
classification strategy. Brit J Surg 2001;88:1234- 7. STEAM- Stimulated Echo Acquisition Mode
40 8. PRESS- Point Resolved Spectroscopic Sequence
112. Thomas MA, Binesh N, Yue K, DeBruhl N. 9. STEAMSV- single-voxel based STEAM
Volume-localized two-dimensional correlated 10. PRESSSV- single-voxel based PRESS
magnetic resonance spectroscopy of human 11. CSI- Chemical Shift Imaging
Multi-dimensional NMR Spectroscopy and Editing in vivo 109
technical limits of the system. In this context (Positron Emission Tomography), SPECT
a fast imaging technique is one which can (Single Photon Emission Computer
be realized with reasonable technical effort Tomography) and even to ultrasound, all
and ultrafast imaging would be anything of which are based on applications of optical
which pushes the limits of technology. There principles.
is, of course, a strong correlation between In MR spatial information has to be
the application related and the technical scale derived in a fundamentally different way
systems: technical performance will define by encoding the signal in an indirect way.
the range of problems, which can be In practically all MR imaging techniques in
addressed. Vice versa, once imaging speed use today this is being achieved by
will allow to adress a new range of translating the spatial domain into a
applications, one will immediately try to frequency domain while the application of
push the envelope further to a point where linear gradient (Fig. 9.1, Plate 4).
performance is not a limiting factor anymore. This basic principle has been introduced
In the following, a discussion will be given by Paul Lauterbur1 in 1972 who together
about fundamental limitations of MR with Sir Peter Mansfield received the 2003
imaging followed by examples of fast and Nobel Prize for this outstanding achieve-
ultrafast imaging in various fields of ment. Although tremendous progress has
applications. been made in the last 30 years in developing
all kind of new MR sequences with very
FUNDAMENTALS OF FAST AND different performance characteristics, all of
ULTRAFAST IMAGING these are based on this common principle
of spatial encoding using gradients. Without
The fundamental challenge for using going into details of any specific pulse
magnetic resonance for imaging purposes is sequence some very fundamental limitations
related to the fact that the wavelengths of on gradient based imaging can be derived
the radiofrequency field useful the from signal theory:
observation is far longer than the desired Data acquisition in MR imaging is
spatial resolution. The Larmor frequency at performed using the principles of Fourier
1.5 Tesla is 64 MHz corresponding to NMR. This means MR signal is acquired
wavelengths of a bit less than 5 m in air. sequentially in time. The frequency spectrum
The strong dielectric moment of water encoding the spatial information is derived
compresses the wavelengths by roughly a by Fourier transformation from the time
factor of 9. But the ~60 cm in tissue is still signal. In order to acquire data for an image
by far outside the range which would allow of matrix size N × M one therefore needs
imaging based on wave optical principles. to sample N × M data points in time. The
The Rayleigh criterion for imaging by wave sampling speed defined by the dwell time
optical principles states that the limit of DW between successive (complex) data
spatial resolution is roughly equivalent to points is related to the bandwidth BW of
the wavelength of the radiation used. This the spectrum via the sampling theorem:
applies not only to photographic imaging DW=1/BW ...(1)
using light, but also to conventional X-ray The total ‘pure’ data collection time is
imaging, Computer Tomography, PET then given by:
112 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 9.4: Vehicles for moving around k-space: application of gradients will lead to a trajectory, the direction of
which will be defined by the gradients, the gradient amptitude will define the velocity of moving around
k-space (left). A refocusing pulse will bring spins to a position at the other side of k-space symmetrically
around the origin. The origin is indicated by a cross and is normally at the center of the k-space acquisition
window.
rate of field change of ΔB/Δt = 20T/s at is measured, is rather long. The current IEC-
Δxlin= 20 cm, but ΔB/Δt = 30 T/s at Δxmax. standards are based on average exposure
State-of-the-art gradient power supplies over 6 min, with separate provision for short
today easily deliver sufficient power to term peak exposure, where three times the
exceed the physiological nerve stimulation average power can be applied over 10 s.
limits. The technical challenge in gradient These time constants are rather awkward
design today thus is not so much to further in comparison with the typical acquisition
increase gradient power, but rather to design times used in MRI. Taking the limits literally,
gradient coils such, that ΔBmax does not the average 6 min-‘dose’ (1 W/kg body
exceed ΔB lin overly much. For specific weight~ 75 W for a 75 kg patient) could be
purposes like diffusion imaging of the head, applied over 2 min (~ 225 W average
shorter gradient coils designed specifically power), provided, that no RF is applied over
for that purpose can increase the tolerable the rest of the 6 min period. Likewise and
performance of a body coil by a factor of 2 somewhat worrisome, there is no lower limit
or more. for the exposure time. Consequently, one
How these limits translate into perfor- could apply the permitted 225 W short term
mance of ultrafast imaging sequences, which exposure limit over 10 s over any arbitrarily
use gradients to cover k-space will be short period like 2.25 kW over 1 s, 22.5 kW
described later. over 0.1s or a staggering 225 kW over 0.01s.
With respect to radiofrequency (RF) Although no hard data exist for such
power, the physiological limits are defined ultrashort exposure, one would be hesitant
by the amount of energy, which is absorbed to apply such excessively high RF power to
by the patient and which will lead to a patient. So fortunately it is at current
heating. The specific absorption rate (SAR) technically infeasible to reach such high
depends on the energy submitted by the power levels.
patient, but also on the frequency of RF The actual RF power (RFP) necessary for
and the size of the patient. The absorbance a RF pulse will depend on a scaling constant
of the human body has a maximum at c depending on the size and design of the
around 120-150 MHz, which corresponds to transmitter coil and the Q-factor of the
3 T field strength. Therefore the increase in (loaded) coil. Variable parameters are the
SAR between 1.5 T and 3 T is rather steep duration TD and a shape factor ‘s’ related
and scales with roughly the square of the to the shape of the RF pulses used and–last
field strength. The further increase at even but not least–the flip angle α of the pulse:
higher frequencies is somewhat less
dramatic, but in any case, SAR will continue RFP = c s α2/TD ...(6)
to go up with the field. A conservative value for a 180°-pulse of
Different exposure limits are valid for 1 ms duration at 3 T applied by the whole
whole body transmission compared to body transmitter coil is about 5 kW or 5kW
exposure of only parts of the body (head). × 1ms = 5 Ws per pulse. In other words,
Since thermo regulation is assumed to be the 450 such pulses could be applied over 10 s.
relevant mechanism to deal with RF heating, It is therefore clear, that SAR is not a serious
the time constant, over which RF exposure consideration, if a single image is to be
Ultrafast and Parallel Imaging 117
acquired within the 10 s averaging time, for Its pulse program and k-space trajectory are
a single image with 128 pulses the pulse shown in (Fig. 9.7). Originally sinusoidal
duration could be shortened to 0.28 ms. gradient waveforms have been used for the
For such an implementation, the readout gradient GR and a constant gradient
limitations would be given by the maximum GP was used in order to minimize the
voltage tolerated by the RF coil rather than power requirements on the gradient
by SAR considerations. Acquiring a single systems. With current state-of-the-art
image within 10 s is, however, not a very technology, a rectilinear sampling with
sensible way to do fast imaging and what blipped phase encoding gradient is,
be called sudden imaging rather than fast however, more commonly used. As shown
imaging. In most applications fast imaging by the k-space trajectory, EPI requires two
implies short repetition times. Figure 9.6A distinct modes for gradient operation: while
(Plate 6) shows the number of pulses np, data are sampled in each line, a constant
which can be applied over 10 s as a function and high gradient GR is required to optimize
sampling speed, whereas very fast switching
of the flip angle for a pulse of 0.5 and 1 ms
is necessary at the end of each k-line.
duration. The complimentary display in
Technically high and constant gradient
Figure 9.6B (Plate 6) shows the minimum
amplitude requires high current in the
pulse spacing, if pulses are applied conti-
gradient coil, whereas fast switching
nuously. It is quite clear, that for sequences
necessitates high voltage. In terms of nerve
requiring continuous application of RF pulses
stimulation only the switching periods are
(like gradient-echo sequences), the maximum relevant, since a constant gradient does not
allowed flip angle will get rather small at create any stimulation problems.
very short TR. Alternatively, for sequences In order to optimize data acquisition for
requiring high flip angles (like spin-echo EPI one needs to balance two opposing
sequences), additional waiting times have requirements: From equations (2) and (3) it
to be built in order to limit the total number is clear, that the sampling speed is increased,
of pulses to the SAR limits. It should be when gradients with high amplitude are
emphasized, that these diagrams are for being used during data sampling. High
demonstration only and relate to a gradients will require faster switching at
conservative ‘worst-case’ scenario in body the end of each k-line, when the readout
imaging at 3 T. For smaller transmitter coils gradient has to be reversed. Fig. 9.8 gives
and partial body exposure (like in a some examples for the performance of EPI
transmit/receive head coil) and at lower that given gradient slew rates. It is clear,
fields SAR is much less of a problem. that eventually the time necessary for
switching will dominate and the overall
From Theory to Practice: Gradient Based acquisition time may even increase with
Fast and Ultrafast Imaging Sequences higher gradients. Using non-rectilinear
sampling, data acquisition can be performed
Echo-planar imaging has been introduced even during the gradient reversal. In the
by P. Mansfield in 19774 as the very first ultimate limit, the minimum achievable
ultrafast imaging sequence. Even today EPI acquisition time will then be given by the
still is the ‘workhorse’ for most ultrafast switching times alone. Even then and with
imaging applications especially in the brain. extremely fast switching (slew rate 200 T/
118 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 9.7: Pulse program (top left) and k-space trajectory (bottom left) for an idealized EPI-experiment with
rectilinear sampling of k-space using constant gradient GR for sampling each k-line along kx and a blipped
gradient GP to advance by one line during reversal of GR. The figures on the right show a non-rectilinear k-
space trajectory with sinusoidal GR and a constant gradient GP
m/s) the acquisition time will be longer than As an alternate and in several ways more
~20 ms. Typical acquisition times used today efficient way to cover k-space, spiral imaging
are in the order of 50-60 ms for a 64 × 64 has found new interest over the last years.
image. Even with a slew rate of 1000 T/s/ As shown in Fig. 9.9, the k-space trajectory
m and 1000 mT/m gradient amplitude the is much smoother compared to EPI. In
acquisition time would still be in the order practical terms this means, that the demand
of 10-15 ms. This illustrates, that the ‘cost’ on the gradient system is evenly distributed
of increasing imaging speed with EPI in along the data acquisition. The alternation
terms of gradient power and switching between fast switching and sampling and a
behavior grows dramatically. Accelerating constant gradient in EPI is thus avoided. In
data acquisition by brute force thus cannot the literature several approaches have been
be expected to significantly reduce the EPI described, how to optimize the spiral
acquisition times much below the current trajectory within the boundary conditions
20-50 ms. defined by the gradient slew rate and
Ultrafast and Parallel Imaging 119
very fast Fast Fourier reconstruction the k-space center at the beginning of data
algorithm. Many approaches to optimize the acquisition. Spiral images do not show
quality and speed of image reconstruction distortions, since both T2* and off-resonance
for spirals and other nonrectilinear effects will lead to blurring rather than to
k-space sampling strategies have been a displacement. This can be good news or
suggested.8-14 Only in recent times computers bad news: if the acquisition time is very
have become sufficiently fast to perform short, no distortions will occur and only
image reconstruction within reasonable minor blurring will be visible. At longer
reconstruction times. For applications in Tacq, however, image quality will
fMRI, where several hundreds or thousands deteriorate.
of images are acquired within a few seconds The distortion caused by EPI at typical
or minutes, image reconstruction times are acquisition times can be quite pronounced.
still a practical limit on the standard For EPI-based fMRI of the brain the
computer platforms of current MR systems. displacement can easily reach 1 cm or more.
It can be expected, that this problem will Recently stable algorithms based on
vanish in the future, when even faster measurements of the local field distribution
computers will be introduced. A more subtle have been suggested, which can be applied
problem relates to the artifact behavior. The to correct for these effects. 15-19 The
most common source of imperfections in misalignment in uncorrected EPI as well as
single shot sequences are the signal decay the quality of correction performed
due to T2* effects and off-resonance effects. according to19 is demonstrated in Figure
Both of these mechanisms will develop along 9.11, Plate 6. It should be emphasized, that
the k-space trajectory leading to a strong the distortion correction is performed fully
dependence of the resulting artifacts on the automatic during the scan without any user
actual trajectory used. interaction. For spirals, distortion correction
Figure 9.10 shows simulations of the is more tricky and can only be performed
resulting artifacts on a typical range of on the raw k-space data, whereas the
parameters. Typical observations for EPI displacement matrix necessary for EPI-
are: correction can be easily applied to the final
• a signal attenuation with 1/T2* due to images.
the fact, that the center of k-space is It should be noted, that other k-space
sampled at a finite echo time (25 ms in trajectories have been suggested for
the example shown) different purposes. Radial trajectories offer
• at shorter T2* edge artifacts will appear. some quite interesting possibilities with
Note, that the images look pretty good, respect to their tolerance to radial under-
as long as the overall acquisition time is sampling and are quite attractive for
in the order of the T2* of the tissue. imaging at ultrashort echo times.20-24
• off-resonance effects will lead to a shift The main application for EPI is in BOLD-
in the phase encoding direction. This will based fMRI, where its ‘natural’ sensitivity
create typical distortion artifacts at areas to T2* combined with the possibility to cover
of local field inhomogeneities. the whole brain in less than 1 s are ideally
Spiral imaging overall signal intensity suited to observe the hemodynamic res-
remains constant due to the acquisition of ponse to cortical activation. Spiral imaging
Ultrafast and Parallel Imaging 121
Fig. 9.10: Artifact behavior of EPI vs. spiral imaging based on a total acquisition time of 50 ms respectively.
The first row shows the excellent representation of a 4x4 box in the ideal case of no signal decay and perfect
on-resonance of the signal. With shorter T2* EPI images will eventually develop edge artifacts. Additionally a
pixel shift will occur for off-resonance spins. Both signal decay as well as off-resonance effects will lead to
blurring in spiral imaging
has also been used for fMRI.25-27 In order to found considerable interest in the neuro-
generate T2* -sensitivity, this requires either science community,30 although its robust
a delayed start of data acquisition or the clinical application has still to be established.
use of ‘outside-in’ sampling, where the
center is sampled at the end of data Fast Imaging with Multipulse Sequences:
acquisition . Gradient Echoes
Outside of fMRI, EPI (and to some extend Gradient-echo sequences apply a periodic
spirals and other sequences) are mainly used sequence of multiple pulses at a pulse
as imaging modules in contrast modified spacing, which typically is much sorter than
sequences. Probably most important is the the T1 and T2 of the tissue. Depending on
combination with diffusion preparation for the refocusing behavior on transverse
mapping of the apparent diffusion magnetization created by preceding pulses,
coefficient. For early detection in stroke this gradient echoes are know to occur in
is probably the clinically most useful different varieties: Spoiled gradient echoes
application of such techniques. In addition (spoiled FLASH, GRASS, FFE), where only
to that, the measurement of diffusion the available z-magnetization is observed,
anisotropy for neuronal fiber tracking has un-spoiled gradient echoes, where part of
122 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the transverse magnetization is retrieved and field animal imaging because of the good
fully balanced gradient echoes (balanced image contrast and the quite robust image
SSFP; trueFISP, FIESTA, balanced FFE), behavior.
where all magnetization is fully refocused. For short TR and TR<<T2 the signal in
An overview of the intrinsic signal behavior fully balanced gradient echo sequences can
is given in.31 be shown to be
In spoiled FLASH the steady state trans-
verse magnetization Mtr depends on the Mtr = Mo sinα/((T1/T2+1) –
flip angle α, T1, T2 and the repetition time cosα (T1/T2-1)) ...(8)
TR: The optimum flip angle is then given by
Mtr=M o sinα (1-exp(-TR/T1)) exp(-TE/ cosαSSFP = (T1/T2-1)/(T1/T2+1); ...(9)
T2)/(1-cosα exp(-TR/T1)) ...(7) The signal intensity for αSSFP is then given
For tissues with specific T1 and T2, the by
maximum signal is achieved at the Ernst- Mtr ~ ½ M0 √(T2/T1) ...(10)
angle αE:
Remarkably, the signal intensity does not
cosαE = exp (-TR/T1) ...(8)
depend on TR, therefore a constant signal
Mtr then yields:
amplitude can be maintained even at
Mtr(αE) = Mo exp(-TE/TR) sinαE/(1+cos arbitrary short TR. Since the acquisition time
αE) Tacq will always be shorter than TR, faster
Fig. 9.12 shows, that for TR<<T1 the imaging will require higher bandwidth and
Ernst angle will be very small and the steady thus yield lower SNR according to Eq.[4].
state signal will be only a few percent of A serious consideration for balanced
the available magnetization. Therefore SSFP is the fact, that it takes quite some
ultrafast imaging with T1-weighted FLASH time (roughly in the order of T1) until the
carries a heavy penalty in signal-to-noise. signal steady state is reached. Before that,
Nevertheless it is used especially for high- signal fluctuation will occur, which may
seriously affect image quality. An initial
pulse with flip angle α/2 and preceeding
the periodic sequence by TR/2 will stabilize
the signal for on-resonance spins, but not
for off-resonance magnetization. Various
approaches have been published, how to
improve the initial signal behavior. 32
Nevertheless, balanced SSFP is not really
suitable for single shot imaging. The main
are of applications are based on 3D-versions
and/or repetititive measurements during
continuous acquisition.
Practical limitations for balanced SSFP
will be determined by SAR. Optimum flip
Fig. 9.12: Steady state signal intensity lral (left vertical
scale) and Ernst angle α (vertical scale right) for data angles are quite appreciable (αSSFP = 90° for
acquisition with spoiled FLASH as a function of T2/T1=1). According to the earlier discussion
TR/T1 (see Fig. 9.6B) the practical limit of TR will
Ultrafast and Parallel Imaging 123
clear, that quite appreciable waiting times The necessity to come back to the same
have to be accepted between successive point in k-space before each refocusing pulse
applications of HASTE especially when 180°- plus the finite duration of such pulses leads
refocusing pulses are being used. Luckily, it to a quite significant ‘overhead’ in terms of
has been shown early on, that the refocusing the time efficiency of the experiment.
flip angle can be quite considerably reduced Therefore it is clear, that RARE (TSE,
with only a minor penalty in SNR. For FSE…) is not well-suited for ultrafast,
constant refocusing flip angles SNR will still repetitive measurements. On the other hand,
be 50 percent at α = 60° or 1/9th of the the extremely benign signal behavior leads
original RF power (see Eq.6). to very high image quality even at
The very benign and robust signal moderately fast implementations with
behavior and the excellent tissue contrast acquisition times in the order of 250-500
make RARE (TSE, FSE…) very attractive ms. This very robust signal behavior allows
especially in the rapidly developing field of the application everywhere in the body and
high field imaging at 3 T or even higher. At not just for head imaging.
the same time, SAR dramatically increases
with field strength. Consequently, the BEYOND THE LIMITS: PARALLEL
development of strategies to optimize RARE IMAGING
(TSE, FSE…) without any signal loss has
found widespread interest. The hyperecho- From the earlier discussions it seems like
technique has been introduced first as a very the development of fast MRI has reached a
generic way to recover fill magnetization status of completion, where even dramatic
after arbitrary pulse sequences.34 Applied further increase in gradients and RF
to RARE (TSE, FSE…) it can be used to performance will lead to only minor
retrieve the full SNR at the particular echo improvements in imaging speed. Therefore
used for encoding the k-space center. In image acquisition in times below 10-20 ms
recent years further improvements leading per 2D-slice appear to be infeasible at least
to asymmetric Hyperechoes (called TRAPS for imaging in humans. These limitations
= TRAnsition into Pseudo Steady state) have been shown earlier to be of a rather
have been developed, which allow to fundamental nature, which can not be
dramatically reduce SNR by a factor of 3-5 overcome by further hardware development
for conventional multishot applications and or by inventing just another sequence.
up to a factor of 10 for single shot HASTE/ Whenever fundamental limits are
RARE.35 By calculating flip angles such, that reached, it is wise to question the underlying
the envelope of the echo train assumes a concepts. The basic cornerstone of MR
Gaussian shape, this will not only improve imaging is spatial encoding by gradients and
SAR, but also lead to a better point-spread sampling the signal sequentially while going
function and thus to improved image around k-space. It has been known for quite
quality.36 It has been shown, that an overly sometime, that the spatial selectivity of coils
fast variation of the refocusing flip angles can be used to discriminate signals from
will lead to a loss in image resolution,37 but different parts of an object even during
this can be easily avoided by use of a careful simultaneous data sampling. The real
and proper implementation. breakthrough to parallel imaging started,
Ultrafast and Parallel Imaging 125
however, with the introduction of the changes affect only a fraction of the total
SMASH-technique by Sodickson.38 The basic data. An even more extreme example is
concept is rather simple at least in found in mapping of the magnetic field by
retrospective. If coils with approximate sequential acquisition of EPI-images with
sinusoidal sensitivity are put together, than different echo times, where parallel
different linear combinations of the signals reconstruction factors by a factor of 4-8 can
sampled separately can be generated, which be easily realized without any loss in
correspond to different lines in k-space. The information content.19
reduction in the number of lines, which have In imaging applications the improvement
to be consecutively sampled, is reduced in in imaging speed the reduction factor
proportion to the number of separate coils. depends on the number of independent
In the basic concept the field of view is coils, which is always smaller than the
determined and fixed by the hardware of number of coils actually used. Strong
the coil arangement. Following the original overlap between the sensitivity profiles of
developments, various improvements like the coils will lead to a redundancy in the
GRAPPA,39 autoSMASH40 and others have data and thus to errors in the coefficients
been found leading to a more general way used to separate the acquired signals. This
to simultaneously acquire multiple k-lines will lead to increased noise but also to
without the inherent limitations of SMASH. artifacts caused by signal misattribution. On
On a parallel track and immediately the other hand, strong separation between
following the original presentation of sensitivity profiles will improve signal
SMASH, the SENSE-approach has been separation, but at the cost of reduced SNR.
introduced by K.Pruessmann.41 SENSE also At the higher frequencies at higher fields,
is based on data acquisition using separate the separation of the coil sensitivies can be
coils with different spatial sensitivity. Data shown to improve and higher reduction
recombination is, however, performed in the factors become feasible. In general parallel
image domain rather than on the k-space imaging will suffer from a penalty in SNR,
data. Various improvements have been which at best is proportional to the square
found for SENSE also.842-45 Maybe the most root of the reduction factor. In a very
important current development in parallel elegant paper, the theoretical limits of SNR
imaging is related to the fact, that parallel to be gained by parallel imaging have
data acquisition strategies can not only be already been established.48 These limits are,
applied in the spatial domain. The however, based on data acquisition with
algorithms can also be used to improve equal weights for all data points. Except for
sequential data acquisition in a technique steady state sequences like FLASH and
called k-t-BLAST.46 It can indeed be shown, trueFISP, many fast imaging techniques used
that parallel techniques are expecially well today are based on the acquisition of a
suited to be applied along data, which do signal, which decays with T2* (EPI, SPirals)
not show excessive changes along one or T2 (TSE). Parallel imaging will reduce
particular domain in the dataset. In k-t- the length of the echotrain. As shown in
BLAST this scarcely sampled domain is the Fig. 9.15 this will lead to discarding low
time domain for the sequential observation intensity signals at the end of the echotrain.
of images of moving objects, where typical For an echo train length corresponding to 2
126 Biomedical Magnetic Resonance: Proceedings of the International Workshop
T2, reducing the acquisition by one half will is of particular interest especially in high
already lead to a slight increase in SNR. field MR as discussed above.
Depending on the decay constant, SNR can Similar arguments also apply to other
thus even improve with parallel imaging single shot techniques like EPI and spiral
approaches. This is illustrated in Fig. 9.16, imaging, where the reduced acquisition
where parallel acquisition for hyperecho TSE window leads not only to an improvement
with considerably reduced number of data in SNR, but also to much better image
leads to improvement in SNR by more than quality as shown in Fig. 9.10.
50 percent.47 In practical terms the reduced The number of coils to be used in parallel
number of data will not only improve SNR, imaging will increase with decreasing coil
but lead to a further reduction in SAR, which size compared to the observed field of view.
In the extreme case, N × M independent
and very small coils can be used to acquire
a N × M image, where the signal in each
pixel is uniquely defined by the signal in
one coil. Such a massive parallel imaging
approach has already been demonstrated in
principle.49,50 The penetration depth of such
coil arrays will be in the order of the size
of a single coil, so medical applications may
be limited to the surface of the body. In
terms of the ultimate technical limits, the
Fig. 9.15: Effect of reducing the length of the echotrain imaging speed of such massive parallel
by parallel imaging. SNR is proportional to the square
imaging arrays are determined by the
root of the are under the curve. At sufficiently fast
signal decay, SNR can thus increase, when the less sampling speed. Theoretically a ‘shutter
important data at the end of the echotrain are speed’ in microsecond range could be
discarded (shaded area) achieved by reconstructing one image per
sampling point. The SNR of such
implementations will, however, be a severe
challenge.
CONCLUSION
The limits of imaging speed are in the order
of a few 10th of milliseconds, when
conventional principles of gradient encoded
Fourier imaging are being used. Parallel
imaging approaches allow further reduction
of the image acquisition time. In the ultimate
Fig. 9.16: TSE image with full acquisition (left) limit using as many RF coils as there are
compared to an image acquired with GRAPPA at a
reduction factor of 2 (right). Image resolution is
data points in the image, imaging times in
identical. For deep gray matter SNR is improved by > the microsecond regime could be realized
50% with parallel imaging but with coil arrays being sensitive only at
Ultrafast and Parallel Imaging 127
their immediate surface. The ultimate and to a large rectilinear matrix: A practical solution
realistic limits of fast imaging will be for routine systems. J Magn Reson Imaging
1999;10:84-92.
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13. Rasche V, Proksa R, Sinkus R, Bornert P, Eggers
further ‘lateral thinking’ will be required to H. Resampling of data between arbitrary grids
escape the boundary conditions of current using convolution interpolation. IEEE Trans
imaging techniques. Hyperpolarization with Med Imaging 1999;18:385-92.
the possibility to increase SNR by several 14. Sedarat H, Nishimura DG. On the optimality
of the gridding reconstruction algorithm. IEEE
orders of magnitude may offer totally new
Trans Med Imaging 2000;19:306-17.
opportunities to redefine imaging speed in 15. Morgan PS, Bowtell RW, McIntyre DJ,
future MR applications.51,52 Worthington BS. Correction of spatial distortion
in EPI due to inhomogeneous static magnetic
fields using the reversed gradient method. J
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Reson Med 2001;46:6-12.
Hyperechoes Combined with Partial Fourier
35. Hennig J, Speck O, Scheffler K. Optimization of
Parallel Imaging. Proc. Intl. Soc. Mag. Reson.
the signal behavior in the transition to driven
Med, XII, Kyoto, 2004;539.
equilibrium in steady state free precession
sequences, Magn Reson Med 2002;48:801-09. 48. Wiesinger F, Boesiger P, Pruessmann KP.
36. Hennig J, Weigel M, Scheffler K. Multiecho Electrodynamics and ultimate SNR in parallel
sequences with variable refocusing Flip Angles: MR imaging. Magn Reson Med 2004;52:376-90.
Optimization of Signal Behavior using smooth 49. DG Brown, MP McDougall, and S.M. Wright,
Transitions between Pseudo Steady States Proc Intl Soc Mag Reson Med 2002;10:863.
(TRAPS), Magn Reson Med 2003;49:527-35. 50. MP McDougall, SM Wright. Coil Phase
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angle in FSE sequences. Magn Reson Med Kyoto,2004;330.
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acquisitions (GRAPPA). Magn Reson Med NMR. Proc Natl Acad Sci USA 2003;100:
2002;47:1202-10. 10158-163.
10
fMRI: Neuroscientific and
Clinical Applications
Wolfgang Grodd
sented, an electronic protocol can be gene- the other fingers yielded a somatotopic
rated. In addition, cardiac and respiratory representation of the individual fingers and
responses may be collected to indicate concluded that somatotopy within the hand
whether a certain picture or stimulus has area of M1 is represented as “quantitative
been recognised during an examination. predominance of certain movement or digit
representation embedded in an overall joint
APPLICATIONS hand area”. Using PET Grafton et al 18
reported distinct activation maxima in M1
fMRI of Sensorimotor Somatotopy of shoulder, elbow, wrist, and finger
In 1873 Jackson9 was the first to postulate movements if an individual analysis was
that distinct cortical activation centers serve performed, but this distinction was lost after
for different types of movements after grouped analysis. Therefore we performed
having observed isolated movements of an fMRI study to map foot, elbow, fist,
different body parts during epileptic convul- thumb, index finger and lip movements in
sions. He hypothesized that the size of the 30 healthy subjects. For each movement type
cortical area which represents movements, confidence intervals of representational sites
is not proportional to the size and strength in M1 were evaluated.19 In order to improve
of the muscle group involved but to the the precision of anatomical location and to
degree of differentiation and specialization optimize the mapping of cortical activation
for the type of movement. Since then sites, we compared the assessment of
numerous experimental studies were locations in the conventional 3D-system and
performed and a functional differentiation an own 2D-projection method. In addition
of pre- and post-central gyri was achieved to the use of activation maxima the location
by Sherrington and coworkers.10 In 1936-37 was determined by the center of gravity of
Foerster as well as Penfield and Boldrey the activation cluster within the precentral
reported a somatotopic order of movements gyrus (Fig. 10.2, Plate 8). Both methods
in the human precentral gyrus comparable showed a high overlap of their confidence
to the somatotopic representation of somato- intervals but only the 2D-projection method
sensory stimuli in the postcentral gyrus.11,12 revealed statistically significant distinct
Later more precise electrical stimulation in locations for the elbow and index finger
monkeys13,14 suggested that the concept of movements and for index finger to thumb
a somatotopic organization of the primary movements. Increased degree of complexity
motor cortex (M1) is probably only adequate of finger movements resulted in a spreading
in a gross approximation. The representation
of the location in the direction of the arm
areas of finger and hand movements in M1
(Fig. 10.3, Plate 8).
show strong overlap indicating that
movements of different representational
fMRI of Speech Production
sites share atleast some neural substrates.
Similar functional imaging studies An important neuroscientific field is the
showed a high overlap of the represen- study of language processing and compre-
tational sites in M1.15,16 Kleinschmidt et al17 hension in the cognitive and speech motor
showed that subtraction of maps of domain. Commonly cortical areas like of the
individual finger movements from those of left inferior frontal lobe (Broca´s area), the
132 Biomedical Magnetic Resonance: Proceedings of the International Workshop
left superior temporal lobe (Wernicke´s and similar timing (early 3rd trimester of
area), and the primary motor cortex bila- pregnancy) by fMRI.22 Twelve young adult
terally are primarily attributed to language patients with congenital hemiparesis (Fig.
functions. But by disentangling syntax, 10.5) due to unilateral lesions in the
semantic and prosody aspects in respect to periventricular white matter and without
speech motor control yielded in a more any history of epileptic seizures were studied
refined view. Penfield and Rasmussen as well as 10 age-matched controls. The goal
already reported on arrest of vocalization was to examine the localization of the motor
and speech after left as well as right hemis- system and of language processing especially
pheric electrical stimulation.20 To evaluate in respect to its motor part, the speech pro-
lateralization of speech production at the duction.23 The severity of structural damage
level of the primary motor cortex an fMRI to hand motor projections of the cortico-
examination was performed during a speech spinal tract was assessed on semi-coronal
task (continuous silent recitation of the MRI reconstructions along anatomical
names of the months of the year). As control landmarks of cortico-spinal tract (Fig. 10.6).
conditions, non-speech tongue movements The functional integrity of these crossed
and silent singing of a well-known melody cortico-spinal projections in the affected
with the syllable ‘la’ as its carrier were hemisphere, as well as the presence of any
considered.21 Tongue movements produced abnormal ipsilateral projections to the paretic
symmetrical activation at the lower primary hand, was examined by transcranial mag-
motor cortex. During automatic speech a netic stimulation (TMS). Cortical activation
strong functional lateralization to the left during simple voluntary hand movements
hemisphere emerged within the same area. was studied by functional magnetic
In contrast, singing yielded a predominant resonance imaging (fMRI).
right-sided activation of the motor region
(Fig. 10.4). Functional lateralization of speech Motor Function
production therefore seems to include the
The severity of damage to cortico-spinal
pre-central gyrus as well as Broca´s area.
projections of the pyramidal tract, as
assessed on semi-coronal reconstructions,
Cortical Reorganization in Unilateral
correlated with motor impairment of the
Brain Lesions
paretic hands, and also with the type of
Reorganization of motor and language cortico-spinal tract reorganization. Patients
functions after early brain injuries is not with small lesions (SL; n = 4) and only mild
only determined by the maturational stage hand motor impairment possessed intact
of the central nervous system at the time of crossed cortico-spinal projections to the
the insult (“timing”), but also by the struc- paretic hand (TMS), whereas in those with
tural properties, location, and extent of the large lesions (LL; n = 6) and more severe
lesion. To address the impact of different hand motor impairment, no motor response
lesion extents on the type of reorganization could be elicited by TMS of the affected
induced we examined a cohort of patients hemisphere. Evidence for compensatory
with lesions of uniform structure and recruitment of the unaffected hemisphere
location (unilateral periventricular defects) was found in both subgroups: In the SL
fMRI: Neuroscientific and Clinical Applications 133
Fig. 10.4: Top: Axial T1-weighted images with individual activation clusters at the level of the basal ganglia in
one representative subject during left aprosodic (automatic) speech, middle vertical tongue movement and
right during syllable singing. Note the left hemispheric activation during speech (regions 3,4 and 5), and the
right hemispheric activation during singing (area 4 and 5). Bottom: Corresponding group results of the
distribution of activated voxels in left and right area 4 and 5 from 10 subjects
group, fMRI demonstrated ipsilateral combined features of both groups (SL, LL).
activation of premotor areas, without any
abnormal projections to the paretic hand Language Function
originating from these sites (TMS). In the In the second part of our study seven young
LL group, such abnormal ipsilateral adult patients were studied. 24 All had
projections to the paretic hand were indeed attended main-stream schools, without any
found (TMS), and fMRI confirmed cortical obvious cognitive or language deficits. The
activation of an abnormal ipsilateral hand cortical organization of language was
motor representation in the primary assessed by fMRI using two different
sensorimotor region of the unaffected activation tasks: To monitor language
hemisphere (Fig. 10.7). Two patients with perception, a narrative story was presented.
intermediate-sized lesions presented For the assessment of language production,
134 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 10.5: Axial T2-weighted images of 12 patients included in this study with right-sided
hemiparesis due to left-sided periventricular leukomalacia
Fig. 10.6: Reconstruction of semi-coronal plane along the course of hand motor projections, adopted from [2].
(A) Axial and b) parasagittal image with the hand knob identified by its omega shape (axial) and its hook sign
(inset in (B). Both sign were used for the upper landmark and the anterior third of the posterior limb of the
internal capsule (dashed line in (C)) as the lower landmark. (D) The semi-coronal plane of this patient shows
enlargement of the right lateral ventricle (black arrow) and hypointensity of the adjacent white matter, which
corresponds well with a moderate hand motor impairment (score 2)
fMRI: Neuroscientific and Clinical Applications 135
Fig. 10.7: Top: Individual structural and functional MRI results (paretic hand movement) for two representative
patients from each group. Left side: Semi-coronal reconstructions (from T1-weighted 3D-data) following
anatomical landmarks of pyramidal tract somatotopy, illustrating the severity of structural damage to hand
motor projections. A white “figure-of-eight coil”-symbol indicates hemispheres from which MEP’s in the paretic
hand could be elicited. Right side: Individual fMRI activation patterns (SPM99, p <0.05 corrected) during
paretic hand movement. The functional data are displayed on individual surface reconstructions calculated
from normalized 3D-data sets. Bottom: Schematic illustration of cortico-spinal tract organization in the three
subgroups of patients (P = paretic hand), and TMS results from one representative patient from each group.
Vertical dashed line = time of the TMS stimulus
the subjects were instructed to silently next word starting with the last letter of
generate word chains, the first word starting the previous word, and so on. Hemispheric
with a letter given by the examiner, the asymmetries of fMRI activation for each task
136 Biomedical Magnetic Resonance: Proceedings of the International Workshop
were assessed by determining the number Representative examples for the activation
of activated voxels (SPM99, p<0.05, patterns in one patient and one control are
corrected for multiple comparisons) in each given in Figure 10.8B, Plate 9. The degree
cerebral hemisphere. From these voxel of rightward asymmetry during language
counts, asymmetry ratios were calculated production correlated with the total lesion
as (R-L)/(R+L), yielding a range from –1 (= volume (r = 0.750, p = 0.026, Spearman corre-
exclusive left-hemisphere activation) to +1 lation rank) and, more strongly, with the
(= exclusive right hemisphere activation). lateral extent of the lesion in the recons-
The relationship between the lesion and the tructed semi-coronal plane (r = 0.893, p =
facial portion of the pyramidal tract, we 0.003, Figure 10.8C, Plate 9).
used the same approach as depicted above
to correlate pyramidal tract lesions to motor CONCLUSION
impairment. Neurons projecting to the lower
The presented fMRI results on functional
face are located in the lower half of the anatomy of the motor system and of cortical
central region; in the internal capsule, their networks engaged in language production
axons pass through the genu (Fig. 10.8, Plate as well as the detected cortical reorganiza-
9). Therefore, the course of facial pyramidal tion after acquired brain lesions should
tract projections can be depicted on a coronal convince the reader that the fMRI method
reconstruction that includes these landmarks can reliably be used to address neuroscienti-
(see Fig. 10.8D, Plate 9). The severity of fic questions and safely be applied in a
damage was quantified by measuring the clinical setting. With the latter prospect we
distance from the most lateral part of the might gain a deeper insight in neuronal
lesion to the interhemispheric fissure; again, mechanism of functional reorganization and
the correction for different brain sizes was compenzation after localized brain damage,
achieved by dividing this value by the which will be of relevance for the design of
new therapies and rehabilitation regimens.
distance measured from the lateral wall of
the lateral ventricle in the intact hemisphere REFERENCES
(x/y in Fig. 10.8D, Plate 9). Figure 10.8A
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displays the asymmetry ratios for our nation-sensitive contrast in magnetic resonance
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for language production (mean –0.930, SD 2. Turner R, Le Bihan D, Moonen CT, Despres D,
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respect to the asymmetry of language Kennedy DN, Hoppel BE, Cohen MS, Turner R
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Taylor J (Ed.) Selected writings of John Hugh-
B, Grodd W. fMRI-evaluation of somatotopic
lings Jackson. Basic Books, New York 1958.
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10. Leyton ASF, Sherrington CS. Observations on
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20. Penfield W, Rasmussen T. Vocalization and
and gorilla. Q Jl Exp Physiol 1917;11:135-222.
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11. Foerster O. The motor cortex in man in the
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2918-47. Mann I. The pyramidal tract in congenital
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138 Biomedical Magnetic Resonance: Proceedings of the International Workshop
11
Appropriate Functional Magnetic
Resonance Imaging for the
Study of Evolving Yogis
N Kochupillai, V Nandini
yogic tranquility, to borrow an expression meters. On the basis of known and emerging
from Shakespeare! Obviously, Mr Chakyar, functional anatomy of brainstem techno-
through life long yoga and sadhana, scientific competence for such sensitive and
successfully erased the voluntary— discriminant study of the living brain stem
involuntary dichotomy in facial expression would help unravel a variety of interesting
and added a 3rd aesthetic dimension to the variations from the ‘normal’ in early
expression language of face by transforming evolving yogis. Examples of such yogis
it to artistic experience! Here we have an include the Tum-mo yoga trainees with the
excellent paradigm to study the yogically neuro-biologically altered bodily thermo-
engineered anatomy and physiology of genesis and practitioners of pranayama
human facial expression, in all its psycho- dhyana based Patanjali yoga and related to
physical and even spiritual dimensions. But psycho-physical training and evolution it is
before we can venture into it by studies not contextual or relevant here to elaborate
involving ‘koodiyattam’ artists at different details in this regard. Suffice it to state that
stages of their professional training, we need the major impact of these practices on our
to bridge gaps in the techno-scientific mind-body system has been scientifically
approach to it, particularly in the realm of documented to some extent. Therefore,
functionally and anatomically definable sub-
imaging living and functioning human brain!
cortical systems involved in these yogic
Brainstem: The Psychosomatic phenomena can be studied appropriately if
Generator-cum-Switch Board of technical requirements of the type referred
Yogic Transformation to above are achievable to facilitate mini or
micro-functional imaging of brainstem of
Those who go through yogic progression evolving yogis. It is earnestly hoped that
with neurobiological insights can discern that imaging science would soon come up with
psycho-physical practices like pranayama appropriate science and technology to permit
and dhyana are both energizing and scientific studies on one of the most
transforming in a psycho-somatic sense— enigmatic facets of the mind body gestalt
and that the major anatomical substrate for of man!
such transformation is brain stem. Therefore,
a area that need to be studied to unravel REFERENCES
the biology of yoga would be the brain stem.
1. Logothetis NK, Guggenberger H, Peled S, Paul
On the basis of known anatomy of the
J. Functional imaging of the monkey brain.
various functionally and anatomically Nature Neurosci 1999; 2: 555-62.
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say that pixel size requirement to study the magnetic resonance imaging of human brain
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discrimination are not met, in a technical Proc Natl Acad Sci USA 1992; 89: 5675-79.
sense by the currently available imaging 3. Grodd WG. Recent advances in functional MRI
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MR Imaging and Spectroscopy. New Delhi:
specificity/requirement in this regard may Jaypee Brothers Medical Publishers (P) Ltd,
vary from few microns to several milli- 2005.
Perinatal MR Studies in Animal Models 141
12
Perinatal MR Studies
in Animal Models
RJ Ordidge, DW Carmichael, X Wang, DM Peebles
pre-birth injury process associated with Studies in the adult rat and neonatal
hypoxia in brain in vivo without the piglet show a close correlation between
confounding effects of maternal physiology. reductions in apparent diffusion coefficient
Intrauterine growth restriction is associated (ADC) and Adenosine tri-phosphate (ATP)
with newborn encephalopathy5 and may (which is essential for cellular energy
increase susceptibility to perinatal hypoxia- metabolism), measured by 31P spectroscopy
ischemia. Infection has also been shown to or biochemically. 13,14 Hence, diffusion-
be a major etiology in fetal brain injury5,6. weighted MR imaging (DWI) was used to
Therefore, the synergistic effects of hypoxia measure the ADC of brain water before,
and either growth retardation or pre- during and after the hypoxic episode to
treatment with a bacterial endotoxin are provide a putative marker of cellular energy
evaluated using this model. failure.
T2*-weighted MRI is commonly used to
HYPOXIC INJURY IN CHICKEN monitor cerebral deoxyhemoglobin levels in
EMBRYO BRAIN activated human and animal brain regions
Effective hemodynamic and metabolic using the blood oxygenation level depen-
compensation mechanisms are thought to dent (BOLD) contrast effect.15 Therefore,
make the fetal brain less vulnerable to HI T 2 *-weighted MR imaging was used to
than at later stages of development.7,8 These measure changes occurring during the
mechanisms are important because their hypoxic episode.
failure, for example during severe HI during
labor, can result in brain injury with the Materials and Methods
long-term consequence of cerebral palsy.3 Fertilized white leghorn chicken eggs were
The fetal hemodynamic response to incubated at 38.5º C and 50 to 60 percent
hypoxemia, defined as an insufficient humidity. Studies were performed on
delivery of oxygen to the brain, has been incubation day 19 (hatching occurs on day
well characterized in humans, and more 21).
recently, the chick embryo.9,10 However, less
is known about fetal cerebral metabolic NMR Experimental Procedure
responses.
Therefore, proton magnetic resonance The chick embryo were anesthetised with
spectroscopy (1H MRS) was used to measure either 5.0 mg ketamine, dropped onto the
changes in cerebral metabolite concentration chorio-allantoic membrane through a hole
during acute hypoxia in the chick embryo in the shell, or immoblized in a similar
at Day 19 of incubation. MR visible meta- fashion using 0.5 mg tubocurarine one hour
bolites of interest are the N-acetyl aspartate prior to the start of the insult. A non-
(NAA) resonance which is only present in magnetic thermistor wire (Luxtron) was
viable neurons11 and, therefore, provides positioned for continuous monitoring of
an estimate of the concentration of such temperature in vivo. The egg was placed
cells, and the Lactate (Lac) resonance which into a plastic bag, adapted with an inlet
is a product of anaerobic metabolism and and an outlet tubing system for gas supply
also an alternative metabolic substrate for and exhaust, through which room air was
the developing brain.12 circulated (5 litres per minute). Inside the
Perinatal MR Studies in Animal Models 143
bag, the egg was placed onto a 7 × 5.5 cm cerebral metabolic response and ADC/T2*
(elliptical) surface coil and temperature was changes that accompany hypoxia. 1H-MRS
regulated to maintain the eggs at approxi- were obtained using PRESS with an echo
mately 35 to 36°C (either by circulation of time (TE) of 135 ms, repetition time (TR) of
heated air or heated water through piping). 2 s, and the spectra were obtained from a
The arrange-ment was then placed within 4 × 4 × 4 mm3 volume.
the magnet bore of a 7 Tesla Bruker Biospec ADC measurements were obtained using
imaging spectrometer (Bruker Medical spin-echo EPI (four images, one with no
GmbH, Ettlingen, Germany). diffusion weighting (b~0) and three with
diffusion weighting (b = 875) applied along
MRI and MRS the three Cartesian axes; TE = 110 ms, TR
= 2.5 s, 128 × 128 matrix; 3.5 cm field of
Multi-slice gradient - echo MR scout images view). ADC maps were calculated by
were obtained in three orthogonal planes performing a log/linear fit for each image
to determine the position of the chicks head pixel and combined to obtain the trace ADC
within the egg. Baseline MR measurements value.
were taken prior to the insult, then data Absolute T2* values were measured using
acquired throughout the duration of the the fast low angle single shot (FLASH)
insult and post-insult. The egg and bag were method with echo times of 6.3, 23.4 and
then transferred to a 2.5 cm diameter 33.4ms (128 × 128 matrix, TR = 100 ms, field
circular surface coil with the head placed of view = 3.5 cm; total scan time for one
immediately over the coil center. Global image = 12 s; one average for 6.3 ms echo
shimming was performed and further time, three averages for 23.4 and 33.4 ms
gradient-echo scout images acquired to echo times).
establish geometrical locations for the The spectra were analyzed using an
subsequent acquisitions. Volume-localized automated linear combination modeling
1
H MRS spectra were acquired using a point- method, whereby in vivo spectrum are fitted
resolved spectroscopy sequence (PRESS).16,17 with a weighted sum of spectra from in
vitro solutions of the compounds (the basis
Protocol A set). The basis set was acquired using the
This was used to collect high quality 1H same pulse sequence, echo time, and under
MRS. PRESS was implemented with an echo the same conditions as in the in vivo spec-
time (TE) of 270 ms, repetition time (TR) trum, to ensure that effects of J-modulation,
eddy currents, and partial volume are
5 s, and the spectra were obtained from a
6 × 6 × 6 mm3 volume containing mostly modelled correctly.18,19 The spectral data of
tissue from the cerebral hemispheres. A choline (Cho), NAA, Lac, alanine, taurine,
signal average of 256 echoes was used for beta-hydroxy butyrate, asparatate, gluta-
mate, glutamine, inositol, taurine and GABA
the baseline measurement (total acquisition
time 21.3 minutes), and 128 echoes was used (gamma amino butyrate) were analyzed.
in subsequent acquisitions (total acquisition
time 10.7 minutes). Hypoxia and Recovery
Fig. 12.2: Metabolite response versus time of cerebral lactate/creatine and NAA/creatine ratios for one
hour of hypoxia (shown by bar) with FiO2 = 4% (n = 10)
146 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 12.3: Temporal response of cerebral trace ADC Fig. 12.4: Temporal response of change in cerebral
(lower graph) within the embryonic brain, before T2* (lower graph) within the embryonic brain, before
during and after hypoxia (one hour; FiO2 = 4 percent; during and after hypoxia (one hour; FiO2 = 4%; n =
n = 10). The upper three images show actual trace 10). The upper three images show T2*-weighted
ADC maps with a visible decline during the hypoxic images (TE = 23.4 ms) with a visible reduction in
period signal amplitude during the hypoxic period
Histology did not show any significant changes compared with 1H MRS. In neonatal
neuronal damage in chicks that had been piglet brain, a correlation was demonstrated
subject to hypoxia with FiO2 = 4 percent between reduced cerebral ADC and loss of
(although a small amount of apoptosis high energy phosphorous metabolites. 21
occurs naturally in perinatal brain). Therefore, reduced ADC is likely to offer a
similar surrogate index of cellular energy
Conclusion failure in this model.
1
Compared with a milder insult, mild/mode- H MRS OF TRANSIENT CEREBRAL
rate acute hypoxia caused a decrease in HYPOXIA IN A CHICKEN EMBRYO MODEL
ADC, compatible with a depletion in high- OF FETAL GROWTH RETARDATION
energy metabolites and T2* values suggest
Intrauterine growth restriction may increase
hyperperfusion post-hypoxia. However, the
cerebral damage susceptibility to perinatal
results from MRI/MRS suggest while acute hypoxia-ischemia. On the basis of the
hypoxia (with FiO2 = 4 percent) caused previous study, where 1H MRS was shown
metabolic disturbances and cellular energy to be a sensitive marker for hypoxic injury,
deficits it constitutes a relatively mild insult. the chicken embryo model was used to study
Histology data confirmed that the brain cells the effects of chronic hypoxia and protein
could repair and recover from this injury, insufficiency during incubation upon 1 H
as no significant neuronal damage was metabolite levels. In vivo MRS was also used
observed. to compare the cerebral metabolic response
The advantage of acquiring ADC trace to acute hypoxia in growth-restricted (GR)
maps is that they provide excellent spatial with that observed in normally-developed
resolution that could better define regional chicks.
Perinatal MR Studies in Animal Models 147
Fig. 12.5: Right hand graph: Cerebral NAA/creatine and inositol/creatine ratios for normal (n = 8) and growth
restricted (n = 11) chick embryos. Left hand graph: Cerebral β-hydroxybutyrate/creatine ratios for normal and
growth-restricted chick embryos (levels of min, IQR, median and max are shown by horizontal lines in
ascending order)
148 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 12.6: Cerebral lactate/creatine ratios before, during and after hypoxia (40 minutes, FiO2 = 8%) for normal
(n = 8) and growth restricted (n = 11) chick embryos. The period of hypoxia is shown by a bar. Significant
differences are indicated by a star(*) next to the data; p < 0.01 Mann-Whitney Rank Sum test
assigned to β-HB. However, β-HB may act effect is mediated by changes in cerebral
as an alternative energy substrate and metabolism.
elevation would be consistent with enhanced Chick embryos (19 days incubation)
ketosis consequent to impaired nutrition. received either 0.4 ml of saline (control
The acute hypoxic insult caused an elevation group) or lipopolysaccharide (LPS, 3mg)
of cerebral lactate (presumably due to from Salmonella typhimurium. Four hours
increased anaerobic glycolysis) which was later they were subject to either normoxia
attenuated in the GR group. The reason for (for embryos that would subsequently be
the attenuation is as yet difficult to elucidate.
sacrificed for histopathology) or hypoxia
However, such a response may suggest that
(FiO2 = 4%) for one hour (for embryos to
GR embryos find it less easy to recruit
be studied by MR). 1H MRS and trace ADC
anaerobic glycolysis in order to replenish
ATP in response to a hypoxic insult. and T2* were repeatedly-measured before,
during and for 2 hours post-insult according
TRANSIENT CEREBRAL HYPOXIA IN A to Protocol B. Data was then analyzed and
CHICKEN EMBRYO MODEL WITH combined to obtain the trace ADC.
BACTERIA INFECTION MR data shows that compared to base-
line values brain Lac/Cr ratios increased
Having established the synergetic effect of dramatically by the end of hypoxia and then
growth retardation and hypoxia in the
returned to baseline by 2 hours (Fig. 12.7).
previous study, this section aims to investi-
In contrast, ADC fell during hypoxia, indica-
gate whether prior exposure to endotoxin
worsens the degree of cerebral injury ting a change in cell membrane homeostasis,
observed in chick embryos in vivo after an and then recovered (Fig. 12.8). However,
acute global hypoxic insult and whether this these changes were not significantly
Perinatal MR Studies in Animal Models 149
Fig. 12.7: Cerebral lactate/creatine ratios plotted against time for chick embryos that had received hypoxia +
LPS (n = 9) and hypoxia alone (n = 10). The hypoxic period is shown by a bar (one hour; FiO2 = 4%)
Fig. 12.8: Cerebral trace ADC values plotted against time for chick embryos that had received hypoxia + LPS
(n = 9) and hypoxia alone (n = 10). The hypoxic period is shown by a bar (one hour; FiO2 = 4%)
150 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 12.9: Change in cerebral T2* values plotted against time for chick embryos that had received hypoxia
+ LPS (n = 9) and hypoxia alone (n = 10). The hypoxic period is shown by a bar (one hour; FiO2 = 4%)
resonance. However, due to the relatively and hypoxia suffer secondary cerebral
long echo time used in our study (270 ms) energy failure due to their weakened state
and the shorter relaxation time of PCr resulting in cellular death, but those that
compared to Cr (T2 = 143 ± 41 ms for PCr have just received the insult make a full
and T2 = 297 ± 41 ms for Cr 31 ) significant recovery with no detectable permanent
dephosphorylation should have been damage.
observable but was not. Taken together with T2 * values dropped by upto 31 ± 19
the results of histopathology, our data shows percent during the hypoxic insult (Fig. 12.4),
that at both levels of FiO2 (8 and 4%) the which reflects increased blood flow to
hypoxic insult on its own was severe enough the brain coupled with the expected
to affect energy metabolism but mild increase in deoxyhaemoglobin. The T 2 *
enough not to cause lasting cellular damage. changes were more rapid than ADC
In addition to being a product of changes, and slightly elevated values post-
anaerobic glycolysis, lactate is known to insult may indicate hyperperfusion with
provide an alternative metabolic substrate return to normal oxygenation levels. Whilst
for the developing brain.12 We conclude that T 2* -weighted imaging may be useful to
the increase in Lac could be a component of follow the time course of hemodynamic
a co-ordinated metabolic response to match changes, it is not a prognostic indicator of
cellular energy demand with production the insult outcome. However, in the embryos
rather than representing a failure of cellular that suffered both LPS and hypoxia, the
energy metabolism. reduced decline in T2* values may indicate
In neonatal brain, reduction in ADC is that brain meta-bolism was already impaired
observed during and after cerebral hypoxia- by LPS induced fever.
ischemia in both animal and human The data of transient cerebral hypoxial
studies.21,32-34 The exact mechanism is unclear in chicken embryo model shows the
but is thought to be related to a net shift in expected finding that in the growth retarded
water from the extracellular to the embryos, neuronal density is reduced (as
intracellular space during hypoxia-ischemia, estimated by NAA). Such changes would
as a result of cell membrane depolarization also be expected to be visible in embryos
due to energy failure and a reduction in that have suffered neuronal damage by
intracellular water mobility. Reductions in severe hypoxia, although the changes are
ADC were measured in embryos that not immediate and the hypoxic insult
received hypoxia with FiO2 = 4 percent and delivered in our studies is relatively mild.
all the embryos that received both hypoxia However, paradoxically the GR embryonic
and bacterial infection, where significant cell brain shows a reduced increase in Lac during
death was confirmed by histopathology. The hypoxia compared with normally-developed
Lac and ADC changes in these two groups embryos. The observation of an increased
were indistinguishable. The lack of progno- peak, tentatively assigned to β-HB, in the
stic discrimination may be due to the time GR embryos is significant. β-HB may act as
difference of 2 days between MR studies an alternative energy substrate and
and histopathological analysis of the hatched elevation would be consistent with enhanced
chick. During the intervening period we ketosis due to impaired nutrition (protein
speculate that those embryos exposed to LPS insufficiency and reduced ambient oxygen
Perinatal MR Studies in Animal Models 153
concentration during incubation) and the 8. Newman JP, Peebles DM, Hanson MA. Adeno-
associated low levels of blood glucose. The sine produces changes in cerebral haemo-
low levels of glucose may then make it less dynamics and metabolism as assessed by near
infrared spectroscopy in late gestation fetal
easy for GR embryos to use anaerobic
sheep in utero. Pediatr Res 2001: 50; 217-21.
glycolysis to replenish ATP in response to 9. Sutterlein MW, Seelbach-Gobel B, Oehler MK
hypoxia. et al. Doppler ultrasonographic evidence of
intra-partum brain sparing effect in fetuses with
CONCLUSION low oxygen saturation according to pulse
oximetry. Am J Obstet Gynecol 1999; 181:216-
We have demonstrated the value of in vivo 20.
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but when applied in combination with other 87.
11. Salibi N and Brown MA. Clinical MR spectro-
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Magnetic Resonance in the Developing Brain 155
13
Magnetic Resonance in
the Developing Brain
David G Gadian
own contributions have included the intro- spectra often show abnormalities, indicating
duction of 1H MRS1,2 and of T2 relaxometry3 that there is relatively widespread pathology
for the detection of temporal lobe patho- that is additional to any more focal damage
logy, both in adults (in collaboration with to the hippocampi. This diffuse pathology
colleagues at the Institute of Neurology, is not necessarily evident on MRI.6 The pre-
London) and in children. Together with sence of such MRS abnormalities in areas
volumetric measurements of the hippocampi that appear normal on MRI is intriguing,
(see, for example Van Paesschen et al4), and some of the implications are discussed
these techniques add greatly to the below, in relation to our understanding of
sensitivity and specificity of conventional the cognitive impairments that may be
visual inspection of MRI scans, even when present in these children.
these scans are optimized for visualization Functional MRI studies can also be
of the hippocampi. For example, T2 relaxo- invaluable in the study of epilepsy. In some
metry (a procedure whereby the T2 value is cases, fMRI can play a role in helping to
computed from a series of MR images identify the seizure focus through the
acquired at different echo times) has proved detection of abnormal cortical activation that
to be an objective and highly sensitive means may take place at sites of seizure activity.7
of detecting abnormalities of the hippo- In addition, the use of fMRI to identify brain
campus.3,5 1H MRS provides complementary regions that are activated with motor or
information; in particular, it commonly language or memory tasks can aid in
detects the presence of additional, relatively neurosurgical planning of candidates for
widespread temporal lobe pathology than epilepsy surgery8,9, and can provide impor-
can be extremely difficult to detect on MRI. tant neuroscientific information about any
The dominant signals seen in 1H spectra reorganization of brain function that may
of the brain are normally from N-acety- occur in response to brain injury.9
laspartate (NAA), creatine plus phospho-
CHILDREN WITH MEMORY IMPAIRMENTS
creatine (Cr), and choline-containing
compounds (Cho). While much remains to The methods that we have adopted for the
be learned about the functions of NAA, it investigation of temporal lobe abnormalities
is believed to be present in the brain in epilepsy are useful for studying memory
primarily within neuronal cells, and so its impairments also, given the importance of
signal provides a means of reporting on temporal lobe structures in memory function.
neuronal loss or damage. Consistent with An important conclusion from our
this, a reduction in the NAA signal (or in extensive studies of patients with temporal
the ratio of the NAA signal to the Cr and lobe epilepsy is that many of them, including
Cho signals) is commonly seen in disorders some with well-lateralized seizures, have
where neuronal loss or damage can be bilateral pathology. This has implications for
expected. In our studies of temporal lobe our understanding of the cognitive impair-
epilepsy, we have acquired spectra from a ments observed in such patients. An
2 × 2 × 2 cm region within each temporal illustration of this is provided by a study in
lobe, positioned so that there is little which we found unexpected deficits in
contribution from the hippocampus.1,2 These verbal learning and recall amongst a series
Magnetic Resonance in the Developing Brain 157
of adult patients who had undergone right resulting images into grey matter, white
temporal lobe surgery for relief from child- matter and cerebrospinal fluid; spatial
hood-onset epilepsy.10 Given our obser- smoothing; and finally statistical analysis of
vations of bilateral pathology in pre-surgical group data on a pixel-by-pixel basis. In the
cases of temporal lobe epilepsy, we hypo- children with developmental amnesia, voxel-
thesized that the unexpected deficits in the based morphometry identified bilateral
patients with right-sided resections may reductions in grey matter, not only in the
reflect previously unsuspected pathology of hippocampus, but also in the regions of the
the left hemisphere. Subsequent MRS and putamen and the ventral part of the thala-
T2-mapping studies showed that this was mus13 (abnormalities of the putamen and
indeed the case.10 thalamus were not visible on conventional
We have seen particularly striking effects neuroradiological assessment of the scans).
of bilateral temporal lobe pathology in a This pattern of neuropathology of putamen,
number of children with a severe memory thalamus, and hippocampus is consistent
disorder that we now refer to as develop- with some of the known effects of acute
mental amnesia. Our initial report11 descri- perinatal hypoxic-ischemic injury, and
bed our findings in three young patients, strengthened the argument that the
all of whom showed a substantial reduction pathology and associated memory impair-
in hippocampal volume bilaterally. These ments in these children can be attributed to
patients had a profound impairment of the hypoxic-ischemic episodes that these
episodic memory with relatively preserved children had suffered early in life. Presum-
semantic memory, a pattern of function that ably, the degree of hypoxia-ischemia had
provoked extensive interest and debate been sufficient to produce selective damage
within the cognitive neuroscience commu- to these particularly vulnerable regions of
nity. Interestingly, 1H MRS showed much the brain, but was not sufficient to result in
more modest abnormalities than those seen the more severe neurological and cognitive
in many of our cases of temporal lobe deficits that can follow hypoxic-ischemic
epilepsy; this is consistent with the notion injury.
that the pathology is quite focal, and in turn
AN INHERITED SPEECH AND
is consistent with the selectivity of the
LANGUAGE DISORDER
resulting functional deficits.
We went on to show that this pattern of Voxel-based morphometry has also made a
memory impairments may occur as a critical contribution to our investigations of
previously unrecognized consequence of structure-function relationships in members
perinatal hypoxic-ischemic injury.12 Some of of a family with an inherited speech and
the evidence for this came from another language disorder. The disorder is caused
method of analysis of MRI scans, namely by a mutation in the FOXP2 gene14, and
voxel-based morphometry.13 This method manifests itself as a pronounced verbal and
provides an automated means of comparing orofacial dyspraxia. In the affected members
brain images from different groups of of the family, voxel-based morphometry
subjects. Several steps are involved: these revealed bilateral brain abnormalities in a
include normalization of the images to a number of motor- and language-related
standard template; segmentation of the regions of the brain, including the caudate
158 Biomedical Magnetic Resonance: Proceedings of the International Workshop
nucleus (which also showed bilateral reduc- visuospatial deficits and grey matter density
tions in volume on volumetric measure- in right extrastriate cortex.21 More recently,
ments), putamen, cerebellum, and inferior we have gone on to show that preterm
frontal gyrus.15,16,17 Further studies were children are at risk of declining IQ over-
carried out using functional MRI to examine time even if they have not suffered obvious
regions of the brain that were activated with neurological damage and that the decline is
covert verb generation and with overt verb associated with specific neural regions.22 On
generation and word repetition. In the the basis of all of these studies, it is evident
affected family members, many of the that voxel-based morphometry provides a
regions that were morphologically-abnor- powerful approach to identifying the subtle
mal were also found to be functionally- differences in brain structure that underlie
abnormal on functional MRI, with a pattern the various types of cognitive impairment
of activation that was more posterior and that may be associated with very premature
more extensively bilateral.18 Together, these birth.
structural and functional MRI studies have
provided some fascinating insights into the CHILDREN WITH SICKLE CELL DISEASE
links between the genetic abnormality and While stroke is primarily a disease of
the resulting speech and language disorder. adulthood, it can also occur in children.
Certain groups of children are at particular
CHILDREN BORN PREMATURELY
risk; for example in children with sickle cell
Advances in perinatal care have led to the disease, stroke is 250 times more common
survival of increasing numbers of children than in other children, with an incidence
born very prematurely. However, these similar to that for a general population of
children have an increased risk of cognitive elderly adults. Diffusion and perfusion
and learning difficulties. We set out to imaging techniques now have a major role
establish whether volumetric measurements to play in the investigation of patients with
and in particular voxel-based morphometry acute stroke, and we set out to evaluate
might enable us to identify the neural whether these techniques, in particular
correlates of such difficulties. Following perfusion imaging, might also play a role in
initial studies in which we showed that a investigating the hemodynamic effects of
sub-group of these children had mild chronic cerebrovascular disease, as seen in
episodic memory impairments impaired with children with sickle cell disease.
modest reductions in hippocampal volume19, An important imaging feature of acute
we went on to use voxel-based morpho- stroke is that the area of perfusion abnorma-
metry to identify a neural correlate of lity is commonly greater than the area of
calculation difficulties in a group of children diffusion change. Given the accepted
born preterm; the technique revealed an interpretation of the diffusion changes that
area in the region of the left intraparietal are seen in acute stroke, such areas of
sulcus where children with calculation diffusion-perfusion mismatch presumably
difficulties had less grey matter than child- reflect a fall in regional cerebral blood flow
ren who did not have these difficulties.20 that is not sufficient to cause energy failure,
We have also shown an association between cytotoxic edema, and inevitable infarction.
Magnetic Resonance in the Developing Brain 159
These mismatch areas may well be at risk subtle morphological abnormalities that may
of subsequent damage, however, and have not be visible on conventional neuroradio-
attracted considerable interest as a target logical assessment of MRI scans. The
for therapeutic intervention in patients with investigations described in this article
acute stroke. In our studies of children with illustrate the complementary roles that the
sickle cell disease, we have concentrated on different magnetic resonance approaches
chronic abnormalities rather than acute play in the study of brain structure/function
changes. Nevertheless, in line with the acute relationships. In addition to helping us to
studies, we also frequently detect regions understand the structural basis of a range
that display perfusion abnormalities but no of neurological and cognitive problems, one
other neuroimaging abnormality.23 On the of our long-term hopes is that early MR
basis of these and additional findings, we findings in high-risk groups of children will
believe that perfusion imaging could make be predictive of functional outcome, and will
important new contributions to our under- as a result provide the opportunity for more
standing of the pathophysiology of sickle timely intervention.
cell disease, and that it could play an
important role in the development and ACKNOWLEDGMENTS
evaluation of strategies for the prevention
This article draws on the results of a multidisciplinary
of stroke in this at-risk population of
programme of research at the Institute of Child
children. Health and Great Ormond Street Hospital for
Children, and I would like to acknowledge all of the
DISCUSSION many colleagues involved in this programme. I also
thank the Wellcome Trust for its support.
As stated at the outset, there are two
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14
MRS in Brain Pathologies
Peter B Barker, Mari Smith, A Gambini, SA Fatemi,
TO Crawford, EH Kossoff, A Horská
Fig. 14.3: Schematic diagram of the data processing scheme for phased-array MRSI. MRSI data from each
channel of the phased-array are processed in the spatial domains before combination with phase- and
amplitude-correction from B1 field maps created from MRI scans. Combined, time-domain MRSI data is then
further processed by filtering, Fourier transformation and magnitude calculation before constructing metabolic
images by integration of peak areas
142 percent depending on location, with the number of approaches for parallel imaging
greater part of the improvement due to the have been proposed. One of the attractive
higher sensitivity of the phased-array coil, features of parallel imaging is that it is a
rather than the higher field strength.8 general way of decreasing scan time, and
can be used with any type of MRSI pulse
SENSE-MRSI sequence.
As is well known from MRI, the non- Parallel encoding has been previously
uniform sensitivity profiles of phased-array described for proton MRSI of the human
coils can be used to decode spatial infor- brain.10 Since MRSI scan times are usually
mation from the MR signal.9 This then allows long (e.g. 15 to 30 minutes), acceleration
the number of phase-encoding steps to be factors of 2 or more offer substantial scan
reduced, resulting in shorter scan times time savings. Since MRSI typically involves
without sacrificing spatial resolution. A phase-encoding in 2 or even 3 dimensions,
MRS in Brain Pathologies 165
Fig. 14.4: Phased-array MRSI of the human brain recorded at 3 Tesla. T1-MRI localizer, and metabolic images
of Cho, Cr and NAA are shown. Selected spectra from deep and superficial voxel locations are shown, and
signal-to-noise ratio improvements compared to conventional MRSI performed at 1.5 Tesla on the same
subject are given. Frontal lobe hypointensity in the Cr and NAA images is due to field inhomogeneity effects
from the sinuses
the use of SENSE-MRSI in multiple dimen- Equation 4 can be solved using standard
sions offers the possibility of very large matrix inversion techniques. Note that s, A
reductions in scan time (e.g. factors of 4 or and b are all of complex data type, and that
even 8). However, minimum scan times will the SENSE-reconstruction in this case is
almost certainly be determined by signal-to- applied on time domain data. While it may
noise requirements (SNR decreases with the also be performed on the frequency domain
square root of the scan time) so very high data, the advantage of applying SENSE to
acceleration factors may not be viable unless the time domain data is that some of the
working under high SNR conditions. Our time domain filtering used on the MRSI data
implementation of SENSE-MRSI involves an may be spatially dependent (e.g. we apply
acceleration factor (M) of 2, used in one a susceptibility correction as a time-domain
direction of a 2D-MRSI scan. Multichannel phase ramp prior to performing a high-pass
data is combined according to water-suppression filter).
Figure 14.6 shows an example of a
S(t, x, y) = A(x, y)–1 b(t, x, y) ...(4)
SENSE-MRSI scan recorded with similar
where b(t, x, y) is a vector of dimension N parameters to that of Figure 14.5; the number
containing the folded MRSI data, A(x, y) is of phase-encoding steps in the left-right
the coil sensitivity profile matrix of dimen- dimension was reduced to 16, and the field-
sion N × M, and s(t, x, y) (the combined, of-view (FOV) in this direction was reduced
output MRSI) is a vector of dimension M. to 12 cm, lowering scan time to 10 minutes,
166 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 14.5: SENSE-MRSI of the human brain recorded at 3 Tesla. T1-MRI localizer, and metabolic images of
Cho, Cr and NAA are shown. Selected spectra from deep and superficial voxel locations are shown, and
signal-to-noise ratio improvements compared to conventional MRSI performed at 1.5 Tesla on the same
subject are given. Some lipid contamination is apparent in the spectra; this is due to the very intense lipid
signals detected because of their proximity to the phased-array coils. The bright rim in the NAA image is also
due to pericranial lipid contamination
with CHESS water suppression and outer- regions (p = 0.02 in basal ganglia, p< 0.01 in
volume lipid suppression 15,16 (TR/TE = white matter). Cho/Cr was unchanged in
2300/280 ms, 4 axial slices, thickness 15 mm, lesions compared either to contralateral
nominal voxel size 0.8 cm3 ). Regions of brain regions or control subjects. In normal-
interest were chosen in both ADEM lesions, appearing white matter (NAWM) in ADEM
and normal-appearing white matter in patients, NAA/Cr and Cho/Cr were signi-
standardized supratentorial brain regions. ficantly reduced (p < 0.01 and p = 0.013)
The same regions were also measured in compared to controls. Patients with multi-
control subjects. Peak areas were measured phasic disease and/or poor clinical outcome
by integration and metabolite ratios were showed a significant reduction of NAA/
Cho (p = 0.02), and also a trend for an
calculated.
increase of Cho/Cr in NAWM versus pati-
The ADEM patients were classified into
ents with monophasic disease.
2 groups depending on clinical outcome; the
“good” outcome group had monophasic
Discussion
disease progression and no permanent
neurological deficit on follow-up exami- At presentation, brain lesions in ADEM pri-
nation, while the “poor” outcome group had marily showed reduced NAA/Cr and NAA/
either multiphasic disease progression (with
at least one relapse), and/or significant
residual neurological deficit on follow-up
clinical examination. Statistical analysis
between groups was accomplished using
student’s t-test with correction for multiple
comparisons. The level of significance was
set at p < 0.05.
Results
Metabolite ratios for lesions and normal-
appearing white matter in ADEM patients,
and control data from the same regions are
presented in Figure 14.6. Figure 14.7 shows
an example of MRI and MRSI data in one
ADEM case; this patient had monophasic
disease and good clinical outcome. In total,
6 patients had a monophasic disease with
good recovery, while 5 had either multi-
phasic disease and/or permanent deficits.
N-acetyl-aspartate (NAA)/creatine (Cr) and Fig. 14.7: FLAIR MRI, metabolic images of Cho and
NAA, and spectra from lesion and contralateral white
NAA/choline (Cho) ratios in lesions within
matter in an ADEM case. There is a large T 2
the basal ganglia and white matter of hyperintense region in the right occipital white matter
patients were reduced both versus controls which is characterized by a prominent decrease in
(p < 0.01) and versus contralateral brain NAA, but without much change in Cr or Cho
MRS in Brain Pathologies 169
Cho ratios, while Cho/Cr was within normal and in normal appearing white matter)
limits. This pattern seems to be consistent would appear to be due to temporal axonal
with lesions that primarily consist of vaso- dysfunction rather than axonal loss, at least
genic edema with neuronal/axonal dysfunc- in those cases that showed improvement on
tion, rather than overt inflammatory follow-up scans and favorable neurological
demyelination (which is often accompanied outcome.
by an increase in Cho in other patho- Finally, ADEM patients with recurrent
logies).17-19 Since inflammatory demyelina- disease or poor neurological outcome
tion is a common pathological finding in showed lower ratios of NAA/Cho (and a
ADEM lesions, the absence of an elevation trend for increased Cho/Cr) compared to
of Cho in ADEM is perhaps surprising. In those with good outcome, suggesting a
MS, elevation of the Cho peak is believed possible prognostic role for proton MRSI in
to be either due to the accumulation of ADEM. We interpret these findings as
myelin breakdown products18 or due to indicating a more severe degree of
inflammation.20 It would appear that the pathology (i.e. greater degree of neuroaxonal
degree of myelin breakdown or inflamma- loss/dysfunction, more intense inflamma-
tion in ADEM (with good outcome) may be tory demyelination) occurring in those
insufficient to cause significant accumulation patients who subsequently develop recurrent
of Cho, and irreversible white matter disease and/or permanent neurological
damage. MRI T2 hyperintensity in ADEM deficits. While these preliminary findings
lesions would, therefore, appear to reflect a will require confirmation in larger studies
greater component of vasogenic edema as with long-term clinical follow-up, they do
opposed to irreversible demyelination. This nevertheless suggest a role for MRSI in
is also consistent with the lack of peri- or offering prognostic information in ADEM.
intra-lesional contrast enhancement, sugges- It should also be pointed out, although
ting a rather mild inflammatory process beyond the scope of the current article, that
without any breakdown of the blood-brain proton MRSI may be helpful in distin-
barrier. guishing ADEM from other pathologies that
Brain involvement may be more exten- may sometimes be considered in the
sive than suggested by conventional MRI, differential diagnosis; for instance, multiple
since NAWM also showed abnormal sclerosis, brain tumors, or mitochondrial
metabolism (low NAA/Cr). These changes, diseases.13,23
apparently due to lower NAA rather than
increased Cr, were found in both white and ACKNOWLEDGMENTS
gray matter regions, and would appear to This work was supported in part by P41RR15241,
indicate the presence of a more diffuse R21CA/RR91798, R21-EB00991 and Philips Medical
pathological involvement in ADEM than Systems.
suggested by conventional MRI. NAA
reductions in regions of normal MRI REFERENCES
appearance have also been reported in MS.21 1. Gillard JH, Waldman A, Barker PB. Clinical MR
In white matter, NAA is believed to be neuroimaging: Diffusion, perfusion and spec-
primarily located within axons. 22 The troscopy. Cambridge, UK: Cambridge Univer-
reduction of NAA in ADEM (both in lesions sity Press, 2005.
170 Biomedical Magnetic Resonance: Proceedings of the International Workshop
15
Application of Proton MRS in the
Study of the Pathology of Human
Cancers Other than Brain
Cynthia L Lean, Peter Russell, Carolyn E Mountford
late 1970s using an AKR mouse model5 that vials containing phosphate buffered saline
MRS could identify changes during tumor in D 2O (PBS/D2O) and frozen in liquid
development, which were not morphologi- nitrogen. The accuracy of the MRS method
cally manifest. This initial finding has now decreases with the length of time the
been substantiated over a period of 25 specimen remains at room temperature.
years.6,7 According to the type of tissue from which
The MRS can detect small populations of the biopsy was taken there is a finite time
abnormal cells with high accuracy, and the biopsy can be stored frozen at –70oC
identify and subcategorize preinvasive states. before MRS examination for diagnostic
MRS often detects changes to cellular accuracies to be maintained. For all organs
chemistry in human tissues prior to these examined to date, MRS analysis within six
changes being apparent under the light weeks of biopsy collection is sufficient.
microscope and, hence, discernable by current When preparing frozen samples for MRS
methods. The MRS methodology is also of analysis it must be considered that tissues
clinical importance in that it can identify the may leak variable amounts of potentially
extent of abnormality in preinvasive states, diagnostic metabolites into the storage buffer
particularly in patients with a predisposition upon thawing (Fig. 15.1).8 In many labora-
to cancer. Accurate diagnosis and characte- tories, preparation of tissue samples for
rization of preinvasive states is at present a
key missing link in the diagnostic chain and
would allow optimal management and
minimal therapy for many patients in whom
cancer has not fully developed.
MRS analysis includes washing thawed tissue processes,2,7,9-25 Many of these biochemical
in fresh buffer to reduce the amount of processes occur in parallel but do not neces-
residual water. This has been shown to result sarily alter in the same direction. Thus,
in the loss of large and unpredictable managing complex databases which include
amounts of possibly diagnostic metabolites large volumes of biomedical data including
prior to MRS and to reduce the reproduci- spectroscopy, pathology and patient statistics
bility of the MR analysis. The problem is requires a special methodology. The lack of
overcome by minimizing the volume of specialist methods to overcome this problem
storage buffer, omitting tissue washing and was recognized by Dr Ian Smith (National
performing the MRS measurements on the Research Council, Canada, NRC) in the late
tissue immersed in the original storage 1970s.
buffer.8 A three-stage statistical classification
strategy (SCS) was developed at the Institute
Statistical Classification Strategy (SCS) for Biodiagnostics (IBD), NRC by Dr Ian
Smith and Dr Ray Somorjai specifically to
Magnetic resonance spectroscopy is known address these issues (Fig. 15.2). The method
to report on pools of chemicals in cells and has allowed automated analysis of both ex
tissues as they alter (many at the same time) vivo and in vivo spectroscopic data generat-
with ageing and the development of disease ing highly accurate and reliable mathematical
Fig. 15.2: Statistical Classification Strategy (SCS):A schematic road-map of how the SCS method is developed
for individual data bases. Abbreviations are GA_ORS: genetic algorithm based optimal region selection; LDA:
linear discriminant analysis; LOO: leave one out (method of cross validation); coeff: coefficients. Reprinted
from Chemical Reviews, 2004;104: 3677-704. Mountford C, Doran S, Lean C, Russell P. Proton MRS Can
Determine the Pathology of Human Cancers With A High Level of Accuracy. American Chemical Society
174 Biomedical Magnetic Resonance: Proceedings of the International Workshop
classifiers for a variety of clinical applica- this study (Fig. 15.3, Table 15.2). Spectra
tions. For a review see Lean et al 2002.1 from tissues with different proportions of
Described below is the current state of adenocarcinoma present (5 % and 50%)
1H MRS on biopsies of the prostate, breast,
were also found to be spectrally unique (Fig.
liver, thyroid, oesophagus and in vivo for 15.3). Resonances from lipid, amino acids,
the prostate, breast and thyroid. citrate, choline and creatine were assigned
in the spectra of specimens containing
PROTON MRS ON BIOPSIES adenocarcinoma and PIN, albeit at different
levels (Table 15.2). The spectra of BPH
Prostate specimens from glandular and stromal areas
Prostate carcinoma is the most common of the prostate were different with citrate
cancer in men in Australia26,27 and the United (the marker for apparently healthy tissue)
States.28 Histological examination is the defi- not identified in the spectrum from stromal
nitive standard for diagnosis and classifi- BPH. It can be seen that there is a gradation
cation of prostate neoplasms.29-31 in the spectra from benign stromal and
The IBD/NRC undertook the first MRS glandular BPH through PIN to frankly
study (8.5 T) of prostate biopsies and malignant tissue with increasing volumes of
analysed the data using the SCS method tumor was reported.
(Table 15.1). The accuracy for distinguishing The Swindle study33 further divided the
benign prostatic hyperplasia (BPH) from specimens into BPH from patients with and
cancer was 96.6 percent.32 It was notable in without cancer elsewhere in the prostate
this study that no prostate intraepithelial and compared both groups with
neoplasia (PIN) or proportional volumes of adenocarcinoma. Histologically confirmed
disease states in tissue specimens were carcinomas were determined by MRS with
reported. As the accuracy of the SCS method a sensitivity of 100 percent and a specificity
is dependent upon correct and detailed of 94 percent but the accuracy decreased
histological data being provided to the rapidly when routine hospital histology
computer-based strategy the possibility that reports were used for correlation. Depleted
variant pathologies may have been included citrate and elevated choline levels alone
in developing this classifier needed to be were not accurate markers of malignancy,
considered. In addition the prostate is a since citrate levels remained high when a
complex organ with four different functional small amount of malignant disease was
zones and mixtures of glandular and stromal present. Citrate resonances were present in
tissues present in varying quantities in a the spectra of tissues containing 95 percent
given tissue biopsy. BPH and 5 percent adenocarcinoma. A
A second study by Swindle et al 33 recently developed mathematical regression
involved MRS spectral analysis of biopsy method34 allows small volumes of carcinoma
specimens that were subsequently serially to be identified with accuracies approaching
sectioned. An error rate of 8 percent in the 100 percent for the prostate cohort.
routine hospital diagnosis was identified Although histological examination remains
due to incomplete sampling of the tissue. the standard in the assessment of human
Distinct proton MRS spectra (8.5 T) from prostate disease, the use of MRS as an adjunct
glandular BPH, stromal BPH, PIN, and to aid in the characterization of prostate
invasive adenocarcinoma were recorded in lesions on the basis of their biochemical
Application of Proton MRS in the Study of the Pathology of Human Cancers 175
Malignant
Brain (n = 206) Control v 23/55 subregions 94.4% 87
Malignant
Astrocytoma High v 0.81-0.85, 1.71-1.75, 2.16-2.20, 2.46-2.50, 95.7% 88
39
Normal v 1.52-1.57; 2.03-2.08; 3.63-3.67 92.1%
Cirrhotic
Ovary (n=56) Normal v Cancer 1.47, 1.68, 2.80, 2.97, 3.17, 3.34 98% 90
Benign
In vivo at 1.5T
3.17-3.42
Soft tissue Normal mesen- 93% 93
Amines
Choline, phosphocholine, Chol -N(CH3)3 3.2
glycerophosphocholine
Spermine, spermidine, PA -NCH2 3.1
polyamines
Creatine, phosphocreatine Cr -NCH3 3.0
Lipids
Triaclglycerol (fatty acyl chain) Lip CH3-CH2-CH2- 0.90
Lip CH3-CH2-CH2- 1.33
Lip -OOC-CH2-CH2 1.6-1.7
Lip CH3-CH2-CH2- 2.02
Lip CH=CH-CH2- 2.08
Lip -OOC-CH2-CH2 2.3
Lip CH=CH- 5.32
Amino acids
Alanine Ala CH3-CH- 1.49
Glutamate, glutamine Glu, Gln -CH-CH2-CH2-COO-/NH3+ 2.2
Glutamate, glutamine Glu, Gln -CH-CH2-CH2-COO-/NH3+ 2.6
Isoleucine Ile -CH3 0.97
Leucine Leu -CH3 0.97
+
Lysine Lys H3N -CH2-CH2-CH2 1.7
Lysine Lys H3N+-CH2-CH2-CH2 3.03
Threonine Thr CH3-CHOH- 1.33
Threonine Thr CH3-CHOH- 4.25
Valine Val -CH3 1.03
Other
Lactate Lac CH3-CH- 1.33
Citrate Cit -OOC-CHAHB-C(OH) 2.4-2.7
Taurine Tau H3N+-CH2-CH2-SO3- 3.25
Taurine Tau H3N+-CH2-CH2-SO3- 3.43
composition may provide additional diagno- 100 newly diagnosed patients per 100 000
stic and prognostic information. inhabitants per year.35 The combination of
physical examination, mammography, and
Breast fine needle aspirate cytology or needle core
Breast cancer is the most common cancer to biopsy (triple assessment) is currently the
affect women in Western countries with 80- preferred method for the preoperative
Application of Proton MRS in the Study of the Pathology of Human Cancers 177
Liver
A common internal malignancy worldwide
is primary hepatocellular carcinoma (HCC).37
In 1998 it was the most frequent tumor in
males38 and the most common in Asia and
Africa. The annual incidence in Western
Fig. 15.4: 1 H MR (8.5 T, 37 o C) Spectra (256 countries is 4 per 100,000.38 HCC almost
acquisitions) of a FNAB taken from an invasive always occurs as a sequel to cirrhosis and is
carcinoma of the breast and normal tissue. By visual increasingly prevalent because of the
inspection the distinction between normal breast and upsurge in cases of chronic hepatitis B and
invasive carcinoma is based on an increase in N-
hepatitis C.
trimethyl resonance at 3.25 ppm normalised to that
of creatine at 3.05 ppm (peaks indicated by arrows) Biopsies obtained from apparently
(Reprinted from Radiology, 1997; 204, 661-666. normal liver, cirrhotic liver and HCC, were
Mackinnon WB, Barry B, Malycha P, Gillett D, Russell examined by 1H MRS at 8.5 T (Fig. 15.6).
P, Lean CL, Doran S, Barraclough B, Bilous M and The correlative histopathology from 54
Mountford CE: Fine needle biopsy of benign breast consecutive patients undergoing partial or
lesions distinguished from invasive cancer by proton
magnetic resonance spectroscopy. With permission
total hepatectomy was obtained from Royal
from the Radiological Society of North America.) Prince Alfred Hospital39 in Sydney where
the Australian National Liver Transplant
potential to revolutionise breast cancer Unit is located.
management by providing both diagnosis Distinct MR spectra were recorded from
and prognostic information prior to surgery. HCC, cirrhotic, and apparently healthy liver.
It is important to note that the entire tissue Resonance assignments were made from two-
specimen needs to be examined histologically dimensional (2D) 1H, 1H COSY (Fig. 15.7)
at section intervals as small as 100 μm for and 1H, 13C HSQC and are summarized in
accurate correlation with MRS data. A good Table 15.2.39
example of this is ductal carcinoma in situ SCS-based classification of 1H MR data
(DCIS) of the breast where differences in from the liver biopsies distinguished normal
pathology may exist in between two adjacent liver from HCC with 100 percent accuracy
tissue sections 100 μm apart (Fig. 15.5, (Table 15.1). The SCS method also distin-
Plate 9). guished cirrhotic liver from HCC with an
Visual inspection of MR spectra from accuracy of 98.4 percent (Table 15.1). The
DCIS found that specimens that had distinction between normal and cirrhotic liver
breached the basement membrane (< 1 mm) was, however, less accurate at 92 percent
in one or more foci (i.e. microinvasion) and (Table 15.1). The histological distinction of
those with extensive comedonecrosis had HCC from tumor look-alikes can, however,
Application of Proton MRS in the Study of the Pathology of Human Cancers 179
A second study22 involved MRS of FNAB In a study of FNAB and tissue specimens
of thyroid tissues with the aim of addressing from 70 patients undergoing thyroidectomy
the inability of the fine needle cytology to for solitary or dominant thyroid nodules, a
accurately discriminate between follicular close correlation between fine needle MR
adenomas and carcinomas. MRS on FNAB spectra and tissue spectra for a range of
offers the prospect of a technique that more benign and malignant neoplasms has been
accurately reflects the actual biology of demonstrated (Fig. 15.9).22 The sensitivity
follicular thyroid tumors, allowing more or probability of correctly identifying
accurate diagnosis of thyroid cancers and thyroid cancer on the basis of MRS assessed
reducing the need for unnecessary surgery on FNAB was 95 percent. However, again
performed solely for diagnostic purposes. these FNAB’s were obtained intraoperati-
It is possible to obtain an accurate 1H MR vely.
spectrum from as few as 106 thyroid cells The application of SCS allowed further
from a FNAB.22 refinement of the discriminatory potential
Application of Proton MRS in the Study of the Pathology of Human Cancers 181
Fig. 15.9: Two directional plots to compare the resonance intensity ratio (1.7/0.9 ppm) obtained from FNB and
resected tissue specimens from the same solitary nodule. A O, Normal thyroid; the difference in the MRS ratio
between FNB and tissue bipopsy is 0.75± 0.79 (mean ±SD. , Malignant thyroid; the difference is 0.19 ± 0.12
(mean ± SD). B: , Follicular lesions; the MRS ratio is 1.1 or more; the difference is 0.31 ± 0.27 (mean ± SD).
, Follicular lesions; the MRS ratio is less than 1.1; the difference is 0.19 ± 0.24 (mean ± SD). (Reprinted from
Journal of Clinical Endocrinology and Metabolism 1995; 80:1306-1311, Lean CL, Delbridge L, Russell P, et
al. Diagnosis of follicular thyroid lesions by proton magnetic resonance on fine needle biopsy. Copyright
1995, The Endocrine Society.)
182 Biomedical Magnetic Resonance: Proceedings of the International Workshop
*Sensitivity and specificity determined as the ability of MRS to predict malignancy based on the presence
or absence of choline (3.2 ppm).
Fig. 15.13: Typical proton single voxel spectrum (135/
2000, 256 signal averages) from a patient with
histologically confirmed invasive ductal carcinoma is
compared with a typical spectrum from one of the three
lactating volunteers and one of the false-positive non-
lactating volunteers (3/40) (2.0-3.7 ppm shown).The
spectra are processed by zerofilling the 2048 data points
to 8192 data points, applying a Gaussian apodization
function of 1.5 Hz with a 15% echo offset before fast
Fourier transformation. In the spectrum from the cancer
bearing patient, resonances can be assigned to creatine-
containing compounds (3.04 ppm), phosphocholine (3.22
ppm) and glycerophosphocholine/taurine/myo-inositol
(3.28 ppm). In the spectra from the lactating volunteer
and the false-positive non-lactating volunteer the
dominant resonance is centred at 3.28 ppm consistent
with glycerophosphocholine/taurine/myo-inositol. The
spectra were referenced to the methylene resonance of
lipid at 1.33 ppm and water at 4.74 ppm. (Reprinted from
European Radiology 2004 online edition September.
Stanwell P, Gluch L, Clark D, et al. Specificity of choline
metabolites for in vivo diagnosis of breast cancer using
1H MRS at 1.5 T. With permission from Springer-Verlag.)
Application of Proton MRS in the Study of the Pathology of Human Cancers 187
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192 Biomedical Magnetic Resonance: Proceedings of the International Workshop
16
P MR Spectroscopy of
31
time via two concurrent processes described Compared to analytical methods, 31P MRS
as relaxation. T1 relaxation (longitudinal or offers the opportunity of measuring
spin-spin relaxation) involves the loss of noninvasively and continuously with high
energy to surrounding nuclei with similar time-resolution, the concentration of
resonance frequencies and roughly phosphorylated compounds involved in
determines signal intensity for a given muscle energetics. In addition, comparison
repetition time. T2 relaxation (transverse or of direct biochemical measurements with 31P
spin-lattice relaxation) results from MRS findings suggests that the two methods
interaction between the excited nuclei and give comparable results.9
A typical 31P MRS spectrum exhibits six
any perturbing magnetic field with no
to seven peaks corresponding to phospho-
transfer of energy. Signal width is inversely
creatine (PCr), inorganic phosphate (Pi), the
proportional to T2. The non-discriminatory
three phosphate groups of ATP (in position
nature of T2 relaxation mechanisms greatly
α, β and γ) and phosphomonoesters (PME)
increases the probability of such interactions
(Figs 16.1A and B). The Pα signal of ATP
thus making T2 much shorter than T1 in
displays occasionally an upfield shoulder
heterogeneous solutions. Signals obtained
corresponding to NAD+/NADH. In between
in 31P MR spectra and 1H MR images are
the PCr and Pi signals, the phosphodiester
highly dependent on both T1 and T2 values signal is sometimes observed. This signal is
together with nuclear density, i.e. the usually assigned to glycerophosphory-
number of free rotational nuclei of the tissue lcholine and glycerophosphoryethanolamine
investigated. For a given experimental time, which can be detected as a small peak in
compounds with shorter T1 will end up with normal muscle spectra (mainly from lower
higher intensities in MR spectra, while signal limb) and as a larger peak in patients with
width in spectra is inversely proportional muscle dystrophy10 indicating membrane
to T2 values. Regarding MR images, T1, T2 breakdown. Given the low sensitivity of the
and nuclear density will determine the technique, the free metabolically active ADP
contrast of cellular and subcellular concentration, which is only a tiny fraction
structures. of its total intracellular concentration, cannot
be measured. It can, however, be calculated
Informational Content of a using the creatine kinase equilibrium where
31
P MR Spectrum the total creatine content is taken as either
Measurement of phosphorylated com- 42.5 mM or considering that phospho-
pounds’ concentrations in living cells is not creatine represents 85 percent of the total
easy. Traditional methods such as percuta- creatine content.11 Similarly, AMP concen-
neous needle biopsy and freeze clamping tration can be calculated using the adenylate
exhibit limitations, especially related to kinase equilibrium. 11 In the absence of
alteration of anatomic integrity and partial biochemical data, ATP is often assumed to
degradation of phosphorylated metabolites be normal and is used as the equivalent of
during extraction and analysis. In addition, an internal standard in order to calculate
repeated measurements cannot be performed the concentrations of other metabolites.
on the same muscle, making impossible the Apart from the dynamic measurements
achievement of high-time resolution kinetics. of high-energy phosphate compounds, 31P
194 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 16.1A and B: Typical series of 31P MR spectra recorded in humans forearm flexor (A) and thigh muscles
(B). In A, MR spectra have been recorded at 4.7 T (Biospec 47/30 Bruker) during a standardized rest-exercise-
recovery protocol with a time resolution of 15 s. In B, MR spectra have been recorded at 1.5 T(Siemens Vision
Plus) after a standardized bicycle exercise protocol with a time resolution of 2 s. On the higher panel of each
figure, is represented a single spectrum with the corresponding assignments. Ref : Reference compound
(phenyl phosphonic acid), PME (phosphomonoesters), Pi (inorganic phosphate), PDE (phosphodiesters),
PCr (phosphocreatine), phosphate groups of ATP in position α, β and γ
MRS offers the only noninvasive way of average of both monobasic and dibasic
assessing intracellular pH. Indeed, under forms. Due to this sensitivity of the Pi
conditions of physiological pH and consi- chemical shift to pH, it is possible, with
dering one of the pKa, i.e. 6.75, two forms of appropriate calibration curves, to translate
Pi coexist (H2PO4– and HPO42–). These two any Pi shift in terms of intracellular acidosis
forms are exchanging so fast that only a single or alkalosis.
Pi signal is detected. However, the chemical The quantitative measurements of high-
shift of this single signal is a weighted energy phosphate compounds and pH allow
31
P MR Spectroscopy of Muscle Energy Metabolism in Humans 195
sequentially the variations of concentrations energy demand and supply.27 A net ATP
in PME, Pi, PCr, ATP as well as intracellular decrease has been only reported when
pH during muscle contraction and recovery. exercise intensity was by far larger28 and
Biochemical reactions taking place in the interestingly such a net ATP consumption
myocyte are then directly reflected by specific has rarely been reported in patients with
perturbations on the spectrum. Muscle energy metabolic disorders.29-31
production results in PCr and pH changes As illustrated in Figure 16.2, the balance
which in turn would modify ADP and Pi between ATP demand and ATP supply is
concentrations both of which ultimately ensured by the three main sources of ATP
stimulate ATP production. Typical series of within the muscle and each of them can be
spectra recorded in exercising flexor muscles accurately quantified from the PCr and pH
(4.7 T) and in thigh muscles (1.5 T) are dis- time-dependent changes in exercising
played in Figure 16.1. During contraction, muscle and throughout the recovery period.
the free energy stored in ATP (the ubiquitous
PCr Changes in Exercising Muscle
substrate for energy production) is converted
into mechanical energy via the interaction of PCr is largely recognized as an intracellular
two muscle proteins, actin and myosin. buffer (some authors have also reported PCr
Muscle work is linked to hydrolysis of ATP. as an energy shuttle between production and
However, in normal subjects, the intra- consumption sites) allowing to compensate
cellular concentration of ATP remains for any shortfall in either non-oxidative or
unchanged during moderate exercise oxidative ATP production. Across the rest
indicating an adequate balance between to work transition, ATP in working muscle
tion of PCr breakdown with respect to tile sites; then, for a given ATP demand,
oxygen availability. A direct proportionality PCr kinetics may remain unchanged
between the kinetics of pulmonary oxygen regardless of oxygen availability. Recent
consumption and PCr hydrolysis has been experiments aiming at analyzing the effects
demonstrated. Changes in PCr at the start of using varied fractions of inspired oxygen
of exercise has been analyzed and found (FiO 2 ) on PCr kinetics have provided
comparable to the onset of oxygen interesting information. During an incre-
consumption kinetics.21,36,37 In addition, a mental exercise, the reduction of oxygen
correlate of the slow component in oxygen availability accelerates the occurrence of
consumption, indicating an increased oxygen fatigue, whereas the extent of PCr reduction
cost for a constant work-rate, has been was not modified. 44 However, for an
reported for the time-dependent PCr identical work output, PCr hydrolysis was
changes further illustrating the similarity larger when oxygen supply was lower
between oxygen and PCr changes.34,38 The suggesting that this greater PCr hydrolysis
steady-state level to which PCr falls, i.e. might have contributed to the earlier time
PCrend is also positively correlated with the to exhaustion. Similar experiments conduc-
steady-state level of oxygen consumption ted throughout a steady-state exercise have
during exercise suggesting that PCr
provided additional information regarding
hydrolysis and more likely inorganic
the time-dependent changes in PCr and
phosphate accumulation could be a potent
oxygen. PCr and oxygen kinetics remain
regulator of oxidative phosphorylation as
comparable regardless of oxygen availability
suggested previously. 39,40 However, this
at least during the primary phase. An
tight coupling between PCr hydrolysis and
additional phase (the slow component) can
regulation of aerobic ATP production could
be observed for both PCr and oxygen unless
be modified by altering oxygen avail-
oxygen supply is made hyperoxic. All
ability.34,41-43 Interestingly, it has been repor-
ted that any change in oxygen availability together, these data indicate that PCr
was immediately translated at the PCr level changes at the onset of exercise are not
in exercising muscle either as a faster modulated by the oxygen driving pressure.
decrease (switch from hyper to hypoxic On the contrary, after this initial phase,
conditions) or a faster increase (switch from reducing oxygen supply can accelerate PCr
hypoxia to normoxia and switch from kinetics and precipitate the occurrence of
hypoxia to hyperoxia). 41 Two opposite fatigue; while increasing the oxygen, driv-
hypotheses could be formulated regarding ing pressure (hyperoxia) eliminates the
the effects of oxygen availability upon PCr additional PCr consumption (slow compo-
changes in exercising muscle. If PCr kinetics nent or phase II) suggesting that PCr kinetics
follow oxygen consumption as previously after the initial phase are regulated by
suggested, PCr changes should be slowed microvascular and intracellular PO2.41
in hypoxia due to the fact that oxygen has
pH Changes in Exercising Muscle,
to be present as the terminal electron
Buffering Components and Glycolytic
acceptor within the electron transport chain.
ATP Production
On the other hand, if PCr hydrolysis is a
process largely dependent upon the On initiation of muscle contraction or at
metabolite signals arising from the contrac- times when the workload or rate is
31
P MR Spectroscopy of Muscle Energy Metabolism in Humans 199
increased, glycogen breakdown is rapidly These values are used for the deter-
activated to provide substrate for glycolysis. mination of the glycolytic rate of ATP produc-
Pyruvate that is not used as a substrate by tion which can be calculated differently
the mitochondria accumulates and the according to the type of exercise. Under
combined production of lactate and ATP ischemic conditions, given that ATP supply
hydrolysis lowers pH.45,46 The extent of the is mainly supported by PCr breakdown and
change in pH is a balance among lactic acid glycolytic ATP production, the glycogenolytic
production proton efflux, ATP hydrolysis flux can be calculated using a two-step
and the pH raising effect of PCr breakdown. method, using measurements of PCr content
Interestingly, all these components could be only.51 In the first step, the total rate of ATP
quantitatively analysed using 31P MRS. turnover is calculated from the derivative of
With a high enough time-resolution such equation 1 at time zero. Then glycogenolytic
that an initial alkalosis (dpH/dt) can be flux can be calculated as the simple difference
measured at the onset of exercise, a composite between the total rate of ATP turnover and
buffering capacity (βc) including bicarbonate the rate actually calculated at any given time-
and non-bicarbonate compounds is point of exercise as indicated in Figure 16.4,
calculated according to equation 4 taking Plate 10.
into account the rate of proton consumption When ATP demand is supported by both
due to PCr breakdown. (γdPCr/dt) non-oxidative and aerobic sources (mixed
represents the proton stoichiometric coeffi- exercise), the calculation of the glycolytic rate
cient of the coupled Lohman reaction, i.e. takes into account each component of the
PCr breakdown coupled to ATP hydrolysis:47 H+ balance, i.e., overall H+ production asso-
ciated with non-oxidative ATP production,
βc = [γd(PCr)/dt]/[d(pH)/dt] ...(4)
H+ efflux, and H+ uptake associated with
The contribution (βx) of inorganic phos- PCr breakdown (Eqn. 6).30,52
phate (Pi) and sugar-phosphate (PME) to βC As previously described,48 the rate of
can be determined using the standard +
H efflux (EfH+) cannot be directly estimated
formula of a buffer dissociation with K being during exercise but has to be analyzed from
the dissociation constant: the coupled analysis of pH changes (usually
an additional acidosis, VpH) and PCr resyn-
βx = [2.303 (H+) K (X)]/[K +(H+)]2 ...(5)
thesis during the initial part of the recovery
where (X) is the concentration of either Pi period following exercise. Indeed, as
or PME and K the corresponding illustrated in Figures 16.5A and B, pH
dissociation constant. The buffering capacity recovers back towards its resting value
of tissue alone mainly including imidazole despite a continuous proton load (H+PCr)
groups of histidine can then be calculated from PCr resynthesis:
as the difference between βx and βc. In an EfH+ = βVpH + H+PCr ...(6)
“open” muscle system, the buffer capacity [CRMBM5]
due to bicarbonate is between 30 slykes at
resting pH and 3 slykes at pH close to 6.48 Taking into account both phenomena, a
Exercising muscle is usually assumed as a first-order rate constant linking the rate of
“closed” system48-50 and buffer capacity due H+ efflux to the extent of pH changes can be
to bicarbonate is set to zero. calculated and used to infer the rate of
200 Biomedical Magnetic Resonance: Proceedings of the International Workshop
REPRODUCIBILITY OF
MEASUREMENTS
For meaningful comparisons data must be
reproducible from study to study and it is
important to assess repeatability in groups Figs 16.7A and B: Typical PCr (A) and pH (B) time-
of subjects and patients. Foremost, one have dependent changes recorded throughout a
to keep in mind that metabolic changes in a standardized rest-exercise (shaded area)—recovery
control population is highly heteroge- protocol in a group of 18 subjects. Measurements
neous.11,15,16,89 The usual standardization (black and white symbols) have been recorded seven
days apart. Results are presented as means ± SD
procedures such as measurements of maxi- and one can observe the very good reproducibility of
mum voluntary contraction does not reduce measurements
this between-subject variability.11,16,89 In that
respect, it is clear that a set of factor which metabolic changes in exercising muscle.89
could influence metabolic changes in exer- Similarly, severe metabolic acidosis induced
cising muscle has to be taken into account by ammonium chloride loading had no effect
and ad-hoc standardization procedures that on changes recorded during exercise.89 Aging
would reduce the inter-subject variability is another potential accounting factor that
have to be used for proper comparisons should be taken into account. The studies
among various groups of subjects. based on measurements of enzymatic acti-
vities have suggested a decrease oxidative
Between-subject Variability
capacity with age.90,91 Results from MRS
In a limited number of subjects, it has been investigations differed regarding that physi-
shown that neither fasting nor carbohydrate cal activity has been taken into account or
loading significantly modified the extent of not.92 Aging has been related to both a 50
206 Biomedical Magnetic Resonance: Proceedings of the International Workshop
percent reduction in the PCr recovery rate program had also no effect on mitochondrial
and a reduced mitochondrial content with function thereby suggesting that a low
no changes regarding the metabolic changes substrate oxidative capacity of skeletal
in exercise.93 When physical activity has been muscle is not involved in the pathogenesis
taken into account, no deficit of aerobic of obesity on the contrary to what has been
capacity has been reported, whereas the PCr suggested earlier.101
depletion was not modified and the pH was
more alkalotic in older subjects94 in agree- Within-subject Variability
ment with previous studies conducted in Beside the between-subject variability, the
sedentary95 and moderately active subjects.96 within-subject variability is also important
These results would imply a primary role of to analyse. Several studies have clearly
disuse in the decline of oxidative capacity demonstrated a very low within-subject
rather than an inherent age-related defect. In variability as illustrated in Figures 16.7A
addition, the recent observation of increased and B and indicating a very high repro-
maximum aerobic capacity in older subjects ducibility.73,89,102,103 For instance, variation
as a result of training would be in keeping coefficients calculated from repeated
with the hypothesis that the decline in experiments were 5-10 percent for metabolic
oxidative capacity would mainly result from indices at rest such as ADP and PCr/Pi and
a reduction in habitual physical activity 3-15 percent for variables measured during
rather than of aging per se.97 However, this exercise and in the recovery period.102
conclusion must be moderated on the basis
of two points. First, the oxidative capacity Standardization Procedures
per mitochondrial volume measured in older
This within-subject variability may also be
subjects after a training period was still
used in order to understand muscle ener-
below the value reported in young adult getics and to initiate ad-hoc standardization
muscle93 and adaptive mechanisms were procedures that could be used as reliable
distinct from what has been reported so far comparative methods between groups of
in young especially regarding resistance subjects. It has been shown in normally
training.97 active adults that an index of PCr consump-
Another factor that should be taken into tion [(PCr + Pi)/PCr] (measured with 31P
account regarding the between-subject MRS) was highly correlated with power
variability is related to high body fat stores. output normalized to the volume of muscle
Indeed, changes in fiber types proportions (measured with MRI) in the plantar flexor
that could affect muscle energetics have been compartment indicating that combined MRS
reported in obese subjects98 and high-energy and MRI measurements could offer a way
phosphate metabolism has been investigated of reliably compare subjects with different
using 31P MRS in order to test this hypo- body size.104 Similarly, expressing metabolic
thesis. Studies conducted in moderately- changes such as PCr breakdown and intra-
obese women (BMI range: 27 to 30 kg/m2)99 cellular acidosis in exercise muscle with
and in prepubertal girls with a familial respect to power output has been reported
predisposition to obesity100 have clearly to offer an interesting standardization proce-
shown that muscle energetics was not dure independent of exercise protocols
altered. In addition, a weight-reduction and anthropometric measurements.105 In
31
P MR Spectroscopy of Muscle Energy Metabolism in Humans 207
Mitochondrial Reduced PCr/Pi No Larger Limited Slow together 29, 72, 107
PFK deficiency Increased PCr/ATP Yes Larger Limited Slow 118, 119
dystrophy phosphorylation
potential
sIBM Increased pH Normal Normal Normal Normal 124, 125
Increased Pi/ATP
Malignant Increased PDE No Larger Excessive Normal 126, 127
hyperthermia
IIM (Poly and Low PCr/ATP No Larger Normal Slow together 108, 128
failure
Congestive heart No Larger Excessive Slow 133, 134
failure
Peripheral vascular No Larger Excessive Slow together 11, 135, 136
(Hyper)
Essential Smaller Normal together 139
thereby providing one or more diagnostic with 85 percent mutated DNA and the other
indices. However, one should be cautious with only 4 percent mutated DNA.109 This
with the interpretation of the results. For result could be interpreted as a discrepancy
instance, two brothers with genetic defects between in vivo and in vitro measurements
of MELAS showed similar reduction in or as indicative of the absence of a clear
maximum aerobic capacity, while one was threshold for the A3243G mutation in
31
P MR Spectroscopy of Muscle Energy Metabolism in Humans 209
skeletal muscle. These results have impor- be suspected as accounting for the greater
tant implications for the understanding exercise-induced metabolic changes. In
of the phenotypic expression of mtDNA addition, considering that both oxidative
disease. and glycolytic contributions to energy
An additional step into the understanding production were increased in absolute terms
of 31P MR metabolic indices resides in the but normal when energy cost was taken
quantitative analysis initially developed by into account, it can be proposed that reduced
Kemp et al.106 With this type of analysis, muscle efficiency rather than impaired
one can further distinguish between reduced aerobic ATP production likely accounts for
muscle mass and/or reduced muscle the greater PCr changes in exercise.30
efficiency and reduced oxidative capacity as
accounting factors of abnormal exercise- FUTURE INVESTIGATIONS
induced metabolic changes. Indeed, for a
given muscle mass and a given mechanical Employing non-MR based techniques simul-
power output, the initial rate of ATP synthesis taneously with MRS can aid in data inter-
(equation 3) is by definition inversely pretation and will certainly broaden the
proportional to metabolic efficiency.110 As scope of muscle investigation. Near infrared
illustrated earlier, both aerobic and non- spectroscopy (NIRS) provides data on state
oxidative contributions to energy production of tissue oxygenation but reliability has still
can be calculated from the analysis of PCr, to be proven mainly on the basis of compa-
Pi, PME and pH dynamics. These changes rative and/or combined analyses using MRS
depend on mechanical work, muscle mass and NIRS.111 Electromyography is another
and metabolic efficiency (the latter two technique of interest which can be used to
variables are combined as effective muscle study correlation between metabolic and
mass).30 Generally speaking, greater PCr electrical changes and provide interesting
and pH changes should be recorded if the features regarding muscle fatigue.112 Com-
ratio of work to effective muscle mass is bined with MRS, MR imaging techniques can
increased or if aerobic ATP synthesis is also help understanding muscle energetics.
decreased. Such a comparison has been For instance, MRS data showing altered
performed in a study of dialyzed uremic metabolites concentrations need to be
patients. 30 Raw data recorded in this interpreted with caution. Indeed, metaboli-
group of subject illustrated a larger PCr tes’ concentrations may be altered, but the
consumption, while exercise duration and presence of fatty or fibrous infiltration can
the corresponding power output was introduce false-positive results in a sense
reduced. pH time-dependent changes were that data might not represent concentrations
normal and the analysis of the recovery in the actual muscle fibers. Functional MRI
period disclosed a 50 percent reduction of can also be of interest for understanding
the maximum aerobic capacity. From the muscle activation during exercise. Based on
initial increase in the PCr consumption rate, T2 changes due to uptake or redistribution
one can infer a reduced effective muscle of fluid within the exercising muscle,
mass. Given that muscle mass (measured functional MRI is considered as a semi-
separately) was similar in both groups of quantitative method of assessing muscle
subjects, reduced metabolic efficiency can recruitment during exercise.113 For instance,
210 Biomedical Magnetic Resonance: Proceedings of the International Workshop
in McArdle’s disease, abnormal glycogeno- 10. Lodi R, Muntoni F, Taylor J, Kumar S, Sewry
lysis is coupled to abnormal exercise-induced CA, Blamire A et al. Correlative MR imaging
and 31P-MR spectroscopy study in sarcoglycan
T 2 changes. 114,115 . Similarly, subnormal
deficient limb girdle muscular dystrophy.
exercise-induced T2 changes increase can be Neuromuscul Disord 1997;7:505-11.
recorded in other metabolic myopathies.116 11. Kemp GJ, Roussel M, Bendahan D, Le Fur Y,
However, muscle functional MRI is still Cozzone PJ. Interrelations of ATP synthesis
underutilized although its use is certainly and proton handling in ischaemically exercising
well suited in the context of muscle ener- human forearm muscle studied by 31P magnetic
getics and for documenting muscle activation resonance spectroscopy. J Physiol 2001;535:901-
28.
pattern in pathological conditions and in
12. Iotti S, Frassineti C, Alderighi L, Sabatini A,
subjects with very highly trained to exercise. Vacca A, Barbiroli B. In vivo 31P-MRS assess-
ment of cytosolic [Mg(2+)] in the human skeletal
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112. Bendahan D, Jammes Y, Salvan AM, Badier M, myotonic dystrophy. J Neurol Sci 1993;116:193-
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spectroscopy study of human muscle fatigue Karpati G, Arnold DL. Intracellular phosphates
during static contraction. Muscle Nerve in inclusion body myositis—a 31P magnetic
1996;19:715-21. resonance spectroscopy study. Muscle Nerve
113. Meyer RA, Prior BM, Siles RI, Wiseman RW. 1998;21:1523-5.
Contraction increases the T2 of muscle in fresh 125. Lodi R, Taylor DJ, Tabrizi SJ, Hilton-Jones D,
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Biomed 2001;14:199-203. skeletal muscle oxidative metabolism in
114. de Kerviler E, Leroy-Willig A, Jehenson P, Duboc sporadic inclusion body myositis assessed by
31P magnetic resonance spectroscopy. Brain
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modifications: Study of healthy subjects and 1998;121:2119-26.
patients with metabolic myopathies with MR 126. Bendahan D, Kozak-Ribbens G, Rodet L,
imaging and P-31 spectroscopy. Radiology Confort-Gouny S, Cozzone PJ. 31Phosphorus
1991;181:259-64. magnetic resonance spectroscopy characteri-
115. Fleckenstein JL, Haller RG, Lewis SF, Archer BT, zation of muscular metabolic anomalies in
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induced MRI enhancement of skeletal muscle in cation to diagnosis. Anesthesiology 1998;88:96-
McArdle’s disease. J Appl Physiol 1991;71:961- 107.
9. 127. Payen JF, Bosson JL, Bourdon L, Jacquot C, Le
116. Patten C, Meyer RA, Fleckenstein JL. T2 mapping Bas JF, Stieglitz P et al. Improved noninvasive
of muscle. Semin Musculoskelet Radiol diagnostic testing for malignant hyperthermia
2003;7:297-305. susceptibility from a combination of meta-
117. Ross BD, Radda GK, Gadian DG, Rocker G, Esiri bolites determined in vivo with 31P-magnetic
M, Falconer-Smith J. Examination of a case of resonance spectroscopy. Anesthesiology 1993;
suspected McArdle’s syndrome by 31P nuclear 78:848-55.
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in high-energy phosphates in rat skeletal muscle Nicolaides A, Amato A et al. Calf muscle
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1995;89:581-90.
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Y, Yamabayashi H. Muscle energy metabolism Bank WJ. Effects of thyroid hormones on skeletal
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Circulation 1988;78:320-6. 7.
MRI and MRS of Nuclei other than 1H 217
17
MRI and MRS of
Nuclei other than 1H
Diana M Lindquist, Richard A Komoroski
exogenous nuclei, such as 7Li or 19F, will be A different method of localization, which
on the order of 10 mM or less. Under these can be used with either volume or surface
conditions, even 19F will have a poor signal- coils, uses gradients and radiofrequency
to-noise ratio (SNR), despite its relatively pulses to choose the volume element, or
good sensitivity (83% that of protons). voxel, that is excited. For simple single voxel
and multivoxel spectroscopy, the read-out
Localized Spectroscopy gradient is off during the data acquisition
period to preserve the spectral information.
Localized spectroscopy refers to the acqui- In these sequences, localization can be
sition of spectral data from a restricted one-, two-, or three-dimensional, depending
region of interest within a subject. There on the available SNR and the study
are several methods by which this can be requirements. An example of a three-
accomplished. The simplest method is to use dimensional localization scheme is the point-
a surface coil, in which case the region resolved spectroscopy sequence (PRESS),
sampled is determined by the excitation which is shown in Figure 17.1. It consists of
profile of the coil. In general, this is an area three RF pulses applied simultaneously with
approximately equal to a hemisphere with orthogonal gradients. Each RF/gradient
the same radius as the coil. Although this combination produces the selective excitation
can be useful if the region of interest is of a “slice” of spins. The excitation of three
near the surface and easily accessible, it such slices orthogonal to each other results
suffers from the inhomogeneous excitation in the selection of the voxel at the inter-
produced by the coil. section of the slices. The resolution
MRI and MRS of Nuclei other than 1H 219
considerations described above for MRI also in tissue, make it nearly ideal for in vivo
apply to MRS. studies of drugs or other exogenous
compounds. Over 150 fluorinated drugs are
Equipment Considerations in use clinically,4 so the potential usefulness
of 19F MRS for in vivo monitoring of these
In general, a clinical MR imager does not drugs is substantial.
include the hardware or software necessary Although no comprehensive review
for heteronuclear studies. This hardware can describing the biomedical uses of 19F MRS
include an additional transmitter and recei- has recently appeared, there have been a
ver, although many modern MR scanners number of focused reviews of studies on
now use broadband receivers that are psychoactive medications, such as fluoxetine,
capable of sampling the heteronuclear signal. and the anticancer drug 5-fluorouracil
Since each nucleus resonates at a different (5-FU). Several mini-reviews5-8 of 19F MRS
frequency, nucleus-specific radiofrequency are included in more general reviews of
coils must be built. The imaging and MRS in psychiatry. The most recent general
spectroscopy software must be modified to review is by Wolf et al4 which describes
include the effects of γ, as discussed above. primarily the usefulness of 19F MRS in
studying 5-FU.
SPECIFIC NUCLEI
Psychiatric Medications
Fluorine-19
Many psychoactive drugs contain fluorine.
Of the non-hydrogen isotopes available for These include the anti-depressant fluoxetine
in vivo study with MR, one of the most and the drugs trifluoperazine and fluphe-
used is 19F. Fluorine-19 is a spin-1/2 nucleus, nazine, which are used in the treatment of
is 100 percent naturally abundant, has a schizophrenia. The concentrations of
relative sensitivity that is 83 percent that of these drugs in brain are on the order of
protons and has short spin relaxation times.3 10-200 μM, which is significantly higher than
These properties, combined with the fact serum levels. Because serum levels of these
that there is no naturally occurring fluorine medications tend to provide little infor-
mation about the clinical response to or side
effects from these drugs, in vivo measure-
ments in the target tissue can be beneficial.
Fluorine-19 magnetic resonance spectroscopy
has been used to study these fluorine-
containing drugs in vivo in both human
subjects and animal models,9-26 and has been
reviewed.5-9
Initial work focused on detecting and
quantifying compounds containing a trifluo-
romethyl group, such as trifluoperazine,
fluphenazine, or fluoxetine.10-16 The struc-
Fig. 17.1: A version of the PRESS sequence used tures of these and similar compounds are
for in vivo localized spectroscopy of rat brain. Shaped shown in Figure 17.2. Figure 17.3 shows an
gradients were used to improve the slice profile68 in vivo spectrum from the head of a patient
220 Biomedical Magnetic Resonance: Proceedings of the International Workshop
on fluoxetine. More recently, work has identified brain metabolites in vivo20-21 and
focused on the pharmacokinetics and correlated brain concentrations with clinical
distribution of these and other response.22,23
medications.17-19 One difficulty with these Trifluoperazine has been the subject of
types of studies is that in general the in vivo several in vivo studies.13,15,20 In unlocalized
spectrum consists of a single peak due to studies we observed signals from 6 clinically
the overlapping reso-nances of the fluorine- responding patients on TFP doses of
containing drug and its metabolites. Despite 10-120 mg/day.15 A good correlation was
this limitation, several studies have found between brain concentration and
Fig. 17.3: Fluorine-19 spectrum from the head of a patient on fluoxetine. The peak labeled ‘standard’ arises
from a vial of 12 mM 2,2,2-(trifluoroethyl)-p-toluene sulfonate in CDCl3. The chemical shift scale is in ppm from
CCl3F.
daily dose. Interestingly, despite repeated suggests that the primary in vivo signal
attempts, we could not observe a signal arises from the parent drug and 5-7 meta-
from a nonres ponder who was on the very bolites. 20,27 Figure 17.4 shows the 19 F
high dose of 120 mg/day. This result spectrum of the extract of brain tissue from
suggests that in vivo 19F NMR may have a a rat dosed with trifluoperazine. Other
role in assessing nonresponse to antipsy- metaboli-tes of trifluoperazine may be
chotic medication. visible in the in vivo spectrum as well.20
More recent work in our lab to identify Fluoxetine metabolizes to the therapeuti-
trifluoperazine metabolites in rat brain cally active compound norfluoxetine in the
Fig. 17.4: In vitro 19F spectrum of the extract of brain from a rat dosed with trifluoperazine.20 See reference 20
for compound names. Unidentified peaks are marked with asterisks. The chemical shift scale is in ppm from
CCl3F.
222 Biomedical Magnetic Resonance: Proceedings of the International Workshop
brain. The 19F chemical shift of norfluoxetine tics in animals.33-36 Fluorine-19 MRI has also
is very close to fluoxetine and the two been used, but with less success.37,38 Lock-
compounds are not resolved in vivo (Fig. wood et al acquired 19 F MRS data from
17.3).10,15 In vitro 19F NMR studies of extracts volunteers to determine the wash-out time
of both human and rat brain confirmed that of isoflurane.39,40 No further human experi-
the in vivo signal arises roughly equally from ments have been done, presumably because
fluoxetine and norfluoxetine.10 A study of of the difficulties involved.39
22 patients on fluoxetine showed that brain
concentrations continued to rise long after OTHER APPLICATIONS
clinical effects were evident and leveled off Fluorine-19 MRI and MRS have been used
after a 6-8 month period. 16 The drug for a variety of other applications, including
accumulated to about 20 times the level in measuring lung volume,41-43 oxygen partial
the plasma in these patients. 16,24 No pressure,44,45 tumor oximetry,46 imaging the
correlation was seen with clinical response, gastrointestinal tract, 47 catheter visuali-
although more recent studies have found zation,48 and metabolism of anti-inflam-
such correlations.22 matory drugs.49
Other psychiatric medications that have
been studied with in vivo 19F MRS include Lithium-7
the trifluorinated drugs dexfenfluramine,25
paroxetine,22 fluvoxamine,17,19,23 fluphena- Naturally occurring Li consists of the 7Li
zine,13 as well as the monofluorinated anti- and 6Li isotopes at 92.58 percent and 7.42
psychotics melperone26 and haloperidol.18 percent abundances, respectively (Table
17.1).2 Like the other alkali metals, both Li
5-fluorouracil isotopes are quadrupolar. The problems of
achieving adequate SNR and resolved
5-Fluorouracil is a primary drug used in the resonances can be exacerbated by quad-
treatment of many cancers. The role of 19F rupolar effects that occur with nuclei that
MRS in studying the metabolism of 5-FU have spins greater than ½. For quadrupolar
was reviewed by Wolf et al4 Since that nuclei, the interaction of the nuclear
review there have been several studies of quadrupole and the electric field gradient
the 5-FU prodrug capecitabine.28-30 Improve- arising from the local electronic environment
ments in localization techniques have usually provides a strong relaxation mecha-
allowed the in vivo detection of 5-FU and nism for the spin states, often resulting in
α-fluoro-β-alanine in tumor and liver tissue short-lived signals with broad resonance
with a time resolution of 4 minutes at lines. This is particularly true in biological
1.5 T.31 The in vivo spin-lattice relaxation systems, where ionic motion is restricted.
However, both Li isotopes are weakly
times (T1) of 5-FU and fluoronucleotide have
quadrupolar relative to 23Na or 39K. Both
been measured recently.32
isotopes yield narrow spectral lines and long
spin-lattice relaxation times (T1). Lithium-7
Anesthetics
(I=3/2) is the isotope of choice for most
Because many inhaled anesthetics contain studies because of its higher natural isotopic
fluorine atoms, 19F MRS has been used in abundance and magnetic moment, and more
several pharmacokinetic studies of anesthe- favorable relaxation properties than 6 Li
MRI and MRS of Nuclei other than 1H 223
(I=1).2 Relatively little biological MRS work the brain Li concentration is of great
has been done with 6Li, because of its low interest.56 The relationship between serum
sensitivity and very long T1s.50,51 and brain Li concentration at steady state is
The chemical shift range of the Li cation an issue that has received considerable
is small because of its simple electronic attention. It has been explored in a relatively
structure and aqueous chemistry. Spectra large group of bipolar patients.57 The mean
usually display a single resonance from all brain-to-serum ratio of Li for that group
environments, which complicates inter- was 0.80 ± 0.19. Over a large serum-
pretation of 7 Li NMR signals from concentration range, the correlation with
heterogeneous biological systems. brain concentration was weak but statis-
Lithium, usually administered orally as tically significant, whereas in the therapeutic
Li2CO3, remains the treatment of choice for range the correlation was not significant.
acute mania and for prophylaxis in bipolar Another report found a weak correlation
(manic-depressive) disorder. Despite its between plasma and brain Li concentrations
widespread clinical use and extensive for longer-term therapy, but no correlation
research into its mechanism of action, it is for shorter-term therapy.58 The above results
not known how Li exerts its clinical effects. suggested that serum Li, the parameter
We currently know little about the environ- usually monitored during treatment, is less
ment of Li in vivo. than ideal as a measure of Li therapy. A
An in vivo 7Li spectrum of brain typically recent steady-state study59 on a 3 T MR
consists of two resonances, the second from scanner confirmed the absence of a strong
a vial of aqueous Li standard with position correlation between steady-state brain and
shifted from the in vivo resonance by a shift serum Li levels.
reagent. Quantitation can be performed Moore et al60 measured steady-state
relative to a separate Li phantom, with the brain and serum Li levels as a function of
small, shifted standard for calibration. High age in 27 children and adults. Consistent
quality spectra can be obtained in about with most previous work, they found a
20 minutes at 1.5 T. Usable unlocalized weak but significant correlation of brain Li
spectra can be obtained in as little as level with that of serum (r = 0.60, p < 0.001).
3 minutes. Most 7Li MRS work in vivo has They also found a correlation of brain-to-
centered on humans. Early work in humans serum concentration ratio and age (r = 0.44,
involved surface-coil-localized in vivo 7Li p < 0.02). Based on this result, they
studies of Li pharmacokinetics, and corre- suggested that children may require higher
lation of brain and serum concentrations. maintenance serum levels of Li to insure
Further work continued to improve quanti- therapeutic brain concentrations.
tation, spatial localization, and measurement In vivo 7Li MRS is powerful tool for the
of spin relaxation times. We direct the study of Li pharmacokinetics.61,62 The half-
reader to earlier reviews for detailed life for Li was 28 hours in brain, and
treatments of both methodology and early 16 hours in serum. It has also been
results.5-8,50-56 demonstrated that brain Li followed serum
Since the magnitude of the pharmacologic Li, in attenuated form, over a daily cycle.
effect of a drug depends on the concen- Figure 17.5 shows the time course of Li
tration at receptor sites in the target tissue, levels after termination of therapy for a
224 Biomedical Magnetic Resonance: Proceedings of the International Workshop
patient.63 Whereas Li was not detected in where dosing is flexible and experimental
muscle 5-6 days after termination, brain Li conditions can be closely controlled. Earlier
was still detectable. The reader is referred work on the development of 7Li MRS and
to previous reviews for detailed discussions MRI in animal models has been reviewed in
of early pharmacokinetic results.5-8,50-56 several places.50,51,55
The Li distribution in human brain may Recently our laboratory has built on
shed light on this cation’s mechanism of earlier work to resolve several issues best
action in bipolar disorder, either thera- addressed in an animal model. Our previous
peutically or regarding neurotoxicity. We work in rats emphasized low-resolution 7Li
previously reviewed progress made in the MRI to map the relative distribution of Li
development of 7Li MRI and spectroscopic in brain and muscle tissue.65-67 Such studies
imaging (SI), both in humans and in required substantially higher Li concen-
animals.51 Since then Girard et al64 reported trations than used clinically in humans. These
a 7Li SI scan of human head at 4.3 cm high concentrations were generally in the
resolution in the axial plane (with no slice toxic range and their clinical relevance was
selection) taken in 55 min. The distribution limited. The core therapeutic range of Li in
of Li in the head appeared relatively serum for humans is now considered to be
uniform. However, spatial resolution in all 0.6-1.0 mM.
three dimensions will need to be signi- We very recently reported the appli-
ficantly improved for the technique to be cation of 7Li MR point-resolved spectroscopy
clinically or mechanistically useful. Sensiti- (PRESS) to measure Li concentrations and
vity improvements from higher magnetic spin-relaxation times in rat brain. 68 We
fields and better RF coils may brighten this improved signal-to-noise ratio by using a
picture. more sensitive RF coil and by sampling a
Many Li studies are best carried out in larger voxel than that in our previous
a convenient animal model like the rat, imaging studies, allowing measurements
over the entire range of therapeutic Li
concentration. The method involves use of
an external standard phantom and takes into
account T1, T2, and 7Li MR visibility.68 For
spin-3/2 nuclei such as 23Na, studies of many
tissues have shown that ion concentrations
determined by MR are less than those
determined by other analytical methods.
Lithium-7 is generally considered less likely
to display reduced MR visibility. Previous
7Li-MR estimates of brain Li concentrations
10-25 percent.67 The detailed origin of the studies than for humans, including reliable
reduced 7Li visibility is not known. drug delivery over a relatively wide dose
Using that approach,68 we investigated range, as well as controlled diet, age, and
the relationship of brain and serum weight.
concentrations in the rat at two treatment The average brain/serum Li ratio was
times.69 Measurements were made at about 0.98 ± 0.24 at 6.6 days and 0.93 ± 0.16 at
7 days to insure steady state and at about 16.1 days. Most previous studies found
16 days because that roughly corresponds average ratios of 0.5-0.8, although a recent
to the delay in onset of Li’s therapeutic study60 found 0.92 for adult patients, a value
efficacy after initiation of treatment. No very similar to that found by us for rats.
significant change was observed for either Beyond the fact that our values are for rats
brain or serum concentration, or their ratio, and all previous values are for humans, the
at 16.1 days vs. 6.6 days. However, the major difference is that in rats the reduced
7
brain Li concentration at 16.1 days did Li MR visibility was taken into account in
correlate significantly with the corres- estimation of the brain Li concentrations.
ponding concentration at 6.6 days (r = 0.61, Spin relaxation times are of interest both
p = 0.001, N = 26). for accurate quantitation and as probes of
Brain Li strongly correlated with serum the cellular environment. In vivo spin
Li at both 6.6 days (r = 0.95, p < 0.000001, relaxation of 7Li has been reviewed.51 The
7Li T in vivo ranges from about 3 to 8 s,
N = 34) and 16.1 days (r = 0.95, p < 0.000001, 1
N = 25). Figure 17.6 shows a plot of brain depending on conditions. Our recent loca-
Li concentration vs. serum Li concentration lized, spin-relaxation studies of 7Li in rat
at 6.6 days. Both brain and serum brain under controlled conditions found a
concentrations of Li at both treatment narrower range for T1 (2.4 – 5.1 s, mean
durations correlated with dose. There are a 3.3 ± 0.9 s) at 4.7 T.68 Very few previous
number of factors responsible for the results of any sort are available for T2.51
substantially better correlations in animal Previous T2 results in rats were unlocalized
and hard to interpret. Since biexponential
behavior from the presence of both intra-
and extracellular Li might be expected for
T2, measurements ideally should be loca-
lized to a single tissue type to eliminate
apparent biexponential behavior from multi-
ple tissues with differing T2s. In practice, it
is difficult to detect unambiguously the pre-
sence of biexponential decay. In our recent
study in rats,68 we found T2 values in the
range of 53 to 113 ms (mean 82 ± 20 ms).
Very recently Ramaprasad70 obtained 7Li
Fig. 17.6: Plot of brain Li concentration vs. serum Li spectroscopic images of 3.1 × 3.1 × 10 mm3
concentration at 6.6 days in the rat. The solid line
resolution on rat brain and muscle at 7 T.
represents the regression equation, [Li] in brain =
0.01078 + 0.97315 × [Li] in serum. The dashed lines In vivo 7 Li MRS is likely to remain
represent the 95 percent confidence interval.69 primarily a research tool, or be used to
226 Biomedical Magnetic Resonance: Proceedings of the International Workshop
measure Li levels in conjunction with MRS “optical pumping,” have proven very
of other isotopes such as 1H or 31P.71 successful in increasing the polarization to
upto 70 percent (an increase by a factor of
Sodium-23 about 100,000), which allows these
Sodium-23 MRI is potentially a powerful hyperpolarized gases to be used for MR
technique for visualizing endogenous, imaging. This prepolarization of the nuclei
naturally abundant sodium in vivo. Appli- not only increases the signal that can be
cations of the technique have been reviewed, obtained from the MR experiment, but also
although not recently.72 Of critical impor- imposes some restrictions on the techniques
tance is the distribution of Na between the used to measure that signal. For example,
intra- and extracellular environments. One since the polarization is not the result of
approach to this question has been via the magnetic field, the available signal
multiple quantum techniques.73 The reader disappears with each RF pulse. Thus, small
is directed to recent applications of 23Na flip angle experiments are used to maintain
MRI to myocardial infarction, 74 brain as much of the signal as possible for as long
tumors,75 cerebral ischemia, 76 and other as possible. Fortuitously, the relaxation
organs.77 times of both 3He and 129Xe are long in
vivo, which means that it is possible to
Hyperpolarized Gases—3He and 129
Xe acquire 3 He or 129Xe images using fast
imaging techniques.
An MR experiment has inherently low Two methods are used to achieve
sensitivity compared to other spectroscopic hyperpolarization: Spin exchange optical
techniques, such as optical spectroscopy. pumping and metastability exchange optical
This is due to the small population difference pumping. In both techniques, the noble gas
between the nuclear energy levels, even at is placed in a chamber with the exchanging
the highest magnetic field strength. This atoms. The spin exchange method uses alkali
population difference in a static magnetic metal vapors whereas the metastability
field is given by method uses metastable atoms. The chamber
containing the noble gas and the exchanging
N hB 0
...(2) medium is placed in a magnetic field and
N -
2k BT illuminated with laser light of the appro-
in which N+ is the number of spins parallel priate frequency for optical absorption by
to the field, N– is the number of spins anti- the metal vapor or the metastable atoms.
parallel to the field, h is Planck’s constant, This causes spin polarization of the vapor
B 0 is the magnetic field strength, k B is or the metastable atoms. This energy is
Boltzmann’s constant, and T is the abso- transferred through collisions to the noble
lute temperature. gases, which causes them to become spin
The spin polarizations for 3He and 129Xe polarized. For a more complete description
at 1.5 T and room temperature are 7.8 × of the process, refer to the review by
10-6 and 2.8 × 10-6 respectively. Recently, Kauczor et al78
optical methods of increasing the spin Hyperpolarized 3He and 129Xe have been
polarization of 3 He and 129 Xe, termed used primarily for visualizing spaces, such
MRI and MRS of Nuclei other than 1H 227
as the lungs, that are poorly imaged with in vivo. Isotopic enrichment is a necessity,
protons. The first human studies using both to improve SNR and to follow distri-
hyperpolarized helium were done by bution of label along metabolic pathways.
Kauczor et al.79 Xenon was first used by Despite the difficulty, however, 13C has been
Albert et al.80 In general, helium is preferred used in several studies of brain,91 muscle,92
over xenon because of its larger magnetic and liver 93 metabolism. The challenges
moment, smaller relaxivity, and greater involved in, and the applications of, 13C in
polarization.81 In addition, helium has no brain have been reviewed by Gruetter et
side effects, other than reduced oxygenation al.91 Carbon-13 has been used in the clinical
due to reduced oxygen levels in the inhala- setting to examine various disorders,
tion mixture, whereas xenon has known including Alzheimer’s disease and epilepsy.94
anesthetic properties, which may not be However, the complexity of the exam limits
desirable.81 its availability to research sites.
Recently, hyperpolarized helium has been Similar considerations apply to 15N,
compared to Xe-133 scintigraphy to measure which has been used in vivo to follow ammo-
lung defects.82 The authors found that the nia transport and glutamine metabolism.95,96
two methods gave similar results. Hyper-
polarized helium has been used for MR OTHER NUCLEI
microscopy of the rat lung, providing A number of other nuclei have been used
images with resolutions on the order of for in vivo imaging or spectroscopy. Several
5 × 10–3 mm3.83 The relaxation time of the involve very specific applications in limited
hyperpolarized helium can be used to situations, whereas others hold promise for
measure the alveolar pO 2 . 84 Other more general applicability. Other applica-
applications of hyperpolarized helium in tions include 27Al for measuring gastro-
humans have been reviewed recently85 and intestinal transit times, 47 10B for locating
include spin density imaging, dynamic boron neutron capture agents,97 17O for
ventilation imaging, and diffusion weighted imaging cerebral metabolic rate of oxygen,98
87
imaging. Rb for measuring ion transport in tissue,99
133Cs for probing the intracellular environ-
Unlike helium, xenon is very soluble in
lipids and water and has a large chemical ment,100,101 195Pt for monitoring the fate of
shift change when dissolved or adsorbed.86 the anticancer drug carboplatin in tissue,102
These properties have been exploited in and 2H for following water diffusion and
studies of lung function,86 perfusion,87 blood flow, and lipid metabolism.103,104
oxygenation, 88 and tumors. 89 Hyper-
polarized xenon has also been used to image FUTURE PROSPECTS
tissues, particularly brain.90 Limitations on the clinical applications of
MRS of heteronuclei are slowly being
Carbon-13 and Nitrogen-15
alleviated. Multinuclear capability is now
In vivo 13C MRS can provide a great deal of more commonly available on clinical
information about metabolic pathways in the scanners. This trend is driven largely by
brain and other tissue. However, the low the desire for 31P MRS capability, and will
natural abundance and low sensitivity of improve prospects for other nuclei. High-
13 magnetic-field clinical scanners, now in
C make its MRS technically challenging
228 Biomedical Magnetic Resonance: Proceedings of the International Workshop
distribution in vivo of fluvoxamine and 29. van Laarhoven HW, Klomp DW, Kamm YJ,
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18
MRI/MRS to Predict and Monitor
Response to Anticancer Therapies
Robert J Gillies, Natarajan Raghunand
multitude of possibilities, the right choice dependent phase upon the recipient spins.
of experiment and acquisition parameters If spins do not move during the evolution
requires educated forethought taking into time, they are completely rephased upon
account the drug’s mechanism of action and application of the second pulse. However,
the question to be asked. It is our contention if they do move, they will fail to refocus
that the right choices can be better made and signal is lost. The rate at which signal
with appropriate preclinical studies in is lost is dependent on g, δ and Δ, such
appropriate animal models. This will be that65
discussed in the section below, entitled S = So e–bD
2 2 2
“MRI/MRS to monitor therapeutic where, b = δ γ g (Δ – δ/3)
response”. In practice, gradients of increasing strength
are used, which increases the strength of
MRI/MRS TO MONITOR the diffusion weighting, or b-factor. Signal
THERAPEUTIC RESPONSE decreases exponentially with increasing b-
factor (Fig. 18.1).
The rationale driving this aspect of MR In pure water, the signal will decay with
research is that MR imaging or spectroscopy a single exponential and fitting this will
can provide early, identifiable and yield ca. 3.2 × 10-5 cm2 sec-1, which is the
quantifiable endpoints in response to drug true diffusion coefficient of water, D at 37oC.
actions.16 These endpoints are often, but not In tissues, however, water is restricted by
always, mechanistically connected to known numerous membranous boundaries, which
drug targets. Because this has historically are more numerous and closer together in
been a major focus of research activity, no the intracellular space. Hence, the measured
attempt will be made to comprehensively values do not reflect true diffusion, but the
review the field. Instead, readers are amount of diffusional restriction. Hence, the
directed to recent reviews on the sub- measured value is termed the apparent
ject.17,21,25,49,56 In this section, we will diffusion coefficient, ADC. Since diffusional
describe some of our own work in using restriction is more pronounced in the
diffusion MRI, dynamic contrast MRI, and intracellular space, the ADC will decrease
MRS to monitor response to therapies in with increasing cellularity. 64 Hence,
humans and animal models. diffusion MRI is sensitive to changes in cell
volume fraction. Such changes occur with
Diffusion MRI
multiple modes of cell death, including
In the simplest experiments, diffusion MRI apoptosis.8
involves the use of balanced gradient pulses, Diffusion MRI of animal tumors was first
which serve to refocus spins that are shown to be affected by chemotherapy in
physically stationary. This is shown in the mid-1990s by Ross and Chenevert at
Figure 18.1, wherein two gradient pulses of Michigan 62 and by Evelhoch’s group at
strength, g, and duration, δ, are separated Wayne State.76 Since that time, there have
by an evolution time, Δ. Since the gradient been a number of studies to investigate this
is applied along a direction, the effect of phenomenon in both preclinical animal
the first pulse is to impart a spatially- models and in humans.21,37 In the following
MRI/MRS to Predict and Monitor Response to Anticancer Therapies 235
Fig. 18.1: Standard diffusion weighting protocol. Diffusion weighting is imparted by a matched pair of pulsed
field gradients that will refocus the phase of stationary spins. The magnitude of diffusion weighting is quantified
in the ‘b-factor’ which embodies terms for the gradient strength, g, duration, δ, and evolution time, Δ. Signal
decays exponentially with increasing b-value. The faster the decay with b, the higher the apparent diffusion
coefficient, ADC. The in vitro curve is shown for a pure water solution. The in vivo curve is shown for a prostate
cancer xenograft. See reference 34.
sections, we describe our published and unequivocal increases in the ADC within
unpublished results investigating the effect two days of treatment in those tumors
of chemotherapy on the apparent diffusion destined to respond to drug (MCF-7/s
in clinical and preclinical tumors. Each treated with taxol) and no change in ADC
paragraph is identified by the lead for those tumors that did not respond (MCF-
investigator on the project, and their contact 7/D40 treated with taxol or vehicle, MCF-
information is given at the end of this article. 7/s treated with vehicle). Representative
results are shown in Figure 18.2. This figure
Preclinical Studies shows histograms of the ADC values for
taxol- and vehicle-treated MCF-7/s tumors
Jean-Philippe Galons
two days after initiation of therapy. As
We began examining this approach in 1998.22 shown, there are significant and substantial
These early studies investigated xenografts increase in the ADC by this time point. The
of MCF-7 human breast cancer tumors that use of drug-resistant tumors was significant
were treated with taxol or with vehicle because it showed that the response was
alone. Both drug-sensitive (MCF-7/s) and dependent on tumor response and did not
drug-resistant (MCF-7/D40) tumors were result from systemic effects of drug on the
examined. These studies showed clear and host.
236 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 18.2: ADC histograms in response to taxol. Fig. 18.3: Changes in ADC and TGD in response to
Diffusion data can be represented as histograms Taxotere TM. Changes in the mean ADC were
wherein the percent of pixels are plotted at different measured 2 and 4 days following treatment of LnCaP
values of ADC. As shown in this graph, there is a prostate cancer xenografts in response to different
movement to higher b-values upon treatment with doses of TaxotereTM. Additionally, tumor response was
taxol in drug-sensitive tumors. See reference 22. measured as the tumor growth delay. As shown, ADC
increased at all doses, and increased further between
days 2 and 4. In contrast, TGD increased mono-
Dominique Jennings tonically with dose. See reference 34.
missed significance (p = 0.07). We hypo- histological analyses reveals that the tumor
thesized that analyses would be improved cells are relatively disperse. Hence, the
with better image registration, as motion magnitude of ADC changes would be
between scans prevented pixel-by-pixel fits. expected to be variable and somewhat less
Hence, we have developed an automated that those observed in the liver. To improve
segmentation scheme based on region matters, though, diffusion images from bone
growing followed by fuzzy Sobel edge can be acquired at higher resolution
detection and a modified Kass snake.42 Such compared to the liver (Figs 18.4A and B).
analyses compared favorably to radiologist- Since they are self-registered, images can
identified ROIs, and reduced the time be analyzed on a pixel-by-pixel basis to
required by orders of magnitude. An generate ADC maps. Although these studies
extension of these analyses will allow are only preliminary with only 8 patients,
segmentation and pixel-by-pixel fits, which data have shown that responding patients
should yield more detailed interpretations have small but statistically significant
of the ADC changes. increases in their ADC values by day 11,
and non-responding patients do not.
Dominique Jennings
Because of the aforementioned motion Mechanistic Studies
problems, and because breast cancer often Because of the importance and utility of
metastasizes to bone, we have recently diffusion MRI in cancer and in monitoring
turned our attention to analyses of diffusion ischemia, a number of researchers have
in bone lesions. The prognosis for such an examined the mechanisms behind the ADC
approach is tempered by the fact that bone changes.32,44,64 We have been investigating
lesions can be lytic or blastic, and this both by correlations to mechanisms of
Figs 18.4 A and B: Diffusion weighted images of breast cancer metastases. Diffusion weighted
images were obtained from breast cancer patients with metastases to either liver or bone. In
the liver, due to motion, images were collected during a single breath hold using echoplanar
imaging (see reference 70). In the bone, images were collected with a fast spin echo sequence,
to yield higher resolution (unpublished observations)
MRI/MRS to Predict and Monitor Response to Anticancer Therapies 239
cell death and by the use of a novel in vitro susceptibility-induced shift in the extra-
bioreactor system. cellular water, which uncovers a resonance
identifiable as intracellular. Hence, the
David Morse apparent diffusion coefficient of intracellular
In the first of these studies, we demon- water can be measured directly, and
strated that, under our experimental monitored in response to ischemia, osmotic
conditions, docetaxel kills breast cancer shock or chemotherapy. Although scalar
xenografts via both lytic necrosis and mitotic ADC values are completely dependent on
catastrophe, and not apoptosis.48,50,51,52 The the acquisition conditions (specifically, Δ),
drug resistant line, MCF-7/D40, is not measured values on cultured C6 glioma cells
resistant to docetaxel, and was killed by are consistent with predicted values (ca. 1 ×
the same mechanisms as the other two cell 10–5 cm2 sec-1).
lines used in this study (MCF-7/s and MDA-
mb-231). Furthermore, the spatially variant Dynamic Contrast-enhanced MRI
changes in ADC within these tumors could Dynamic contrast-enhanced (DCE) MRI
be registered to spatially-variant modes of involves monitoring the time-dependent
cell death, showing that, in this system, the signal intensity increases following the bolus
increases in ADC were not due to apoptosis, injection of a contrast reagent. If the arterial
but were related to other modes of cell input function (AIF) is known, these
death, namely lytic necrosis and mitotic intensity changes can be modeled and used
catastrophe. These were shown to involve to extract valuable parameters, such as
the destruction of plasma membrane extraction fraction or transfer constants.71
integrity, which would be consistent with If the AIF is not known and the injections
and increase in ADC. This is significant are reproducible, data can be analyzed using
because previous reports had ascribed the an area-under-the-curve approach.15 Too
diffusion change to apoptosis,30 and it was much has been written on the application of
necessary to determine if the diffusion DCE-MRI to monitor therapeutic response
changes were specific for this mode of cell to be comprehensively reviewed here.
death or not. Instead the readers are referred to the work
of Padhani, 57,58,59 Knopp 31,39,40 and
Jean-Philippe Galons and Brasch9,27,61 for application in the clinic, and
Theodore Trouard to Bhujwalla, 4,5,7 Neeman, 1,9,53,54 and
Nalcioglu66,67,68 for preclinical applications.
Cells grown in an NMR-compatible hollow-
fiber bioreactor (HFBR) can reach tissue
Bénédicte Jordan
densities in the extra-fiber space.3,26 Because
of the orientational geometry in these Our approach to this problem has been to
reactors, magnetic susceptibility gradients incorporate DCE-MRI acquisitions into the
exist that can be exacerbated through the armamentarium of techniques used to
addition of susceptibility reagents, such as investigate MR visible parameters in animal
those containing gadolinium. These gradi- models prior to clinical trials of new drugs.
ents are anisotropic and addition of This paradigm has been applied to a novel
Gd-DTPA to the perfusate results in a class of redox drugs.
240 Biomedical Magnetic Resonance: Proceedings of the International Workshop
correlation between either DCE-MRI and/ expected that, if a drug is efficacious, those
or diffusion MRI and ultimate clinical res- patients with the intended target will have
ponse to this drug has not been established. a higher response rate, compared to those
At a more profound level, however, it who do not.
can be postulated that patients with high- Of all of the MR-visible parameters, the
VEGF tumors will exhibit a greater response one currently with the highest connectivity
than those with more indolent, non-VEGF to a molecular target is DCE-MRI and
expressing tumors and that permeability to VEGF. This is illustrated in Figure 18.5,
a macromolecular contrast agent is a bio- which shows the HIF-1α axis. As shown,
marker for VEGF. Hence, high permeability there are a number of activities that can
from DCE-MRI could be used as an inclusion lead to stabilization of this transcription
criterion in clinical trials with this drug. Such factor besides hypoxia. Elevation of HIF-1α
segmentation of patients would lead to should induce expression of VEGF in most
higher efficiencies in clinical trial design, systems, and elevation of VEGF should
with substantial savings in terms of time increase permeability. However, going
and money. This is discussed further in the backwards, it must be realized that high
following section. permeability can be affected by other factors
besides VEGF, such as nitric oxide, NO.20
MR IMAGING TO PREDICT Thus, permeability would be predicted to
THERAPEUTIC RESPONSE be a sensitive, yet non-specific, biomarker
The entire field of pharmacogenomics is for VEGF. Consequently, permeable tumors
predicated on identifying patients whose would be expected to have a higher
cancerous lesions contain an intended drug proportion of VEGF-expressing phenotypes,
target prior to treatment. 45,47,72,75 It is compared to the general population.
Fig. 18.5: The HIF-1α axis. HIF-1α is stabilized by hypoxia, as well as (counter-clockwise from 12 o’ clock):
alterations in microtubule distribution (ìtubes), cyclo-oxygenase-2 (Cox-2), activities of insulin-like growth
factor (IGF) or human EGF receptor-2 (HER-2), hypoxia, phosphatidylinositol 3-phosphate kinase (PI3K), heat
shock protein-90 (HSP-90), histone deacetylase (HDAC) and thioredoxin (TRX). These can be inhibited by
drugs indicated in italics. One of the proteins induced by HIF is vascular endothelial growth factor, VEGF,
which increases vessel permeability
242 Biomedical Magnetic Resonance: Proceedings of the International Workshop
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19
In vivo Cellular and
Molecular Imaging of Cancer
Zaver M Bhujwalla, Ellen Ackerstaff, Dmitri Artemov, Kristine Glunde,
Arvind P Pathak, Venu Raman and Meiyappan Solaiyappan
Fig. 19.1: Schematic of the biosynthesis (solid lines) and hydrolysis (dashed lines) of phosphatidylcholine
(PtC), including all enzymes involved in choline phospholipids metabolism. Phosphorylation of choline to
phosphocholine (PC) by choline kinase is the first step in the biosynthesis of PtC. PC is then converted to PtC
via intermediates involving the rate-limiting enzyme CTP:phosphocholine cytidyltransferase and
phosphocholine transferase. Hydrolysis of PtC is affected by three major PtC-specific enzymes, phospholipase
C, phospholipase D and phospholipases A1 and A2. MR-detectable metabolites are glycerophosphocholine
(GPC), phosphocholine (PC), and free choline. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine
triphosphate; CDP, cytosine diphosphate; CMP, cytosine monophosphate; CTP, cytosine triphosphate; PPi,
pyrophosphate. Adapted from 24
In vivo Cellular and Molecular Imaging of Cancer 249
Figs 19.2A and B: Representative fully relaxed 31P NMR spectra obtained from (A) an MDA-MB-435-Vβ tumor
(empty vector) and (B) an MDA-MB-435-1β tumor. Peak assignments are (1) 3-APP (2) phosphomonoester
(PME), (3) Inorganic phosphate (Pi), (4) phosphodiester (PDE), (5) phosphocreatine (PCr), (6) γ-nucleoside
triphosphate (NTP), (7) α-NTP (set to -10 ppm), (8) β-NTP. Adapted from 19
Overall, these as well as other data will some extent because of the uncertainty of
be presented to demonstrate that diverse how these molecules cause an inhibitory
molecular alterations such as nm23 expres- effect within a cellular environment. In
sion, p53 expression, and malignant addition, the expression of antisense RNA’s
transformation arrive at a common endpoint or ribozymes does not guarantee an
in choline phospholipid metabolism. These inhibitory effect. It is possible to circumvent
data are evidence of the important role of these problems with the use of small
choline phospholipids and their metabolites interfering RNA (siRNA) of 21-23 nucleo-
in cancer, and provide a strong rationale tides (nt) which mediate effective degrada-
for targeting enzymes in choline phospho- tion of the endogenous complementary
lipid metabolism of cancer cells. homologous RNA. 22 The availability of
Antisense RNA’s20 and ribozymes21 have siRNA technology to target specific gene
been used widely to inhibit gene expression. expression has proved very successful,23 and
The design of these probes is arbitrary to provides new opportunities to target genes
In vivo Cellular and Molecular Imaging of Cancer 251
in the choline phospholipid as well as other 1).27 HIF-1 consists of two subunits, HIF-1α
critical pathways identified by MR methods. and HIF-1β. While HIF-1β is a common
During the course of the lecture examples subunit for several transcription factors,
will be given of siRNA targeting of the HIF-1α is unique to HIF-1 and its expression
enzyme choline kinase, which is one of the is highly regulated by cellular oxygen
enzymes over-expressed in breast cancer concentration.28 Under nonhypoxic condi-
cells,24 and its effect on MR spectra.25,26 tions, HIF-1α is subject to ubiquitination and
These data will demonstrate how MR of proteasomal degradation. Under hypoxic
transgenic models can be used to identify a conditions, ubiquitination of HIF-1α is
key target, to image the effectiveness of the dramatically reduced and the activity of its
delivery of therapy, and to use imaging to transcriptional activation domain incre-
ases.27,29 HIF-1 can act as a transcriptional
determine the outcome of treatment.
activator for VEGF, inducible nitric oxide
synthase, heme oxygenase 1, glucose
SOME EXAMPLES OF MR IMAGING transporter (GLUT1), and the glycolytic
APPLICATIONS TO TRANSGENIC enzymes aldolase A, enolase 1, lactate
MODELS dehydrogenase A and phosphoglycerate
A tumor is a complex organization of cells kinase 1. Each of these target genes contains
with different genotypic as well as hypoxia response elements (HREs) which
phenotypic characteristics. One of the most include one or more HIF-1 binding sites
verified and widely accepted concepts about with the core sequence 5’-RCGTG-3’.30 Thus,
HIF-1 mediates the adaptive response of
cancer is that the malignant cell arises from
cells to hypoxia.
genetic alterations. However, it is possible,
The presence of a HIF-1 binding site is
that several of the subsequent lethal
necessary but not sufficient to constitute a
phenotypic traits of cancer, such as invasion
functional HRE. By placing green fluorescent
and metastasis, may arise from the unique protein (GFP) expression under the control
physiological environment of solid tumors of an HRE, it is possible to indirectly detect
which is frequently characterized by areas HIF-1 activity in tissues. Recently Shibata et
of poor blood flow, hypoxia, high lactate al31 described a vector where the HRE of
levels and low extracellular pH (pHe). MR human VEGF-A was isolated and ligated to
imaging and spectroscopic imaging of the luciferase reporter gene. Luciferase
transgenic tumor models provide unique activity in cells transfected with this vector
opportunities to characterize the dynamics increased over 500-fold in response to
between vascularization and metabolism and hypoxia, reaching levels comparable to that
understand their role in cancer invasion, obtained by a full length CMV promoter
metastasis and drug resistance. We have (which results in constitutive overexpression
studied, with combined MRI and MRSI and of luciferase) linked to the luciferase reporter
optical imaging, a transgenic prostate cancer gene. This vector, with the luciferase
model which expresses green fluorescent reporter replaced with an enhanced green
protein under hypoxia. fluorescent protein (EGFP) gene (schematic
In normal and cancer cells, a critical shown in Figure 19.4, Plate 11), was kindly
response to hypoxia is the induction of the provided by Dr J Martin Brown from
hypoxia-inducible transcription factor (HIF- Stanford University.
252 Biomedical Magnetic Resonance: Proceedings of the International Workshop
the first examples of the use of MRI to detect significantly increased the versatility of MR
gene expression, Weissleder and colleagues in studying transgenic models of cancer.
used 9L glioma cells genetically engineered Although MR techniques provide a wealth
to express the modified transferrin receptor, of structural and functional information,
ETR, with a knocked-down negative sensitivity remains an issue. Developing
feedback regulation domain. Tumors strategies to improve the sensitivity of
derived from these cells exhibited significant detection of molecular targets in MR
shortening of T2* following administration molecular imaging of cancer is, therefore,
of transferrin-monocrystalline iron oxide critically important. One of the most exciting
nanoparticles.38 aspects of MR is the ability to perform multi-
parametric imaging. Multi-parametric
Detecting the expression of a specific
imaging in combination with the ability to
reporter is another example of the appli-
visualize gene and receptor expression of
cation of MR imaging to trangenic cancer
transgenic models of cancer cells and tumor
cells and tumor models. Data will be
models will provide major advances in our
presented to demonstrate an MR based
understanding of cancer.
approach developed by Artemov et al39 to
detect HER-2/neu receptor expression in a
transgenic HER-2/neu overexpressing tumor ACKNOWLEDGMENTS
model. A two-step labeling procedure Work from the authors program was supported
permits separation of the targeted contrast through funding by NIH grants 2R01 CA73850, 1R01
agent complex into two components with CA90471, 2R01 CA82337 and P50 CA 103175.
relatively low molecular weight (160 kDa
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Body MR Imaging 257
20
Body MR Imaging
Raju Sharma, Shiva Gamanagati
GRE imaging is being investigated as a T1- cysts and hemangiomas with long T2 times.
weighted sequence to provide accurate 3D HASTE is not effective for tumor detection
segmental and vascular anatomy for surgical due to poor contrast resolution for lesions
planning, including assessment prior to liver with moderate T2 times. It should not be
resection and transplantation. used as the primary sequence for liver tumor
T2-Weighted sequences: Breathing-averaged T2- detection.
weighted conventional or fast spin-echo, In-phase, out-of phase sequence: This is useful to
with optional fat suppression, is the most look for fatty liver, focal fat or focal sparing.
commonly used T2-weighted sequence. If Dynamic gadolinium-enhanced sequence: This
the respiratory pattern is irregular, turn off can be performed as either in-phase FLASH
respiratory triggering and add fat satura- or as out-of-phase FLASH with the addition
tion. Phase correction is helpful. Flow of fat saturation to eliminate artifact at fat-
compensation is also helpful but reduces the organ boundaries. Repeat the scan multiple
number of slices that can be acquired in a times with breath holding as follows:
given respiratory interval. Fast spin-echo • Pre-injection.
(FSE) techniques are compatible with breath- • Arterial phase (inject gadolinium, flush
hold acquisitions by using long echo train immediately with 30 cc saline and start
lengths of approximately 20 and reduced scan halfway through the flush during
matrix array size. By using a high band- breath holding).
width acquisition (e. g., 32 kHz), acquisition • Portal venous phase.
times are further reduced. Using this • Delayed (If cirrhotic liver, then a delay
method, approximately eight axial sections of 2 to 3 minutes is adequate; if there is
can be acquired throughout the liver in 20 a lesion that might be hemangioma or
to 30 seconds. Fat suppression improves the cholangio CA then repeat every 2 to 3
quality of both conventional and FSE T2- minutes out to 10 minutes).
weighted sequences. There are two reasons Use of contrast media in MRI of the liver:
for this. First, moving fat during respiration The liver may be assessed using para-
causes phase artifacts that are propagated magnetic contrast agents such as gadolinium
through the entire image. By using fat agents, which increase the intensity of
suppression, the signal of the moving normal hepatocytes or superparamagnetic
anterior abdominal wall is suppressed, and agents such as superparamagnetic oxides,
thus it reduces respiratory artifacts. Second, which decrease the intensity and appears
suppression of the high signal fat (especially dark.
with FSE or turbo spin-echo) allows for a To be effective contrast agents should
greater dynamic range of signal intensities exhibit selective or differential accumulation
to be displayed in the images. This increases within the different tissue compartments
conspicuity of focal lesions in the liver. being assessed.
If FSE T2 is of poor quality, use axial Extracellular gadolinium chelates: Gadolinium
single-shot FSE (SSFSE) or Half-Fourier chelates are non-specific extracellular
acquisition single-shot turbo spin-echo contrast agents that rapidly equilibrate
(HASTE) with breath holding. HASTE has between the intravascular and extracellular
been shown to be effective for characterizing spaces after injection. Gadolinium is
Body MR Imaging 259
delivered in the liver through the hepatic The hepatocyte-specific contrast agents
arteries and then by the portal vein, and are ionic metal chelates with a relatively
distributes fairly, rapidly in normal and pronounced hydrophilicity, a weak protein
abnormal tissues. This fast distribution binding and a residual capacity of lipophi-
requires rapid imaging in order to maximize licity. The gadolinium formulations, Gd-
differential enhancement between normal BOPTA and Gd-EOB-DTPA, enter the
liver parenchyma and lesions. On high field hepatocytes by way of the adenosine tripho-
scanners, using 2D or 3D SPGR sequences, sphate (ATP) dependent, membrane bound,
the whole liver could be assessed in one multispecific organic anion transporters
breath hold. Increased SNR is achieved located in the sinusoidal and canalicular side
using phased array coil, whilst CNR is of the hepatocytes. Both are cleared rapidly
improved using fat suppression.5,6 from the plasma with a varying degree of
The portal venous phase images are hepatobiliary elimination. Mn-DPDP is a
obtained approximately 60 seconds after the manganese derivative and demonstrates a
injection of contrast material. During this more complicated pharmacokinetic path-
time, the hepatic parenchyma returns a high way. Its hepatocellular uptake involves a
signal, because the portal vein supplies 75 complex process of dissociation, transmetal-
to 80 percent of the blood to the liver. This lization, selective manganese uptake and
phase will decrease conspicuity of elimination. On the basis of the paramag-
hypervascular lesions as both lesion and netic properties of gadolinium and
normal tissues are filled with contrast. As manganese ions, the above mentioned
most liver tumors are hypovascular relative hepatobiliary contrast agents enhance the
to the liver parenchyma, they will be T1 signal.
visualized as hypointense lesions relative to The superparamagnetic iron oxide (SPIO)
the enhancing liver on the venous phase. consists of nanoparticles typically composed
Delayed or equilibrium phases are of an iron core of 3 to 5 nm in size. The
obtained 3 to 5 minutes after the injection core is covered with a dextran that increases
of contrast media. Most liver lesions become the particle size between 50 nm and
less conspicuous on these images, but some 150 nm. After IV administration the particles
tumors with interstitial space such as are cleared from the plasma by the reticulo-
cholangiocarcinoma will accumulate more endothelial system of the liver (80%) and
contrast material than the liver and return spleen (12%). The particles are eliminated
a high signal. in the lymph nodes and bone marrow. The
plasma half life of the nanoparticles is 8 to
Liver-specific Contrast Agents 10 minutes. Nevertheless the superparamag-
netic effects last for several days, providing
These are divided into hepatocyte-selective an imaging period of several hours after
and Kupffer’s cell-selective contrast agents. administration. The super-paramagnetic
The former encompass the chelated com- properties of the iron core are responsible
pounds Mn-DPDP, Gd-BOPTA and Gd-EOB, for a significant disturbance of the local
whereas the latter consist of the group of magnetic field around the particles resulting
superparamagnetic iron oxide particles.7-9 in a pronounced shortening of both T2 and
260 Biomedical Magnetic Resonance: Proceedings of the International Workshop
A B
Figs. 20.1A and B: Polycystic liver disease
A. Axial T1W image showing multiple lowsignal intensity focal lesions. Note: largest lesion in left lobe is
isointense
B. Axial T2W image showing that focal lesions are hyperintense. Note: largest lesion in left lobe is showing low
signal intensity area inside suggestive of bleed inside the cyst
A B
C D
Figs 20.2A and D: Hemangioma of liver
A. Axial T1W image showing low signal intensity lesion in right lobe
B. On axial T2W image, lesion is uniformly hyperintense
C. Dynamic contrast study, arterial phase image showing, peripheral nodular enhancement of the lesion
D. Delayed phase image showing, centripetal filling in of the lesion
central hypointense region (Type 3). The although lesions may grow to more than 10
Type 1 enhancement pattern was observed cm in diameter. Lesions are solitary in about
only in small tumors. Type 2 and Type 3 70 percent of patients. Multiple lesions of
enhancement patterns were observed in FNH have been described in patients with
tumors of all sizes.13-15 certain brain neoplasms and vascular
malformations of various organs.16 FNH is
Focal Nodular Hyperplasia (FNH) a nonencapsulated firm nodule of normal
FNH may occur in 2 to 5 percent of the hepatocytes with a distinct central scar and
population. It is rarely symptomatic, thin radiating fibrous septa containing
Body MR Imaging 263
A B
C D
Figs 20.3A to D: Hepatocellular carcinoma of liver
A. Axial T1W (FLASH) image showing, isointense focal lesion within the caudate lobe in background of
cirrhotic liver
B. On T2W image, focal lesion is isointense to mildly hyperintense to liver parenchyma
C. Arterial phase image of dynamic study showing, early uniform enhancement of the lesion. Note:
inhomogeneous enhancement of liver parenchyma
D. Venous phase image showing, contrast wash out and peripheral well defined capsule
Kupffer’s cells and primitive bile ductules. HCC. On MR images, FNH is typically
Intratumoral calcification, fat, hemorrhage, isointense to liver on T1 images, and slightly
and necrosis are extremely rare. FNH hyperintense on T2 images. On T1 images,
should be differentiated from adenoma, an internal low signal area that corresponds
because the two lesions occur in a similar to a central scar is present in more than 50
patient population. An additional important percent of cases. The scar is usually
differential consideration is fibrolamellar hyperintense to liver on T2 images, but may
264 Biomedical Magnetic Resonance: Proceedings of the International Workshop
be hypointense. A central scar, best seen on sharply defined masses with smooth
T 1 images, is highly suggestive of the borders. Unlike FNH, adenomas are usually
diagnosis. Other than the central scar, the heterogeneous due to the presence of
lesions are homogeneous on both T1 and T2 hemorrhage, necrosis, or fat. Hepatic
images. FNH lesions are highly vascular. adenomas may infrequently under-go
Unlike the central scars in hemangioma, the malignant transformation.19 In addition, as
central scar in FNH is vascular and is they enlarge, they may be the source of
surrounded by collagenous fibers. Following spontaneous intra-abdominal hemorrhage.
contrast administration, because of their For these reasons, surgeons will typically
prominent vascularity, FNH lesions tend to resect solitary liver adenomas. The MR
enhance early, and may be isointense to liver appearance of adenomas is somewhat
as early as 1 to 2 minutes after contrast variable, but the typical tumor demonstrates
injection, except for the central scar. slight increased signal on T 1 images
FNH can be confused with fibrolamellar (probably due to fat content) and moderate
hepatocellular carcinoma (HCC). Fibro- increased signal on T2 images. As opposed
lamellar HCC is a rare form of HCC seen in to FNH, they are more frequently hetero-
young patients without underlying liver geneous in their internal signal. This is, in
disease. Fibrolamellar HCC, which has a part, due to the tendency of this tumor to
better prognosis than conventional HCC, is undergo spontaneous internal hemorrhage
typically a large mass with a central scar with areas of calcification and fibrosis.
that is low signal intensity on T2W image
(T2WI). After gadolinium administration, Biliary Cystadenoma
there is intense heterogeneous enhancement This tumor presents typically as a sympto-
on the arterial phase and the central scar matic large hepatic mass characterized by
may enhance on the delayed images. If there large cystic spaces surrounded and separa-
is any question that a lesion may be ted by thickened, nodular, enhancing walls.
fibrolamellar HCC, biopsy is mandatory.17,18 Although imaging features might overlap
with a pyogenic or parasitic abscess, this is
Hepatocellular Adenoma usually not a clinical dilemma. MR imaging
Adenoma is a rare hepatic neoplasm that studies clearly demonstrate cystadenoma as
occurs almost exclusively in the liver that an ‘‘aggressive’’ and complex neoplasm.20
has abnormal metabolism due to exogenous
steroids or, less commonly, congenital Biliary Hamartoma
abnormalities of carbohydrate metabolism. Biliary hamartomas are benign biliary
Over 90 percent of cases occur in women of malformations. They are estimated to be
reproductive age, and lesions may regress present in about 3 percent of patients. They
with withdrawal of oral contraceptives or can be solitary or multiple, but their size is
other steroids. Adenomas are composed of almost invariably less than 1 cm in diameter.
cords of hepatocytes that occasionally form On MR imaging, biliary hamartomas are
bile. Adenomas lack bile ductules, portal always small in size (usually 1 cm) and well
tracts, and hepatic veins, which result in defined. They show low signal on T 1 -
necrosis and hemorrhage. Adenomas are weighted images and high signal on
Body MR Imaging 265
Well-differentiated HCC has minimal grows, the hilum of the liver may be
arterial enhancement that washes out on involved, resulting in biliary obstruction.
portal venous images. As stated earlier, well- The most prominent feature with central
differentiated HCC contains Kupffer cells cholangiocarcinoma on MR is usually
and will take up ferumoxides. Moderate and intrahepatic biliary duct dilatation. Morpho-
poorly differentiated HCCs show marked logic changes may occur late in the disease
hypervascularity and therefore enhance on process, with atrophy of the left lobe of the
the arterial phase. In a cirrhotic liver, any liver compared with the right lobe. Dynamic
abnormal enhancement should be gadolinium-enhanced imaging demonstrates
considered suspicious for HCC. HCCs are a characteristic pattern of slow, gradual
highly vascular lesions and may infrequently enhancement best seen on the equilibrium
present with spontaneous hemorrhage. Most phase. Intrahepatic cholangiocarcinoma is a
commonly, lesions are well circumscribed rare primary malignant liver neoplasm.
with the appearance of a well-defined These lesions usually present as a large
‘‘capsule’’27-29 (Figs 20.3A to D). solitary mass without biliary obstruction.
Fibrolamellar HCC is a distinctive type of HCC Dynamic gadolinium imaging demonstrates
that occurs in younger patients (mean age peripheral enhancement on the arterial
of 20 years). Although it is a malignant phase, and incomplete central enhancement
lesion, the prognosis is better than that for on the delayed images. Ten percent of
typical HCC, with 25 percent of patients patients with sclerosing cholangitis may have
having resectable lesions. Alpha-fetoprotein associated cholangiocarcinoma.31
levels are usually not elevated. Fibrolamellar
HCC is typically a well circumscribed lesion Diffuse Liver Diseases
that is hypointense on T1 images and hyper- Fatty Liver
intense on T2 images. A central scar may be
Fatty liver has many causes, including
present. Central calcifications are present in
obesity, diabetes mellitus, malnutrition, or
one third of lesions. The differential
exposure to ethanol or other chemical toxins.
diagnosis includes adenoma or FNH.30
In fatty liver, fat accumulates in hepatocytes,
Cholangiocarcinoma either focally or diffusely. On CT or ultra-
sound, focal fatty change can be difficult to
Cholangiocarcinoma is a malignant tumor differentiate from a neoplasm. With MRI,
arising from bile duct epithelium. Risk the confidence of diagnosing fatty liver is
factors include Clonorchis sinensis infection, greatly improved by using chemical shift
prior exposure to Thorotrast, primary techniques. The most useful technique
sclerosing cholangitis, and Carolis disease. involves comparison of in-phase (water +
The prognosis is poor and patients typically fat) and opposed-phase (water – fat) images.
die within months of diagnosis. Although Fatty liver, on the other hand, will have
not technically considered an intraheptic lower signal intensity on opposed-phase
neoplasm, the most common type of cholan- images because of cancellation of water and
giocarcinoma is hilar cholangiocarcinoma or fat signals within voxels. Fat-suppressed
Klatskin tumor. These tumors may be small images can also be helpful in cases of
and obstruct the biliary tree. As the tumor moderate to severe fatty infiltration. Fatty
268 Biomedical Magnetic Resonance: Proceedings of the International Workshop
liver will have lower signal intensity with carcinoma, diabetes mellitus, and myocardial
fat suppression, although the drop is not as dysfunction. Magnetic resonance imaging
much as on opposed phase images. In the detects low signal intensity on GRE T2*WI
setting of diffuse fatty infiltration, neoplasms as evidence of excessive hepatic iron in
are more difficult to detect by CT or preclinical hemochromatosis without
ultrasound. On the contrary, a diffuse fatty cirrhosis, when treatment is crucial before
liver may help MRI detect a tumor. On non– irreversible damage occurs. As the disease
fat-saturated T1WI, the fatty liver may have progresses, low T2* signal intensity can be
higher intensity than a normal liver, which seen in the pancreas and heart, but normal
increases the contrast relative to the low signal of the spleen is preserved. Magnetic
signal intensity of tumors. On fat-saturated resonance imaging can be used longitudi-
T2WI, the fatty liver has lower intensity than nally to monitor disease progress and to
a normal liver. Again, the contrast between evaluate response to treatment. Liver iron
the liver and high intensity tumors concentration can be measured by calcu-
increases32-34. lating T2 relaxation time or intensity ratios.
Iron Overload In transfusional iron overload, iron deposits in
RE cells of the liver, spleen, and bone
Iron accumulation in liver is seen in hemo- marrow. In contradistinction to hemo-
chromatosis, transfusion overload, hemolytic chromatosis, iron overload in RE is of little
anemias, and cirrhosis. Intracellular iron is clinical significance. Magnetic resonance
usually superparamagnetic. It reduces T2
imaging shows low signal intensity of liver,
and T2* relaxation times, and results in low
spleen, and bone marrow on GRE T2*WI,
signal intensity on both T 2WI and T 2 *-
weighted images (T2*WI). This makes MRI sparing the heart and pancreas. This finding,
a more sensitive and specific method for as well as a clinical history of multiple
diagnosing iron overload; gradient recalled transfusion, helps to differentiate transfusion
echo (GRE) T2*WI have greatest sensitivity. overload from primary hemochromatosis.
Normally, liver has higher signal intensity Paroxysmal nocturnal hemoglobinuria, due to
than skeletal muscle on both T1WI and and renal filtration and tubular absorption of
T2WI. free hemoglobin, they may have renal
Hemochromatosis is an autosomal recessive cortical low signal, which is independent of
disorder with a homozygous prevalence of transfusion. Reduced signal intensity of the
0.25 to 0.50 percent. In hemochromatosis, renal cortex is a characteristic finding in
iron overload results from long-term patients with intravascular hemolysis.
increased iron absorption through the
intestine. Reticuloendothelial (RE) function Cirrhosis
may be deficient. Iron accumulates in liver,
pancreas, and heart, as well as the paren- Cirrhosis is a pathologically-defined entity
chyma of other organs. In liver, the iron is characterized by irreversible chronic injury
incorporated preferentially into the hepa- of the hepatic parenchyma, extensive
tocytes rather than Kupffer cells of the RE fibrosis, and regenerative nodules. The
system.35,36 leading cause of cirrhosis has been alcohol
Hemochromatosis is associated with abuse, followed by chronic viral hepatitis,
increased incidence of hepatocellular hemochromatosis, autoimmune chronic
Body MR Imaging 269
periphery. In chronic disease, signal diffe- seconds (late interstitial phase or equilibrium
rences between the central and peripheral phase) after the start of contrast injection.
part of the liver on T1WI and T2WI and If islet cell tumor is suspected, the first
post-gadolinium contrast images are phase should be obtained in the arterial
minimal. Varices may be present in chronic phase, which occurs approximately 10 sec,
Budd-Chiari syndrome. Curvilinear intra- before the interstitial phase. The dynamic
hepatic collaterals and capsular collaterals study can be performed either by care bolus
are characteristic. In patients with chronic or test bolus technique. A 2D spoiled GRE
Budd-Chiari syndrome, hyperplastic nodules sequence with fat suppression can be used
may develop. These nodules are hyper- instead of a 3D spoiled GRE sequence for
intense on T1WI, variable on T2WI, and en- the dynamic study. With the contrast-
hance early on post-gadolinium images.42,43 enhanced dynamic GRE sequence, one
should try to include both liver, and
MR IMAGING OF DISEASES OF BILIARY- pancreas, so that liver lesions can also be
PANCREATIC SYSTEM studied at the same time. An MRCP
sequence may be added as necessary before
With recent technologic advances, such as
administration of contrast material. MR
breath-hold, dynamic contrast-enhanced cholangiopancreatography is performed
imaging, and MR cholangiopancreatography using half-Fourier rapid acquisition with
(MRCP), MR is increasingly used as a relaxation enhancement (RARE). A thick-
primary imaging modality for the pancreas slab technique is used to provide a global
and biliary system. view of the biliary tree and pancreatic duct.
Thin slice technique is useful for evaluation
MR Protocol of detailed anatomy.
The standard MR protocol includes coronal
half-Fourier acquisition single-shot turbo- The Indications of Pancreatic MRI are as
spin-echo (HASTE), axial in-phase and follows: 45-48
opposed-phase gradient-recalled echo (GRE), • Detection and staging of pancreatic
axial T 2 turbo-spin-echo (TSE) with fat neoplasms to avoid ‘‘understaging’’ on
suppression, or turbo-inversion-recovery the basis of CT
through the liver and pancreas. Two-dimen- • Detection of small non-organ-deforming
sional (2D) spoiled GRE with fat suppression pancreatic ductal adenocarcinomas
and three-dimensional (3D) spoiled GRE • Detection of islet cell tumors
with fat suppression through the pancreas • Evaluation of acute and chronic pancrea-
are also obtained. After administration of titis
gadolinium contrast material at 2 mL/sec, • Characterization of suspected paren-
we obtain breath-hold dynamic 3D spoiled chymal abnormalities found on CT or
GRE sequence in arterial, interstitial, and US (e g, focal fatty infiltration)
equilibrium phases through the pancreas. • Detection of choledocholithiasis, pan-
Dynamic post-gadolinium axial T1-weighted creatic duct calculi, and cholangio-
SGE with fat suppression are obtained at 15 carcinoma
to 20 seconds (arterial–capillary phase), 45 • Possible improved ability to distinguish
seconds (early interstitial phase), and 90 chronic pancreatitis from neoplasm
Body MR Imaging 271
and relative sparse vascularity, there is late in chronic pancreatitis, as opposed to smooth
enhancement of the tumor as gadolinium dilatation of the pancreatic duct in an
diffuses across capillaries. 50,53 In adeno- obstructing adenocarcinoma.64 On the other
carcinomas involving the pancreatic head, hand, the presence of lymphadenopathy and
reduction in T1 signal intensity may be seen liver or peritoneal metastases favor the
distal to the tumor. This is attributed to diagnosis of pancreatic cancer. CT, like MRI,
obstruction of the main pancreatic duct, with has difficulties in the differentiation of focal
subsequent chronic inflammation, leading to chronic pancreatitis from tumor. MRI criteria
progressive fibrosis and parenchymal of unresectability are similar to the esta-
atrophy. In such cases, the distinction blished CT criteria. At most institutions,
between adenocarcinoma and chronic these include involvement of the superior
inflammation of the pancreas can be difficult mesenteric artery, celiac trunk or the
on unenhanced fat-suppressed T1-weighted superior mesenteric vein–portal vein
images. The tumor may be better demons- confluence; and peritoneal or hepatic
trated in the arterial– capillary phase of metastases.53
enhancement as a rim-enhancing low signal
intensity mass in a background of slightly Islet Cell Tumors
greater enhancing chronically inflamed The three most common islet cell tumors
pancreatic parenchyma. 47,48 In addition, are insulinomas, gastrinomas, and nonfunc-
morphological changes such as effacement tioning islet cell tumors. The use of fat-
of the fine lobular contours of the pancreas suppressed T1-weighted, T2-weighted, and
with loss of the well-defined internal archi- dynamic gadolinium enhanced images is
tecture favor the diagnosis of carcinoma. In necessary, because some lesions are seen on
general, the distinction between pancreatic one sequence and not the others. These
cancer and chronic pancreatitis on the basis tumors have long T1 and T2 relaxation times,
of MRI is not difficult. Both entities show a exhibiting low signal intensity on T 1 -
low T1 signal intensity pancreatic mass, with weighted images and high signal intensity
the same degree and delay in enhancement on T2-weighted images. They can be seen
and associated dilatation of the pancreati- as low signal intensity masses on a
cobiliary ducts. This is attributed to the background of high signal intensity pancreas
abundant fibrosis in both conditions. Both on fat-suppressed T 1 -weighted images.
pancreatitis and pancreatic carcinoma can They are well depicted on dynamic contrast
cause peripancreatic changes, including enhanced imaging during the arterial phase
infiltration of the fat planes that surround because of their hypervascularity53,65,66, and
the superior mesenteric vessels with features on the fat-suppressed T2-weighted sequen-
of vessel encasement.60 In cases of masses ces. The enhancement pattern of islet cell
involving the pancreatic head, the diagnosis tumors can be homogeneous, ring-like, or
of chronic pancreatitis is favored if there is diffusely heterogeneous on the immediate
preservation of the morphology of the pan- postgadolinium T1-weighted SGE images.
creas without loss of the usual marbled Functional tumors, predominately gastri-
appearance of the gland. In addition, an nomas and insulinomas, tend to be small at
irregularly dilated main pancreatic duct with presentation, usually less than 2 cm in size.
intra-ductal calcifications is more common Nonfunctioning tumors are usually large at
Body MR Imaging 275
filling defects or papillary projections within MRCP equals or exceeds that of ERCP73,74
the dilated pancreatic duct or cysts and without the associated morbidity or morta-
dilatation of the main pancreatic duct, lity. The risks of ERCP include pancreatitis,
>15 mm for main duct type and any dilation sepsis, hemorrhage, duodenal and bile duct
for branch-duct type.71 perforation, and death. 75 Biliary-enteric
anastomoses and obstructions can also make
Solid and Papillary Epithelial Neoplasms ERCP difficult, if not impossible. After a
Solid and papillary epithelial neoplasm of failed or incomplete ERCP, MRCP should
the pancreas is a rare tumor that occurs be considered the diagnostic test of choice.
mainly in women under 40 years of age. In this group of patients, MRCP has been
The mass is usually large (>10cm) and shown to have a sensitivity of 97 percent, a
commonly exhibits cystic and hemorrhagic specificity of 100 percent, and an accuracy
degeneration from necrosis with a thick rim of 98 percent for the diagnosis of pan-
of soft tissue. On T1-weighted images, the creaticobiliary disease. ERCP has advantages
hemorrhagic component appears hyper- over MRCP, which include direct therapeutic
intense72, and the soft-tissue rim enhances interventional procedures that may be
after administration of gadolinium contrast. performed concurrent with diagnostic
imaging. Unlike ERCP, MRCP produces
MR Cholangiopancreatography images of the ducts in their natural state,
because it does not involve distention of
MR cholangiopancreatography (MRCP) is
the ducts by injected contrast medium.
still a rapidly evolving technique, but has
been already accepted as clinically useful
MRCP Pulse Sequences
and is widely used to evaluate biliary or
pancreatic diseases in a noninvasive way. Bile and pancreatic fluid are rich in protons,
This technique uses MR imaging to visualize resulting in a T2 relaxation time that is much
stationary or slow-moving fluid, such as longer than that of surrounding tissues. On
bile, displaying them as high signal intensity. heavily T2- weighted sequences, fluid within
Heavily T2-weighted sequences are generally the biliary tree, gallbladder, and pancreatic
used for MRCP with single shot echo-train duct is bright, while background tissues are
spin-echo technique achieving the most suppressed. Signal from background fat,
widespread use. which is usually high on fast T2- weighted
sequences, can be decreased with the use of
MRCP Versus ERCP fat-suppression techniques. Magnetic reso-
Direct cholangiography methods (ERCP and nance cholangiopancreatography is usually
PTC) offer certain advantages that cannot performed using one of the hybrid rapid
be reproduced with MR methods. Both acquisition with relaxation enhancement
ERCP and PTC yield images with higher (RARE) sequences or their derivatives. The
spatial resolution, and both techniques have introduction of single-shot RARE techniques
therapeutic as well as diagnostic appli- and half-Fourier acquisition single-shot
cations. However, even with the higher turbo-spin-echo (HASTE) imaging has
spatial resolution of these invasive methods, reduced imaging times to within a single
visualization of bile duct morphology with breath-hold. These breath-hold techniques
Body MR Imaging 277
are best applied in two forms: a single, thick by Giubaud et al80 to be strongly associated
coronal (or axial) slab and a multislice, thin with the presence of biliary atresia children
coronal sequence.76 with biliary atresia confirmed by biopsy also
demonstrated significant high signal inten-
Pediatric applications sity periportal thickening on MR imaging,
which correlated with periportal fibrosis
Magnetic resonance applications in the found at histologic examination. Magnetic
pediatric population are expanding. Mag- resonance cholangiopancreatography may
netic resonance cholangiopancreatography also reveal a triangular-shaped area of high-
rarely requires anesthesia; ERCP and PTC signal intensity within the porta hepatis
cannot be performed on children without caused by cystic dilation of the bile duct
anesthesia. However, conscious sedation is proximally.81
required when MRCP is performed on young
Cystic Diseases of the Bile Duct
or uncooperative patients. Respi-ratory-
triggered MRCP enables evaluation of major Congenital cystic lesions of the bile duct
and minor bile ducts, even in uncooperative, can be classified according to Todani’s
young patients. Images can be produced in classification system.82 type I, choledochal
which the common bile duct and hepatic cyst; type II, diverticulum of extrahepatic
ducts are clearly seen in infants as young as ducts; type III choledochocele; type IV,
multiple segmental cysts (Fig 20.4); and type
3 days old.77
V, Caroli’s disease. MRCP can be effective
and comparable with ERCP for the evalu-
Biliary Atresia
ation of these lesions. Also, the combination
Biliary atresia and neonatal hepatitis are the of MRCP and gadolinium-enhanced T 1-
most common causes of prolonged conju- weighted images is useful to diagnose
gated hyperbilirubinemia in the neonate.78 associated findings, such as gallstone disease
Because the treatment for biliary atresia and and cancer.
neonatal hepatitis is very different (surgical
in the former and medical in the latter),
definitive diagnosis is necessary. Ultrasound,
generally recommended as the initial
imaging test, excludes the presence of a
choledochal cyst, which may also be a cause
of neonatal jaundice. Jaw et al79 found that
the common hepatic ducts and common bile
duct could be readily visualized in control
subjects and infants with neonatal hepatitis,
whereas infants with surgically confirmed
biliary atresia failed to demonstrate a visible
common bile or hepatic duct on MR cholan-
giogram. The failure to visualize the
extrahepatic biliary ducts on MR cholangio- Fig. 20.4: Coronal thick slab (HASTE) image
graphy and presence of high signal intensity showing, fusiform dilatation of the common bile duct
periportal thickening has also been shown suggestive of type I choledochal cyst
278 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Congenital Variants of the Biliary System bile flow within the CD may give false
impression of calculus. Correct diagnosis
MRCP can demonstrate accurately various
usually can be achieved by careful attention
variants, such as a low cystic duct insertion,
to the exact location of these foci and
a medial cystic duct insertion, a parallel
interpretation of thick-slab MRCP or MIP
course of the cystic and hepatic ducts, and
reconstructed images in conjunction with the
an aberrant right hepatic duct.83
thin-slice source images.87,88
Stone Disease
Primary Sclerosing Cholangitis
Biliary calculi are seen as relative areas of
Primary sclerosing cholangitis is charac-
decreased signal surrounded by bright bile.
terized by chronic fibrosing inflammation
Many studies have investigated the use
of the biliary system of unknown etiology.
of MRCP for the detection of choledo-
The diagnosis of primary sclerosing cholan-
cholithiasis, and excellent results have been
gitis is made by cholangiographic findings
reported, with sensitivities of 85 to 100
supported by histologic results. The imaging
percent, specificities of 90 to 99 percent, and
appearance of primary sclerosing cholangitis
accuracies of 89 to 97 percent. 84,85 The
is characterized by multiple, irregular
accuracy of MRCP in diagnosing hepatoli-
strictures and saccular dilatations of the
thiasis is 96 percent.86 Intrahepatic bile duct
intrahepatic and extrahepatic bile ducts
calculi may be seen as well-defined filling
producing a beaded appearance. The
defects or as castlike areas of low signal
conventional imaging modality for the
that conform to the wall of the duct.86 On
diagnosis of primary sclerosing cholangitis
thin-slice source images, stones appear as is ERCP. MRCP has been shown to be useful
signal void lesions and can be detected as for the diagnosis and follow up of primary
small as 2 mm in dilated and nondilated sclerosing cholangitis.89,90 Diagnostic challen-
ducts. ges include that subtle changes of mild
There are several pitfalls and mimickers primary sclerosing cholangitis may be
of stones with MRCP. Intraductal air bub- difficult to detect by current MR imaging
bles (pneumobilia) may mimic the appea-
techniques, and cirrhosis may cause
rance of stones. An important differentiating
distortion of the intrahepatic bile ducts and
feature from stones is that air bubble filling
mimic primary sclerosing cholangitis.
defects lie on the nondependent portion of
the bile duct against the wall on axial images.
Blood clots may appear indistinguishable Strictures
from bile duct stones. Other pitfalls that With MRCP, ducts are seen in their passive
may mimic bile duct stones include (1) state because contrast material does not
tortuosity of the bile duct running in and need to be injected forcefully to opacify
out of the imaging plane; (2) merging of the them. Thus, images reflect the diameter of
cystic duct into the CBD when observed en strictures more closely than they do with
face on coronal images, which may result in invasive cholangiography. Magnetic reso-
a round hypointense focus; (3) metallic clips; nance cholangiopancreatography has been
and (4) extraductal compression from the reported to have a sensitivity of 100 percent
right hepatic or the gastroduodenal artery, and a specificity of 98 percent for the
which may result in a signal void focus (5) diagnosis of strictures.91 However, reduced
Body MR Imaging 279
is direct hepatic invasion. The absence of a contrast imaging includes an axial breath-
gallbladder and the presence of gallstones hold T1-weighted gradient-echo (FLASH)
can be helpful clues to the diagnosis.103 sequence performed in and out of phase.
Infiltrating carci-nomas that replace the Used in conjunction with a frequency-
gallbladder often show irregular contrast selective fat suppressed T 1 -weighted
enhancement with scattered regions of sequence, this also allows differentiation of
internal necrosis on CT and MR.103 fat from hemorrhage, both of which may
Magnetic resonance cholangiopancreato- occur in a renal or adrenal mass. A coronal
graphy is becoming the primary method for breath-hold T2-weighted half-Fourier single-
evaluating patients with a variety of biliary shot turbo spin-echo sequence is performed
and pancreatic diseases. The absence of to help characterize cystic lesions of the
ionizing radiation and safety of this techni- kidney, to assess for hydronephrosis, and
que make MRCP an excellent diagnostic tool. to determine if an adrenal mass is present.
The coronal plane is advantageous in
MR Imaging of the Kidneys
evaluating exophytic lesions that occur at
and Adrenal Glands
the poles of the kidneys. These may not be
The practice of urology has changed greatly optimally demonstrated in the axial plane.
in recent years. Selection of the appropriate To evaluate the renal vasculature, a high-
surgical technique for the individual case resolution breath-hold fat-suppressed three-
depends highly on renal anatomy, tumor dimensional T1-weighted FLASH sequence
size, tumor location, anatomy of obstruction, is performed in a coronal-oblique plane
and the relationship of the diseased organ before and after the intravenous adminis-
to surrounding viscera. The role of pre- tration of gadolinium. Using the proper scan
operative imaging has evolved from pro- delay and acquiring the low spatial fre-
viding diagnostic confirmation to providing quency (high contrast) lines of k-space
a roadmap for selection and application of during peak arterial enhancement are critical
technique. Gadolinium chelates have been to minimize venous contamination and to
shown to be exceedingly safe, may be prevent scanning before sufficient contrast
administered to patients without concern reaches the aorta. Optimization of the
for contrast induced nephrotoxicity, and are arterial phase may be determined by using
well tolerated in those patients with a fluoroscopic triggering , an automated bolus
history of iodinated contrast allergy. Finally, detection technique104, a timing run, or with
the ability of MR imaging to detect both a ‘‘best guess’’ method. Evaluation of the
gross and microscopic fat provides for renal parenchyma is performed with a
accurate characterization of adrenal and second breath-hold three-dimensional fat-
renal masses.
suppressed T1-weighted spoiled GRE sequ-
ence in the axial plane.105 This is performed
Protocol
before and after the administration of
Comprehensive examination of the kidneys intravenous gadolinium. The postcontrast
entails evaluating the renal vasculature, acquisition is obtained after the MR
parenchyma, and collecting system. Pre- venogram, approximately 3 to 5 minutes
282 Biomedical Magnetic Resonance: Proceedings of the International Workshop
after the gadolinium bolus. MR urography extension into the renal vein and inferior
may be performed with T2- weighted turbo vena cava.108 It is important to demonstrate
spin-echo sequences using a thick slab the most cephalad extent of thrombus in
projection technique or with multiple the inferior vena cava, because the surgical
contiguous thin sections. Alternatively, a approach is altered if thrombus approaches
delayed three-dimensional T 1 -weighted the right atrium. For this the multiplanar
GRE sequence after the administration of capability of MR imaging is ideally suited.
gadolinium contrast material can be In addition, it is often possible to differen-
performed106. tiate enhancing tumor thrombus from bland
thrombus. MR imaging does not offer any
Renal Masses advantage when compared with CT for
The most important aspect in analyzing a evaluating retroperitoneal adenopathy.
renal mass is to demonstrate the presence
or absence of enhancement. An enhancing Angiomyolipoma
mass implies a vascular mass, consistent with Angiomyolipoma (renal hamartoma), a
a neoplasm. Once a lesion has been shown benign tumor, is composed of varying
to demonstrate enhancement, it is necessary amounts of fat, smooth muscle, and blood
to characterize it as a surgical lesion (renal vessels. They are uncommon lesions with a
cell carcinoma, oncocytoma, or transitional prevalence of 0.3 to 3 percent. Patients are
cell carcinoma) or a nonsurgical lesion usually asymptomatic and angiomyolipomas
(metastases, lymphoma, or angiomyo- are usually incidentally discovered when the
lipoma). patient is imaged for another reason. When
large, angiomyolipomas may exert mass
Renal Cell Carcinoma effect on the adjacent organs and cause
Renal cell carcinoma is the most common symptoms. In addition, patients with large
renal neoplasm accounting for 80 to 85 Angiomyolipomas may present with acute
percent of all malignant renal tumors and flank pain caused by spontaneous hemor-
for 2 percent of all cancers.107 Renal cell rhage. Angiomyolipoma is the only renal
carcinomas have variable signal intensity on tumor that may be characterized on the basis
T1- and T2-weighted sequences.With regard of its tissue composition and signal charac-
to the background renal parenchyma, they teristics. The relative amounts of fat, smooth
are often slightly hypointense on T 1 - muscle, and vessels within the tumor
weighted images and isointense to slightly establish its MR imaging appearance. The
hyper-intense on the T2-weighted images. diagnosis of angiomyolipoma rests on
Renal cell carcinomas, however, may demonstrating the presence of macroscopic
demonstrate any signal intensity depending fat within the lesion. The hemorrhagic cysts
on their content of hemorrhagic material. may also show similar signal characteristics.
The diagnosis of renal cell carcinoma rests It is imperative to compare the T1-weighted
on demonstrating enhancement within a images obtained with frequency-selective
renal mass. The prognosis of renal cell fat-suppression with those obtained without
carcinoma is related to the tumor stage. MR fat-suppression, to establish the presence or
imaging has been shown to be accurate for absence of macroscopic fat. Some angio-
the staging of renal cell carcinoma and more myolipomas contain only a tiny amount of
accurate than CT for the evaluation of tumor macroscopic fat and a concerted effort
Body MR Imaging 283
with conventional axial images. The capa- nomas is variable and they generally are
bility of obtaining images in innumerable heterogeneous on T 1 - and T 2 -weighted
planes is an advantage of MR imaging. sequences (Figs 20.5A to C). This is
secondary to necrosis and hemorrhagic
Adrenal Adenoma components that are common within these
lesions. After the administration of
The incidence of adrenal adenomas in the
gadolinium, the viable portion of the tumor
general population is estimated at 2 to 8
enhances. Adrenal cortical carcinomas may
percent.116 The adrenal gland is also the
grow to be very large and may directly
most common site of metastases perunit
invade adjacent organs including the kidney,
weight of any organ. Within the oncologic
liver, spleen, pancreas, and diaphragm. At
population, it is common to find an adrenal
times, it may be difficult to determine the
mass, and a frequent clinical problem is to
exact organ of origin, especially when a
determine the etiology of such a lesion. MR
normal adrenal gland cannot be identified.
imaging accurately can distinguish an
Imaging in the coronal or sagittal plane is
adenoma from a metastasis in most cases.
very helpful in showing the relationship of
Various MR techniques have been proposed
the tumor to its surrounding structures and
to characterize adrenal masses as adenoma
demonstrating the suprarenal location of an
or a metastasis. The easiest, fastest, and
adrenal mass. Adrenal cortical carcinoma has
most reliable way to diagnosis an adrenal
a predi-lection to invade the adrenal veins,
adenoma, however, rests on demonstrating
grow into the renal vein and inferior vena
intracellular lipid within the mass (lipid-rich
cava, and extend cephalad toward the heart.
adenoma). By using chemical shift techniques
Gadolinium-enhanced MR imaging can
(breath-hold T1- weighted GRE images in
clearly demonstrate the venous extension
phase and out of phase) it is possible to
of an adrenal cortical carcinoma.
characterize lipid-rich adenomas. These
adenomas contain intracellular lipid and Pheochromocytoma
water protons within the same imaging
voxel. On out-of-phase images, the signal Pheochromocytomas are neoplasms of the
from these protons cancel each other out adrenal medulla that produce catechola-
and result in signal loss when compared mines. Pheochromocytomas are extra-
with the in-phase images. Frequently, signal adrenal, bilateral, or malignant 10 percent
loss on opposed-phase imaging is obvious. of the time. Although patients may be
symptomatic, the symptoms are nonspecific,
Adrenal Cortical Carcinoma and include palpitations, headache, sweat-
Adrenal cortical carcinoma is a rare neo- ing, and hypertension. The MR imaging
plasm that most commonly occurs in the appearance of pheochromocytoma has
fourth to fifth decades.They are typically classically been described as markedly
large (>5 cm) at presentation, may contain hyperintense on T 2 -weighted sequen-
varying degrees of hemorrhage and necrosis, ces117,118 (Fig. 20.6). Subsequently, it has been
and often contain calcium.116 The most demonstrated that pheochromocytomas may
common hormone produced is cortisol, have variable signal on T 2 -weighted
which manifests as Cushing’s syndrome. The sequences, especially when they are greater
signal intensity of adrenal cortical carci- than 5 cm.118 MR imaging is more useful in
Body MR Imaging 285
A B
C
Figs 20.5A to C: Adrenal carcinoma
A. Axial T1W (FLASH) image showing, large mass lesion in right adrenal area with hyperintense areas inside
suggestive of hemorrhagic areas
B. Axial T2W image showing, heterogeneous appearance of the mass lesion
C. Coronal T2W image showing that mass is arising separate from the right kidney
A B
Figs 20.6A and B: Adrenal pheochromocytoma
A. Axial T1W (FLASH) image showing, isointense mass lesion in right adrenal area
B. Axial T2W image showing that mass is markedly hyperintense
can reliably distinguish these two entities gradient-echo images with fat suppression
because a pheochromocytoma enhances and are obtained in the axial plane before, at 20
an adrenal cyst or pseudocyst does not. seconds (arterial phase), and at 70 to 115
seconds (venous phase) after gadopentate
MR Imaging in the Evaluation dimeglumine injection (0.1 mmol/kg). Sagit-
of Bladder Cancer tal or coronal T2-weighted images may be
obtained, if the anteroposterior or infero-
Bladder cancer is a common tumor of the
superior extent of disease needs to be
urinary tract, accounting for 6 to 8 percent
evaluated. Sagittal and coronal gadolinium-
of male malignancies and 2 to 3 percent of
enhanced images are also recommended for
female malignancies.119 The most common
the evaluation of the bladder tumors, when
histologic cell type is transitional cell carci-
the tumor is located in the base or the dome
noma, accounting for 90 percent of all
of the bladder.
primary tumors of the urinary bladder.
Cross-sectional imaging aids in determining
Bladder Cancer
tumor stage, including detection of local
extension of the tumor, lymph node involve- Pathologically, 90 percent of the bladder
ment, and distant metastasis.120 Presently, tumors are epithelial, and 90 percent of the
magnetic resonance (MR) imaging is the epithelial tumors are transitional cell
modality of choice in imaging of urinary carcinomas. At the time of presentation, 30
bladder neoplasms, and staging accuracy of percent of patients have multifocal disease,
MR imaging in bladder cancer ranges from and there may be widespread areas of
73 to 96 percent. These values are 10 to 33 metaplasia and carcinoma in situ.119
percent higher than those obtained with Staging and Management of Bladder Cancer:
computed tomography (CT). Local extension of the tumor (T), lymph node
involvement (N), and distant metastasis (M)
Technique are the indicators used in staging. Treatment
Techniques of Bladder MR Imaging: Lack of options and prognosis depend on the clinical
bladder distension may limit the detection stage of the bladder tumor. It is critically
of small tumors secondary to detrusor important to differentiate between super-
muscle thickening. Overdistension of the ficial and invasive disease.119 Superficial
bladder may result in patient motion and tumors are treated with local endoscopic
can decrease sensitivity for plaque-like resection, with or without adjuvant
intravesical instillations of chemotherapeutic
lesions. Optimal bladder distension is
agents; while invasive tumors are treated
achieved by instructing the patient to void
by curative cystectomy and palliative
approximately 2 hours before imaging.121
chemo- and/or radiation-therapy.122
Currently, a 1.5 T MR scanner with a CT has been a valuable tool for the
phased-array pelvic coil is commonly used evaluation of bladder tumors, but MR
for imaging of the urinary bladder. The imaging has been shown to be superior in
typical urinary bladder protocol includes T1- the detection of superficial and multiple
weighted spin-echo images obtained in the tumors, and in staging accuracy in detecting
axial plane and T2-weighted fast spin-echo extravesical tumor extension and surround-
images also obtained in the axial plane. ing organ invasion, especially with dynamic
Subsequently, fast multiplanar spoiled contrast administration.123-129
288 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Morphologic features: Tumors can arise In the arterial phase of contrast enhance-
anywhere in the bladder, but they are most ment, bladder tumors enhance more than
commonly located in the lateral wall. When the muscle of the bladder.
a tumor is located around the ureteral Based on MR imaging features, it is not
orifices, it may produce partial or complete possible to differentiate between different
blockage of one or both ureters, resulting histologic cell types of bladder tumors
in hydroureter and hydronephrosis. 130
Tumors manifest a variety of patterns of MR Imaging of Prostate
growth, including papillary, sessile, infiltra-
ting, nodular, mixed, and fat intraepithelial Staging of Prostate Cancer
growth. Because the bladder does not have To date, the American Urologic Association
a distinct basement membrane, it is difficult staging system, based on that of Whitmore
to detect the invasion of the lamina propria and Jewett, is the one most widely used in
by imaging. 131 However, the detrussor North America.134,135
muscle can be well evaluated with MR
imaging; therefore, the depth of muscle Technique of Prostate MR Imaging
invasion can be assessed. The fat tissue
around the bladder can be seen clearly on MR imaging and MR spectroscopic imaging
MR imaging, and the interface between the allow a ‘‘one-stop shop’’ for detailed anato-
bladder wall and the surrounding fat can mic and metabolic evaluation of the prostate
be evaluated for extravesical tumor spread. gland. Neither TRUS nor CT can offer this
MR imaging features: Urinary carcinomas have simultaneous coverage and tissue detail. MR
intermediate signal intensity, equal to that spectroscopic imaging is a method of
of muscle on the T1-weighted images. T1- demonstrating normal and altered tissue
weighted images are used to assess metabolism, and is therefore fundamentally
perivesical fat invasion and lymph node different from other imaging modalities that
involvement. T2 -weighted images aid in only assess abnormalities of structure. MR
determining the depth of tumor infiltration imaging, and especially MR spectroscopic
into the bladder wall. On T 2 -weighted imaging, are relatively new technologies in
images, urine has high signal intensity, and continued evolution. T1-weighted MR imag-
the bladder wall appears hypointense. ing T1-weighted images of the pelvis aid in
Bladder tumors have the same, or slightly the detection of postbiopsy hemorrhage,
higher, signal intensity as the bladder wall lymphadenopathy, and bone metastases.
on T2 -weighted images. An intact, low- T2-weighted images in the axial and coronal
signal-intensity muscle layer at the base of planes are the cornerstone of MR imaging
the tumor is classified as stage Ta or T1; a in prostate-cancer evaluation. These images
disrupted low-signal-intensity muscle layer clearly depict the distinction between the
without infiltration of perivesical fat is peripheral zone and the central gland, allow
classified as stage T2b.132 the detection of tumor as low T 2 signal
Urinary bladder carcinomas and their intensity foci in the peripheral zone, and
metastases develop neovascularization133; facilitate staging by demonstration of
therefore, these tumors enhance earlier than extracapsular extension and seminal-vesicle
the normal bladder wall. invasion.136-139
Body MR Imaging 289
noise ratio with more signal averages coronal T 2 -weighted images are then
obtained in a shortened imaging period. obtained because they contain the most
The use of the endorectal surface coil information about the pelvic organs. A pulse
has been the subject of much interest for sequence such as fast or turbo spin-echo,
improving the resolution of MRI prostatic yields the highest resolution images. After
imaging. An improvement in staging all important T2 imaging is obtained, the
accuracy of 16 percent over body coil need for further imaging is variable. In the
technology is described in early experience. case of benign disease of the uterus, no
Criteria for capsular stage C disease were further imaging is necessary. When charac-
infiltration of periprostatic fat, obscuration terizing a pelvic mass or staging a pelvic
of periprostatic veins, involvement of the malignancy, dynamic, axial, or sagittal
neurovascular bundle, disruption of the images of the pelvis should be obtained at
capsule, and a bulge in the capsular contour 20 seconds, 40 seconds, and 3 to 5 minutes
defined as bulging in the glandular surface, following the administration of intravenous
making an angle with adjacent peripheral contrast medium. Fat saturation, although
zone.144-146 not essential, is useful for highlighting areas
of enhancement.
MR Imaging of Female Pelvis
Normal Anatomy
MR imaging is becoming an increasingly
important tool in the diagnosis of benign The uterus has three distinct zones: (1) the
and malignant disease of the female pelvis. high T2 signal endometrium, (2) the low T2
MR imaging is now routinely used in the signal junctional zone, and (3) the inter-
diagnostic work-up of infertility, including mediate T2 signal myometrium. The endo-
mullerian anomalies, and chronic pelvic pain. metrium, composed of high signal glands,
Both CT and MR imaging can be used to may measure upto 1.5 cm in thickness in
determine the origin of and to characterize women of menstrual age and 5 mm in
a pelvic mass. In obstetrics, MR imaging is postmenopausal women. The junctional zone
used to assess maternal complications of has been shown to consist of compacted
pregnancy and to identify or confirm fetal myometrial cells with a high nuclear to
anomalies. cytoplasmic ratio.147 The junctional zone
should measure no more than 11 mm in
MR Imaging Technique thickness.148 In women of menstrual age,
It is also advisable to have the patient void the ovaries should be identified in virtually
before the examination to limit deformation every case. They are ovoid structures
of adjacent organs by an enlarged bladder. containing high signal follicles. In postmeno-
Although imaging using the body coil is pausal women the ovaries are atrophied and
certainly adequate for most diagnoses, the identified in only 40 percent of cases.
highest resolution images are obtained using
Benign Conditions
a phased array. Saturation bands placed
along the anterior and posterior body wall Mullerian anomalies: Although often initially
fat are useful to diminish near field artifact. diagnosed using HSG, MR imaging should
Smaller field-of-view sagittal, axial, and be performed when surgical therapy is
Body MR Imaging 291
considered to characterize more accurately signal mass filling the uterine canal and
the anomaly. It is particularly important to markedly thinning the myometrium. Treat-
differentiate a septate from a bicornuate ment in the young woman often involves
uterus, each of which requires different the formation of a neovagina.
therapy149 (Figs 20.7A and B). Fibroids: Fibroids, or leiomyomata, are
Minimal failure of fusion leads to the present in an estimated 40 percent of women
arcuate uterus with a U-shaped internal over 40 years of age. Symptoms associated
contour of the fundal portion of the endo- with fibroids include bleeding, pain, and
metrial canal. This is an entirely benign urinary incontinence. Their presence can also
variant and has no effect on fertility. Failure contribute to infertility. MR imaging is of
of septal resorption is the cause of the value in the symptomatic patient when
septate uterus. In contradistinction to the uterine salvage therapy is considered. Using
bicornuate uterus, the external contour is axial and sagittal T2-weighted imaging, most
flat or minimally (<1 cm) concave. The fibroids, including pedunculated ones, can
septum may be of variable length and be identified with confidence. They should
contain fenestrations. This anomaly is often be described in terms of size, location, and
treated with hysteroscopic metroplasty. signal intensity. Any fibroid that impresses
Failure of development of the urogenital
on the endometrial canal is considered to
sinus leads to an absence of the caudal two-
have a submucosal component and can be a
thirds of the vagina and an obstructed cervix
source of bleeding. Based on T 2 signal,
and uterus. The differential diagnosis is
imperforate hymen. On axial T2-weighted fibroids can be classified further into groups
MR imaging of the pelvic floor, the vagina depending on their degree of cellularity.150
is absent. A small low T2 signal scar may be The most common group is the ordinary
present. Hematometrocolpos manifests as a fibroids, of relatively homogeneous low T2
heterogeneous, but predominantly high T2 signal and composed of collagenous material
Cervical cancer: Cervical cancer is usually a Over the past 10 to 15 years, magnetic
disease of women of menstrual age that resonance imaging (MRI) has had a major
plateaus at age 40. Any cervical mass that is impact on musculoskeletal imaging. Excellent
greater than 1.5 cm or presumed to extend soft-tissue contrast, spatial resolution, and
beyond the cervix should be examined using multiplanar imaging are among the major
MR imaging. CT scanning lacks the contrast advantages of MRI. Although noncontrast-
resolution On T2 -weighted MR imaging, enhanced MRI is sufficient for the evaluation
cervical cancer is usually hyperintense to of many musculoskeletal conditions, the
the dark cervical stroma. The preservation addition of either intravenous (IV) or intra-
of the black ring of the cervical stroma, no articular gadolinium-based contrast agents
matter how thin, virtually excludes para- is, at times, necessary to improve the
metrial extension. These patients are candi- evaluation of certain disease processes.163
dates for surgical cure (stage IIA). Those in
Bone Tumors
whom the black line is broken and a mass
extends beyond the expected confines of The superior contrast discrimination pro-
the cervix are usually treated primarily with vided by magnetic resonance (MR) imaging
294 Biomedical Magnetic Resonance: Proceedings of the International Workshop
has proved extremely valuable in the assess- of tumor necrosis and tumor response to
ment of bone and soft-tissue tumors.164-167 chemotherapy. For osteosarcoma, the
In the evaluation of tumors, a combi- absence of early enhancing nodules or the
nation of both T 1 and conventional T 2 presence of only one small early enhancing
weighting, fat-suppressed T2-weighted fast nodule suggests greater than 90 percent
spin-echo, or gradient-echo techniques is tumor necrosis, representing a good
essential. With short T1 inversion recovery response to the chemotherapy Masses with
(STIR) imaging sequences, T1 and T2 contrast early and persistent enhancement suggest a
are additive. 168,169 T 1 -weighted images poor response to chemotherapy in both
provide excellent contrast for identification osteosarcoma and Ewing’s sarcoma.
of marrow, cortical, and soft tissue involve- T 2 -weighted images are valuable in
ment. In particular, T1 -weighted images distinguishing muscle from tumor and can
allow differentiation between fat and tumor, increase diagnostic specificity in evaluating
as well as definition of muscle planes and marrow infiltration, seen as areas of low
separate anatomic compartments. signal intensity on T1-weighted images.
Gadolinium has been used to enhance Although each MR examination must be
contrast on T1-weighted images to better tailored to the patient and pathology in
question, certain general guidelines can be
characterize osseous and soft-tissue tumor
followed. The longitudinal extent of tumors
involvement.170-172 On gadolinium enhanced
can be seen on T 1 -weighted coronal or
images, regions of low signal intensity are
sagittal images. T1- and T2-weighted images
thought to represent areas of nonviable
(including fat-suppressed T2-weighted fast
tumor or necrosis. Gadolinium-enhanced spin-echo or STIR sequences) in the axial
images may also be useful for differentiating plane provide the most important images in
peritumoral edema from underlying tumor delineating the relationship of a tumor to
and recurrent tumor from scar or fibrosis.173 adjacent neurovascular structures and
Only the areas of solid tissue show compartments essential information in
significant enhancement. This information preoperative limb salvage planning. Prior
may be quite useful in directing biopsy to to imaging the particular region of interest,
the solid enhancing portions of the lesion a large field-of-view localizer using an
that harbor the diagnostic tissue, as opposed increased diameter surface coil or body coil
to the cystic, necrotic, or hemorrhagic may be necessary to exclude skin lesions or
nondiagnostic components. to accurately determine the proximal and
Dynamic-enhanced MRI: The response of distal extension of a large mass. For lesions
high grade osteosarcoma and Ewing’s of the appendicular skeleton, it is crucial
sarcoma to chemotherapy with dynamic that at least one long axis sequence be
enhanced MRI has been studied by several performed through the entire involved bone
investigators who proposed evaluation to evaluate for the presence of skip meta-
based on rather complicated schemes stases. Skip metastases represent a second
relating to factor analysis of dynamic MRI site of disease in the same bone as the
sequences174 and regions of interest.175-177 primary tumor separated by an area of
Shapeero and Vanel178 have proposed a less normal marrow. The presence of skip lesions
cumbersome method for using dynamic is very important to determine because they
contrast enhancement to evaluate the degree may significantly worsen patient prognosis
Body MR Imaging 295
and must be removed if limb salvage sur- account not only histologic appearance but
gery is to be successful. also radiologic assessment and clinical
behavior. Grade 0 lesions have a benign
General MR Appearances biologic behavior. They may have an
Direct multiplanar imaging facilitates indolent or aggressive course. It is important
improved preoperative and pretherapy to remember that, for these lesions, the
evaluation of the exact extent of a lesion. radiographic appearance may be more useful
It is often not possible to distinguish benign for determining biologic activity than the
from malignant lesions of bone and soft histologic grade.182 Grade 2 lesions are high-
tissue on the basis of MR signal charac- grade malignant neoplasms. Grade 1
teristics alone. Both benign and malignant tumors, classified as low-grade malignant
lesions may demonstrate areas of low signal neoplasms, have a biologic behavior in
intensity on T 1 -weighted images and between these two extremes.
increased signal intensity on T2-weighted
images (Figs 20.9A to D). Malignant lesions Site: The second component of the staging
tend to be more extensive, involving system is site. T0 lesions remain within their
marrow, cortical bone, and soft tissues; but capsule or pseudocapsule and involve only
these criteria do not always distinguish one compartment. Extension of the primary
benign from malignant lesions. No tumor or its reactive zone beyond the
correlations have been shown matching T1 pseudocapsule without extracompartmental
and T2 relaxation times with corresponding spread characterizes T1 lesions. These lesions
histopathology. 179 If the neurovascular are limited by anatomic barriers, such as
bundle is involved, the lesion is likely to be
articular cartilage, cortical bone, joint
malignant. A fluid-fluid level is a nonspecific
capsule, or tendons and ligaments. 182,183
finding in bone and soft-tissue lesions.
Extension of the reactive zone beyond the
Tumors with fluid-fluid levels include
fibrous dysplasia, simple bone cysts, compartment of origin also obligates a T2
malignant fibrous histiocytoma (MFH), designation because this area may often
osteosarcoma, aneurysmal bone cysts, contain satellite nodules or tumors projec-
hemangioma, and synovial sarcoma.180 tions.184 Neurovascular involvement also
defines a lesion as T2.
Staging: Proper staging of osseous neo-
plasms is essential for planning effective and Soft tissue mass: Although CT and MRI can
appropriate therapy. There are two widely both provide information regarding the
used staging systems for primary tumors of presence of soft tissue extension, most
osseous origin: the system developed by studies clearly indicate the superiority of
Enneking and the Musculoskeletal Tumor MRI in defining the precise extent of
Society in 1985, and that developed by the extraosseous disease.176,185 The presence of
American Joint Committee on Cancer. These a soft tissue mass indicates involvement of
systems are very similar and emphasize the a second compartment, which increases the
same basic prognostic factors.181 tumor stage and has significant implications
Tumor grade: The first component of the not only regarding staging but also therapy.
Enneking staging system is tumor grade. In Involvement of major neurovascular struc-
the Enneking system, grade takes into tures is critical information to the orthopedic
296 Biomedical Magnetic Resonance: Proceedings of the International Workshop
A B
C D
Figs 20.9A to D: Pelvic bone chondrosarcoma
A. AP Plain radiograph of pelvis showing large mass lesion involving the right sided pelvic bones associated
with large soft tissue component having chondroid type calcification
B. Coronal STIR image showing true extent of the lesion
C. Axial T1W image showing that mass is hypointense having intrapelvic component as well
D. Axial T2W image showing, heterogeneous appearance of the lesion
oncologist in surgical planning. A fat plane Metastases: The final component of the
of varying diameter surrounds virtually all Enneking staging system is the presence or
major neurovascular bundles, aiding in absence of distant metastases (M0 or M1).
radiologic determination of tumor involve- No differentiation is made between regional
ment. metastases, such as to regional lymph nodes,
Cortical involvement: Early cortical involve- and distant metastases, because they both
ment or localization of a lesion in the cortex carry the same prognosis. 182,183 Skip
is important to recognize. Subtle cortical metastases, as in the case of osteogenic
disruption suggests early extracompart- sarcoma (Figs 20.10A and B), also require a
mental extension and a more aggressive designation of M1 even without associated
lesion. distant metastases The crucial role that the
Body MR Imaging 297
A B
Figs 20.10A and B: Osteosarcoma of femur
A. AP and lateral radiograph of left femur showing, sclerotic lesion arising from distal metaphysis associated
with osseous matrix within the soft tissue component
B. Coronal STIR image showing true extent of the lesion, associated soft tissue component and the cortical
discontinuity
radiologist plays in the staging process, in reported rates of sensitivity and specificity
determination of both the extent of tumor for MR detection of osteomyelitis range from
involvement and the presence of metastases, 86 to 98 percent and 77 to 100 percent,
is obvious. respectively. 186 Standard sequences may
Musculoskeletal infection: The initial imaging include a combination of T1-weighted, T2-
of osteomyelitis typically consists of conven- weighted, and/or short T 1 inversion
tional radiographs; however, this method recovery (STIR) or T2-weighted sequences
is insensitive and may take up to 10 days to with chemical fat suppression. T1-weighted
reveal an abnormality. Radionuclide studies images are generally best for evaluating
can be helpful, but are often nonspecific, marrow infection. T1-weighted sequences
and cannot differentiate neoplasm, infection, will show infection as intermediate or low
and trauma reliably. Non-contrast enhanced signal within the bone marrow. Infection is
MRI is a cost-effective, sensitive, and specific high in signal on T2 -weighted and STIR
study for detecting osteomyelitis. The images.186 Chronic and acute osteomyelitis
298 Biomedical Magnetic Resonance: Proceedings of the International Workshop
lesion contrast than that seen on corres- hyperintensity on T2-weighted images have
ponding conventional T2-weighted images. been found to correlate with metastatic
disease. The halo sign, most commonly seen
Metastatic Disease of the Marrow in osteoblastic metastases from prostate
Marrow invasion occurs by hematogenous cancer, is thought to occur secondary to
dissemination, and the high frequency of trabecular destruction with a resultant fluid-
metastases to pelvic bones and vertebrae is filled gap.201
attributed to the abundant vascular supply
Whole-body MR Imaging
afforded by the vertebral venous plexus,
which serves as a major venous pathway.198 Whole-body imaging can be afforded by
Metastatic disease has been successfully radiographic skeletal survey, whole-body
detected on MR T 1 -weighted and STIR CT, scintigraphy and, more recently, by PET.
images, even when bone scans are negative Each technique has advantages and
or equivocal. MR imaging provides an disadvantages, which include cost, sensiti-
excellent means of assessing soft tissue vity, radiation dose and acquisition time. A
involvement, including adenopathy, and recent modification is the, whole-body
Gd-DTPA enhancement is useful in the turbo-STIR or TRUE-FISP MRI technique, a
assessment of pathologic fractures and powerful screening tool.
epidural spread. Metastatic lesions may
Technique
show isointensity with adjacent vertebral
body marrow on non-fat-suppressed Whole body imaging can be performed
contrast enhanced images.199 The morpho- either by using TRUE-FISP or by turbo-STIR
logy of the posterior vertebral body cortex technique. For the MR examination, the
should be evaluated for convexity, a sign patient is positioned on the rolling table
associated with metastatic tumor extension. platform. The body phased-array coil is
A chronic, benign compression fracture placed at the region of the largest diameter
frequently demonstrates fat marrow signal of the patient with manual movement of
the rolling table platform. Then the surface
intensity (i e, bright on T1-weighted images,
coil is positioned in the isocenter of the
dark on STIR images). In more acute
magnet by using the automatic movement
compression fractures, subchondral reaction
of the original patient table. During the
often parallels the endplate without the more examination, there is no further movement
central or heterogeneous involvement seen of the patient table.202
in metastatic disease. The “bull’s-eye sign” Coronal images are acquired at seven
(a focus of high signal intensity in the center consecutive stations by sequential table-top
of a lower signal intensity osseous marrow movement. Axial images are acquired in a
abnormality, seen on T1-weighted images) similar way by sequential table-top move-
as a specific indicator of normal hemato- ment through up to seven stations. Sagittal
poietic marrow and not metastatic dis- images are also acquired for the spine.
ease. 200 However, both the halo sign (a Sagittal T1-weighted images are acquired to
peripheral rim of increased signal intensity facilitate detection of sclerotic metastases,
around an osseous lesion seen on T 2 - which are often poorly visualized on STIR
weighted images) and diffuse signal weighted scans.
Body MR Imaging 301
evaluate for associated abnormalities and Ultrasound and MRI can be similarly
underlying etiologies that are not as readily complementary in evaluation of sacrococ-
depicted with US. Agenesis of the corpus cygeal teratoma. Ultrasound, with its fine
callosum, in particular, which is commonly spatial resolution, often shows the details
associated with ventriculomegaly, can be a of the association of the lesion with the
difficult second-trimester diagnosis with US caudal aspect of the fetal spine and can also
but is often readily depicted with MRI. MRI show its cystic and solid nature.
has been found to be helpful in evaluating Whether MRI adds value in the routine
posterior fossa cystic lesions and in management of neural tube defects (NTDs)
distinguishing between the Dandy-Walker has been argued. MRI may be more accurate
malformation and variant, posterior fossa than US in the setting of NTDs in the
arachnoid cyst, and mega cisterna magna, presence of maternal obesity. MRI can detect
holoprosencephaly and encephaloceles (Fig. subtle NTDs when US shows only the
20.11).211,213-215 characteristic associated Chiari II malfor-
The fetal scalp, neck, and airway can be mation and fails to show the spinal lesion.
difficult to evaluate fully with US because When in utero surgery is not a consideration,
of artifact related to adjacent bony it is yet established whether MRI changes
structures as well as fetal position. MRI can diagnosis or management in NTD.
be effective in evaluating abnormalities
related to these structures and can provide Prenatal MRI of the Fetal Body
information complementary to US. The fetal After the CNS, the fetal thorax has received
airway is fluid-filled and, therefore, discre- the most attention by those performing fetal
tely bright on T2-weighted images. MRI. Its role has been adjunctive to US,
helping to define the anatomy of large
masses, examine lesions with an atypical US
appearance, and aid in the prognosis of
CDH. Its emerging impact on fetal manage-
ment and counseling for chest masses is
approaching its use for CNS abnorma-
lities.212,216
Congenital chest masses have charac-
teristic, though overlapping, MRI appea-
rances. The appearance of CCAM varies
depending on whether the lesions are
microcystic or macrocystic. The lesions can
have multiple large cysts with discrete walls
or can be solid lesions with scattered small
cysts. Congenital cystic adenomatoid
malformations tend to be higher in T 2-
weighted signal intensity than normal lungs.
Fig. 20.11: Fetal encephalocele. HASTE (T2W) image Bronchopulmonary sequestration can have
showing herniation of brain material to the outside, an appearance similar to CCAM. Indeed,
in occipital region of the fetus combined lesions are common. Pulmonary
304 Biomedical Magnetic Resonance: Proceedings of the International Workshop
sequestration, found predominately in the 6. Hamm B, Thoeni RF, Gould RG et al. Focal
left lower lobe, often appears as a bright liver lesions: characterization with nonenhanced
and dynamic contrast material-enhanced MR
echogenic mass on US and as a well- imaging. Radiology 1994;190:417–23.
demarcated, wedge-shaped region of high 7. Wang C, Ahlstrom H, Ekholm S et al.
signal intensity on T2-weighted MRI. Diagnostic efficacy of MnDPDP in MR imaging
MRI may be particularly useful in the of the liver. A phase III multicentre study. Acta
assessment of pregnancies complicated by Radiol 1997;38:643–9.
8. Vogl TJ, Hammerstingl R, Schwarz W et al.
oligohydramnios, which can limit the
Magnetic resonance imaging of focal liver
diagnostic sensitivity of US. Poutamo et al, lesions. Comparison of the superparamagnetic
performed MRI on 24 fetuses with oligo- iron oxide resovist versus gadolinium-DTPA in
hydramnios or genitourinary anomalies the same patient. Invest Radiol 1996;31:696–
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with superparamagnetic iron oxide in MR
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21
Applications of MR Spectroscopy in
Neuromuscular Diseases
Uma Sharma, NR Jagannathan
carnitine acyl transferase, present on the The α-keto acid thus formed enters TCA
outer surface of the inner membrane of cycle to undergo oxidation and yields CO2
mitochondria. Fatty acid oxidation occurs and H2O. The α-KG is the common acceptor
through two main stages. In the first stage of amino groups from most of the amino
fatty acids undergo oxidative removal of acids. During fasting and in diabetes muscle
successive 2-carbon units through a set of degrades 3-5 fold more branched chain
enzymatically catalyzed reactions in the form amino acids than in normal condition. Ala
of acetyl Co. A. This process starting from yields Pyr directly on transamination with
the carboxyl end of the fatty acid chain is α-KG which enters citric acid cycle through
repeated several times according to the fatty the formation of acetyl Co. A. Threonine
acid chain length. In the second stage, acetyl (Thr), glycine (Gly), serine (Ser) and cysteine
Co. A derived from fatty acid oxidation are
(Cys) are metabolized through the formation
oxidized to CO2 and H2O through citric acid
of Pyr followed by acetyl Co. A. Part of the
cycle in a similar manner as acetyl Co. A
carbon backbone of the amino acids
derived from oxidation of Pyr. Long chain
phenylalanine (Phe), tyrosine (Tyr), Lys,
even and odd number fatty acids both are
oxidized in a similar manner. However in tryptophan (Trp) and leucine (Leu) yield
odd chain amino acid the products of the acetoacetyl Co.A, which is then converted
last pass through of oxidation are propionyl into acetyl Co. A. Arginine (Arg), histidine
Co. A and acetyl Co. A. Propionyl Co. A is (His), Glu, Gln and proline enter the citric
converted to succinyl Co. A through a set acid cycle via α-KG. The carbon skeletons
of reactions which is then oxidized through of methionine, isoleucine (Ile) and valine
citric acid cycle. (Val) are utilized by pathways which yield
succinyl Co. A, an intermediate of Krebs
Amino Acids Metabolism cycle.
During metabolism of amino acids
Amino acids are the building blocks for the
biosynthesis of proteins. However, during ammonia is produced in muscle. Ala plays
fasting or in diabetes when carbohydrates a special role in transporting ammonia to
either unavailable or not properly utilized the liver in a nontoxic form through glucose-
or if in excess of the body’s need for protein alanine cycle. In this cycle ammonia is
synthesis, they undergo oxidative degrada- converted into the amino group of Glu by
tion. In these circumstances the α-amino the action of glutamate dehydrogenase. The
groups of amino acids are enzymatically Glu so formed now transfers its α-amino
transferred from the amino acid to the α- group to Pyr, a readily available product of
carbon atom of α-KG, leaving behind the muscle glycolysis, by the action of alanine
corresponding α-keto acid analog of the transaminase. The Ala so formed escapes
incoming amino acid and in turn amination into blood and is carried to the liver. Here
of the α-KG occurs and is converted into
again through transamination reaction Ala
glutamate (Glu). The reaction is catalyzed
transfers its amino group to α-KG to yield
by enzymes called transaminases or
Glu which then undergoes deamination to
aminotransferases as given below.
again yield α-KG and ammonia. The
L-α-amino acid + α-KG α-keto ammonia so released is converted by the
acid + L-Glu liver into urea and excreted.
Applications of MR Spectroscopy in Neuromuscular Diseases 317
IN VIVO MRS
31P MRS
In vivo phosphorus magnetic resonance
spectroscopy (31P MRS) has been the method
of choice for repetitive, noninvasive dynamic
studies of muscle energy metabolism in
normal and diseased state. Muscle
metabolism has been extensively
31
investigated using P NMR spectroscopy
and several review articles4,16,17 have been
published. Skeletal muscle has the capacity
of changing the rate of metabolism at will
just by exercising at different levels of work.
Information is obtained by measuring the
relevant phosphorylated compounds in Fig. 21.1: 31P in vivo MR spectrum of calf muscle of a
different conditions, i.e. at rest, during normal volunteer using a surface coil with a repetition
time of 3 s (Reproduced with permission from
controlled work and during the subsequent
reference 38)
phase of recovery. One of the major
advantages of 31P NMR is the measurement
of few phosphorus containing metabolites with alteration in pH in muscle tissue and
that offers information on the functionality therefore provides information about the
and regulation of the central metabolic intracellular pH. The peaks due to phospho-
pathways of phosphate which are vital to monoesters (PME) and phosphodiesters
the energy status of the cell. Investigation (PDE) are also often observed in 31P MR
protocols of skeletal muscle therefore in spectra.
general include acquisition of spectrum at Several review articles have been
rest, during exercise and during recovery published documenting the efficacy of 31P
after contraction. MR spectroscopy in the study of muscle
Figure 21.1 shows the 31P MR spectra metabolism in several neuromuscular
recorded from the calf muscle of a normal disease.16,17 In vivo 31P MRS has been used
volunteer in resting state which is simple in the diagnosis and monitoring of muscle
with only few resonances and is easy to metabolism in mitochondrial disease.18,19
comprehend. This is due to the fact that in Several reports exist in the literature in
in vivo MRS only metabolites that are free which large number of patients with mito-
or rapidly exchanging are observed in the chondrial diseases have been studied.7,20-22
spectra. The five major peaks observed in About 35 to 85 percent of patients with
the spectra corresponds to α-, β- and γ mitochondrial encephalopathies have been
phosphates of ATP, phosphocreatine (PCr) reported to have abnormally low PCr/Pi at
and inorganic phosphate (Pi). The area under rest. Under aerobic condition, patients with
each peak is directly proportional to the mitochondrial disease show a more rapid
concentration of the metabolite. The spectral fall in muscle energy state per unit work
distance between Pi and PCr peaks varies than do normal controls consistent with the
318 Biomedical Magnetic Resonance: Proceedings of the International Workshop
limited oxidative metabolism. The most processes like glycolysis and lipid meta-
sensitive variable for mitochondrial bolism in muscle.31-33 Recently Boesch et al
abnormality during exercise was the change reviewed the use of 1H MRS in the muscle
in PCr level.21 tissue.34 Figure 21.2 shows the in vivo proton
Some mitochondrial disorders have lactic MRS from the calf muscle of a normal
acid accumulation and low serum pH. While volunteer recorded in our laboratory. In this
in some a relative resistance to intracellular spectrum various resonances due to residual
acidosis has been observed in exercising water (at 4.7 ppm), terminal methyl of lipids
muscle of patients with mitochondrial (0.8-1.0 ppm), acyl chain methylene [-(CH2)
diseases.7,20,21 The mechanism of this pheno- n-: 1.1 – 1.6 ppm], α- and β- methylene of
menon may be related to adaptation of the lipid chain (2-2.8 ppm) and the oleifinic
intracellular buffering systems (including protons of the polyunsaturated fatty acids
proton extrusion mechanism) in response to (-HC=CH-: 5.5 ppm) are observed. In
chronic overproduction of Lac. 31 P MR addition, the resonances due to N-methyl
studies of the recovery rates of PCr provide protons of Cr and PCr resonate at 3.03 ppm,
valuable quantitative indices of mitochon- while the N-CH3 of carnitine (Car) and Cho
drial dysfunction. The initial rate of recovery containing compounds resonate at 3.22 ppm.
can provide a measure of maximal oxidative Peaks at 7.1 and 8.1 ppm are due to the C-
rate in the tissues and thus a sensitive and 4 and C-2 ring proton of histidyl moiety in
reliable index of mitochondrial dysfunction carnosine.
in in vivo.23 Approximately 90 percent of
patients with mitochondrial myopathies
showed abnormal recovery kinetics.
A high muscle pH at rest has been
observed in patients with muscle dystro-
phies. 24-27 In patients with limb girdle
muscular dystrophy (LGMD), the degree of
fat replacement in calf muscle correlated
inversely with cytosolic pH and directly
with PCr/ATP.26 In patients with Becker
muscular dystrophy (BMD) an early drop
in the level of PCr or PCr/Pi ratio and
reduced acidosis was reported. 25,28 A
reduced glycolytic or glycogenolytic activity
has been assumed as the cause of reduced
acidosis but the cause of defect in glucose
metabolism common to dystrophies and
myotonic dystrophy, remains unclear.25,29,30 Fig. 21.2: 1H in vivo MR spectrum using STEAM pulse
sequence from calf muscle of a normal volunteer
1H MRS observed at an echo time (TE) of 135 ms with
repetition delay of 3s. The voxel size was 20×20×20
Volume localized proton MRS have been mm3 (Reproduced with permission from reference
used by several investigators to study the 38)
Applications of MR Spectroscopy in Neuromuscular Diseases 319
Schick et al have shown that the spectra and in the following section some details
from muscle tissue exhibit two compartments are presented from our study.
of lipid signals with a difference of 0.2 ppm
in their resonance frequencies.33 These two Human Muscle Biopsy
compartments arise from intramyocellular Muscle specimens from patients under
(IMCL) and extramyocellular (EMCL) regional anesthesia were obtained from the
compartments of lipids. In vivo 1H MRS was vastus lateralis muscle of the quadriceps in
used in the study of polio patients by our thigh. A cylindrical piece of muscle meas-
group.35 The spectra were obtained at a long uring 2-3 cm in length was taken and was
echo time of 270 ms to characterize clearly divided into four parts. For NMR studies
the IMCL and EMCL components of the one piece was quickly frozen and stored in
lipid. The study revealed that mildly liquid nitrogen until the perchloric acid
paralyzed patients are comparable with extraction procedure was carried out. Other
control subjects in relation to the presence pieces were used for diagnosis of disease
of IMCL while moderate and severely using histological, enzyme histochemical and
paralyzed patients were comparable in immunohistochemical studies.
relation to the absence of IMCL. In addition, Muscular dystrophy was diagnosed
there is reduction or complete absence of based on histopathological characteristics
Cr, Car and Cho metabolites in severely such as variation in fiber size, ring and
paralyzed patients. whorled fibers, more fiber splitting, degene-
ration and necrosis along with the presence
IN VITRO NMR SPECTROSCOPY OF of lobulated fibers in some cases. Enzyme
MUSCLE TISSUE histochemistry showed that the good fiber
In vitro MR spectroscopy plays an important differentiation was present in 46 percent
role in understanding the biochemical cases. Muscle dystrophies wherein immuno-
processes and pathogenesis of the muscle histochemistry were normal for dystrophin
weakness observed in different neuro- 1,2 and 3 as well as normal for sarcoglycans
muscular diseases. We, in our laboratory, (α, β, γ and δ), merosin and emerin were
are involved in the study of muscular then diagnosed as LGMD in conjunction
dystrophies like LGMD, DMD, BMD and with clinical features (i.e. muscle involved).
metabolic myopathies such as mitochondrial
myopathy (MM). Identification of meta- Perchloric Acid Extraction
bolically pertinent substances from the The PCA extracts from muscle specimens of
perchloric acid (PCA) extracts of human patients with LGMD (n = 11), DMD (n = 9),
skeletal muscle tissue of patients in the BMD (n = 4), MM (n = 5), WNHL (n = 11)
above neuromuscular diseases and normal and normal subjects (controls) who came
controls were carried out using 1- and 2-D for orthopedic surgery (knee and hip
proton and heteronuclear NMR techni- replacement) and who did not show any
ques.36-38 In addition, the concentration of muscle disease were prepared as described
metabolites in these groups of patients were by Payen et al.39 The wet weight of muscle
calculated and compared with the controls tissue taken was in the range of 250 - 400
and among different neuromuscular diseases mg. The frozen biopsy specimen was
320 Biomedical Magnetic Resonance: Proceedings of the International Workshop
pulverized in a liquid nitrogen chilled muscle tissue extracts are assigned to specific
mortar and pestled to a fine powder. The metabolites using 2D DQF COSY, TOCSY,
tissue was homogenized with 0.6 N of PCA spin multiplicities, relative peak intensities.
(10 ml/gm wet weight). After centrifugation In addition, the chemical shifts of meta-
at 10,000 rpm for 20 min at 4°C, the pH of bolites were compared with that reported
the supernatant was adjusted between 7-8 in literature in muscle tissue extracts40 and
with potassium hydroxide using pH strip. also with other tissue extracts9-12 wherever
The precipitate of KClO4 was removed by applicable. The assignments of compounds
centrifugation at 10,000 rpm for 20 min at like Car, Cr and Cho containing compounds
4°C and the resulting supernatant was were made by comparing their chemical
lyophilized. The lyophilized powder was shifts with those of standard compounds.
then dissolved in D2O solvent to carry out Figures 21.3 A to D show the representative
NMR experiments. 1D 1H NMR spectrum of the perchloric acid
extract of the muscle tissue of a DMD patient
NMR Spectroscopy Study
while Figure 21.4 shows the TOCSY
NMR experiments were carried out at DRX- spectrum. In all, about 37 compounds could
400 MHz (Bruker, Switzerland) spectrometer be identified in the muscle tissue spectra of
equipped with 5 mm multinuclear broad DMD36, LGMD patients37 and MM patients.
band inverse probe at 298 K. Sodium tri- However, in some DMD patients 56 meta-
methyl- silyl –[2,2,3,3-H4] propionate (TSP) bolites could be identified and similarly in
was added as an internal standard for few of the LGMD samples 44 metabolites
chemical shift and quantification of concen- could be identified unambiguously. Few
tration. Two-dimensional double quantum resonances still remain to be assigned.36,37
filtered (2D-DQF) COSY and Total Corre- A total of fourteen amino acids were
lation Spectroscopy (2D-TOCSY) experiment identified unambiguously in the spectra of
were carried out using the standard para- patients of most neuromuscular diseases.
meters. In addition, 1H-13C heteronuclear Resonances of many organic acids such as
multiple quantum coherence (HMQC)
Lac, Ace, Succ, α-KG and sugars such as
experiments were also recorded using
Glc were also observed in the spectra of all
standard parameters for confirmation of
the muscle diseases studied. The resonances
assignments.
due to glycerophosphoryl choline (GPC),
Quantification of Metabolites Cho, phosphoethanol amine (PE), phos-
phoryl choline (PC) were also present in all
The concentration of metabolites was the diseases and controls. The N (CH3) and
determined by comparing metabolite fit CH2 protons of Cr and PCr resonate at 3.02
integral with the fit integral of TSP as ppm and 3.93 ppm, respectively. The
described earlier.11 The significance of the N(CH3) of Car appear at 3.23 ppm along
various concentration values was analyzed with N(CH3) of GPC and PC (Fig. 21.3B).
using student ‘t’ test. Probability values of The CH protons of CHOH group of Car at
(5 %) were recorded as significant (p<0.05).
4.56 ppm showed connectivity with its CH2
protons at 2.45 and 3.45 ppm, respectively.
Metabolite Assignments
Resonances of H4, H5 and H6 protons of
Various proton resonances in the spectra of indoxyl sulphate were also observed in
Applications of MR Spectroscopy in Neuromuscular Diseases 321
Fig. 21.3A: 400 MHz 1D proton NMR spectrum FIG. 21.3B: 400 MHz proton NMR spectrum showing
showing aliphatic region (expanded from 0.5 to 3.0 downfield region (expanded from 2.9 - 4.7 ppm ) of
ppm) of the perchloric acid extract of skeletal muscle perchloric acid extract of skeletal muscle tissue from
tissue of a DMD patient in D2O at 298 K (Reproduced DMD patient in D 2O at 298 K (Reproduced with
with permission from reference 38) permission from reference 38)
Fig. 21.3C: 400 MHz 1D proton NMR spectrum Fig. 21.3D: 400 MHz 1D proton NMR spectrum
showing aliphatic region (expanded from 5.1 to 6.6 showing aliphatic region (expanded from 6.4 to 9.1
ppm) of the perchloric acid extract of skeletal muscle ppm) of the perchloric acid extract of skeletal muscle
tissue of DMD patient in D2O at 298 K. The pH of the tissue of DMD patient in D2O at 298 K (Reproduced
sample was in the range of 6 to 7 (Reproduced with with permission from reference 38)
permission from reference 38)
Fig. 21.4: Phase-sensitive two-dimensional total correlation spectrum (2D-TOCSY) (expanded region) of
perchloric acid extract of skeletal muscle tissue from DMD patient at 298 K in D2O (Reproduced with permission
from reference 38)
Table 21.1: The concentration (Mean ± SD, mM/Kg wet weight) of metabolites estimated in the muscle
tissue extracts of normal subjects and patients with neuromuscular diseases
Controls 2.3±1.4 24.6±13.6 4.2±2.4 12.6±6.7 0.9±0.3 2.5±2.4 26.4±10.8 2.4±2.1 3.5±2.8
LGMD 0.7±0.2 12.8±4.9 1.3±0.7 5.3±2.8 0.4±0.3 1.04±0.7 24.2±9.6 1.3±0.7 3.8±2.3
DMD 0.6±0.3 4.7±2.7 1.0±0.6 1.4±0.7 0.3±0.2 0.5±0.3 3.4±2.6 1.6±1.1 0.9±0.6
BMD 2.5±0.7 21.1±0.7 4.1±0.6 16.8±3.2 1.0±0.7 4.1±3.3 19.5±6.4 2.9±1.9 1.5±1.1
MM 3.8±1.5 41.7±14.7 6.1±1.5 22±5.4 1.3±0.6 4.8±0.5 27.5±9.8 1.8±0.8 1.5±1.0
WNHL 3.9±0.2 54.2±19.6 6.2±1.8 19.4±7.8 1.2±0.8 5.1±2.8 25.6±14.3 2.3±1.0 1.9±0.8
P-values
Control vs. LGMD 0.02 0.04 0.009 0.02 0.01 0.1 0.4 0.1 0.3
Control vs. DMD 0.008 0.002 0.005 0.004 0.0006 0.03 0.005 0.2 0.01
Control vs. MM 0.03 0.03 0.05 0.03 0.05 0.03 0.1 0.1 0.1
Control vs. WNHL 0.04 0.003 0.05 0.04 0.05 0.03 0.1 0.1 0.1
these may be used as energy source sparing on the composition of phospholipid as well
muscle protein. 44 In our study we also as the relative proportions of different
observed Ace, propionate, 3- hydroxy phospholipids and the ratio of phospholipids
butyric acid (3-HB) and Succ compounds in to cholesterol. Any alteration in the compo-
the muscle tissue extracts indicating the use sition of phospholipids might profoundly
of fatty acids for energy generation, affect the cellular behavior as these are
however, the concentration of acetate was important structural components of mem-
significantly less which may be the branes and also play an important role in
consequence of decreased Car concentration regulating the activities of membrane bound
in DMD patients. enzymes. 59 Thus, several biochemical
processes appear to play an important role
Gln was also significantly reduced in
in the decrease of glucose concentration
LGMD and DMD patients. Gln is a major
observed in DMD patients.
gluconeogenic precursor,53,54 serves as a fuel
for tissues with a high cell turn over rate,
Skeletal Muscle Metabolism in
and play a pivotal role in the regulation of Mitochondrial Myopathy
protein synthesis.55 In healthy adult human
subjects, increase in the plasma Gln The morphological abnormalities are
concentration was shown to stimulate observed in mitochondrial myopathies but
protein synthesis56 and with experimental changes in mitochondrial structure are not
decrease protein wasting was reported.57 It disease specific and the presence of such
may be hypothesized that the decreased changes in muscle biopsy does not
necessarily indicate impaired mitochondrial
concentration of Gln, besides resulting in
metabolism. Diagnosis of muscle myopathies
decreased Glc concentration, may also be
is often challenging and usually requires
the underlying reason for protein wasting
muscle biopsy for morphologic and
and protein breakdown in the muscle of
biochemical characterization of mitochon-
LGMD and DMD patients.
drial abnormalities. In our study, eleven
Another interesting observation of our patients were suspected to have mito-
study is the decreased concentrations of the chondrial myopathy according to clinical
peak at 3.23 ppm due to GPC/phosphoryl symptoms, but the histological, histochemical
choline (PC)/Car and the peak at 3.20 ppm and electron microscopy techniques per-
due to choline in DMD patients. PC is the formed on muscle biopsies were inconclusive
precursor of membrane phospholipid and no proper diagnosis of myopathy could
lysolecithin and is formed in the cell from be made, these were categorized as WNHL
choline by the activity of enzyme choline (histopathology within normal histological
kinase. While GPC is the catalytic product limits) patients.42 Estimation of the meta-
of lysolecithin which again gets converted bolite levels in patients with proven mito-
into PC by phospholipase activity. Burt chondrial myopathy was carried out and
et al have suggested that water soluble compared with WNHL patients and controls
metabolite GPC regulates phospholipid in order to understand the biochemical basis
composition by inhibiting the enzyme of mitochondrial myopathy and to evaluate
lysolecithinase, of which it is a catalytic the utility of in vitro NMR in distinguishing
product.58 It is known that the properties mitochondrial myopathies from control
of membranes are fundamentally dependent group.42
326 Biomedical Magnetic Resonance: Proceedings of the International Workshop
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330 Biomedical Magnetic Resonance: Proceedings of the International Workshop
22
EPR Imaging for
Biomedical Applications
R Murugesan, N Devasahayam, K Matsumoto,
S Subramanian, JB Mitchell, Murali C Krishna
in the presence of the gradient. EPR spectra, maximum gradient of approximately 50 mT/
collected in a polar grid under the presence m in the three axes.
of static gradients, are then used to generate
Field Modulation Super imposition of a low
the EPR images by employing the projection
frequency sinusoidal modulation on the
reconstruction (PR) method. The schematics
Zeeman field for phase sensitive detection,
shown in Figure 22.1 gives the general layout
required for the CW mode of spectral
of a CW EPRI scanner that operates at 300
acquisition is provided by a set of additional
MHz.14 There are three major modules, the
Helmholtz coils, incorporated within the
magnet-gradient coil assembly, the radio
gradient coil system. The center of the
frequency signal detection bridge, and the
modulation field, origin of the gradients,
data acquisition system. A brief description
and the center of the Zeeman field are
of these modules in our CWEPRI instrument
coincident and require no electrical offset
is as follows.
corrections. The modulation coils are
powered using a custom-built amplifier that
Description of RF CW EPRI Scanner is capable of delivering a modulation of up
to 5.0 G and limited to a frequency of 12.5
Module 1
kHz.
Magnet At 300 MHz, for a free electron
Field Sweep Magnetic field sweep is achieved
system, the magnetic field B0 at resonance
using an IEEE-488 bus by setting the field
is 10.6 mT. This field is provided by a set
to a given value and incrementing the
of air-core, water-cooled coils in a
current as a function of time. However, the
Helmholtz configuration. The coils enclose
number of spectral points in a sweep is
a cylindrical volume of 30 cm diameter and
limited because, for each field increment, a
34 cm length. The magnet current is
‘handshake’ period of 10 ms is required for
provided by a DC power supply capable of
the computer to communicate with the
providing 30 A current at 12 V. Long term
power supply through the IEEE bus. During
current stability of this power supply is
this time, the computer is also tied up.
critical for artifact free imaging. The active
Therefore, either a slow scan with more
volume in the magnetic field is a 3-cm
points for increased spectral resolution, or
diameter spherical volume with field
a fast scan with fewer number of points,
homogeneity of ± 200 ppm, reasonable for
for increased temporal resolution may be
small animal imaging.
opted. However, to achieve rapid field
Gradient The imaging gradient system sweep with minimal spectral distortion, the
consists of a set of three mutually orthogonal magnet power supply is modified to include
gradient coils Gx, Gy, and Gz. A Maxwell a sweep controller which enables all the
pair of coils produces the Gz gradient while sweep instructions to be addressed by the
two pairs of saddle coils orthogonal to each PC, only once, after which the field sweep
other provide the Gx and Gy gradients. The can be set to proceed independently of the
gradient linearity is better than 1 percent computer. With this setup, spectral sweeps
over 3 cm DSV. These coils are powered by of up to 50 G can be performed in 4 s
a set of three digitally controlled bipolar without any detectable spectral distortions.
power amplifiers, capable of generating a For example, a 5–8 G sweep that is required
EPR Imaging for Biomedical Applications 333
for small animal imaging using narrow single (AFC) feature is highly desirable. In addition
line spin probes could be accomplished in 4 to animal motion, thermal drifts of the
s when 256 points are used for digitizing resonator frequency and RF source
the EPR spectrum (projection). Thus, a two- instabilities also affect the bridge balance.
dimensional image data collection with 16 The AFC circuit in our CW EPRI scanner,
projections (~256 points each, 4 s per scan) (Fig. 22.1) uses a low level FM modulation
can be completed in about 1 min and a three of the RF source, which is converted to AM
dimensional image with 81 projections (~256 modulation, and after phase-sensitive
points each, 4 s per scan) can be collected in detection, produces an error signal to adjust
less than 6 min. the RF source. During in vivo EPR
measurements, the animal motion can affect
Resonator An imaging coil must resonate, or
also the resonator coupling. Hence, automatic
efficiently store energy, at the Larmor
coupling control (ACC) is needed to further
frequency. Imaging coils are composed of
improve the quality of projections. In an ideal
an inductor, or inductive elements, and a
situation, signals from both AFC and coupling
set of capacitive elements. The resonant
changes need to be separated from the true
frequency of an RF coil is determined by
EPR signal, and used to provide the feedback
the inductance (L) and capacitance (C) of
loop error signals for appropriate dynamic
the inductor capacitor circuit. We have
corrections. Similarly, implementation of
developed suitable resonators known as
automatic phase control (APC) can enhance
parallel coil resonators [PCR] for RF in vivo
the SNR, because the phase error can have a
EPR imaging for both the CW and time
marked effect on the noise level. Hence APC
domain modalities.13 The resonators are 25
can particularly improve the consistency of
mm in diameter, and either 25 or 50 mm in
the projections of large size objects in lengthy
length. Schematics of the PCR is presented
3D data acquisition schemes.
in Figure 22.2, Plate 12.
Module 2 Module 3
RF Detection Bridge The performance of the Data Acquisition The data acquisition system
EPR imager critically depends on the (DAS) is built around a commercially
functioning of the RF bridge, which acts as available lock-in amplifier and a PC to
a precision reflectometer. The resonator is control the spectrometer/imager. The PC,
loaded with the sample and tuned to the via the IEEE bus addresses all operations
resonance frequency of 300 MHz and the of the spectrometer such as: setting up the
coupling is adjusted to ‘‘critical’’ value. The number of data points, modulation
‘‘balanced’’ bridge condition is perturbed frequency and amplitude, signal
by the EPR resonance absorption. A amplification level, detector reference phase,
sinusoidal modulation of the resonance center field, sweep width, sweep time, the
frequency and subsequent phase sensitive number of gradient steps, the gradient
detection produces the EPR signal. amplitude and orientation, etc. Once these
CW EPRI method is sensitive to parameters are specified, the software
physiological motion of the animal and hence calculates the gradient amplitudes to be set
an efficient automatic frequency control on the x, y, and z axes. Based on these
334 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 22.1: Schematic diagram of 300 MHz EPR imaging system showing the individual submodules consisting
of magnet, gradient coil system, analog sweep unit, modulation unit, RF bridge, RF synthesizer, SR850lock-
in amplifier, acquisition computer, and the communication links.
inputs, the program automatically and Ram-Lak or Shepp Logan filter and the
sequentially sets the required gradient images reconstructed via a single stage PR
currents on the three gradient amplifiers, method. For 3D imaging, the smoothed
acquires projection data, and stores the profiles are subjected to a second derivative
projections in the computer memory. It also filter and are further subjected to a
collects a zero-gradient spectrum to Savitzky– Golay smoothing filter. The
deconvolve the projections, if required, to resulting profiles are then used to
reduce line broadening. reconstruct 3D image using a two-stage back-
projection algorithm. If necessary, it is also
Image Reconstruction possible to deconvolve the projections with
the zero-gradient spectrum prior to the
The image reconstruction sequence involves application of the smoothing filters, for
the following: individual projections are resolution enhancement. To display 3D
base-line corrected, either by averaging a images from the projection data, the surface-
predetermined (user chosen) number of rendering program VOXELVIEW® in a
initial points in the spectrum, or by Silicon Graphics Indigo2 workstation was
averaging the entire first derivative used. The VOXELVIEW software also allows
spectrum. The resulting profiles are slicing of the 3D image through axial,
subjected to a smoothing filter and the coronal, and sagital planes and performing
spectrum is integrated. The integrated distance, area, and volume measurements
profiles are then subsampled to 64, 128, or on the image. Slices of 3D images of normal
256 points. In 2D image processing, the and tumor bearing legs of a 3CH mouse is
filtered profiles are subjected to either a presented in Figure 22.3, Plate 12.
EPR Imaging for Biomedical Applications 335
diplexer is preferred over the circulator, to connected in parallel to obtain large volume
avoid excessive insertion losses. The diplexer coils. These parallel coil resonators offer an
used in our study is of a standard (¼)λ attractive alternative to bird-cage, solenoi-
configuration with a series pin-diode switch dal, and loop-gap resonators. The design is
assembly in the receiver path. These diodes of particular advantage for studying large-
are connected in a back-to-back configuration size objects, because the usable volume can
to minimize the transients in the RF paths. be readily increased and the construction is
Such an arrangement provides high speed inexpensive and relatively simple to
switching (~about 5 ns) necessary for time- fabricate. The resonator bandwidth readily
domain RF FT EPR experiments. Receiver encompasses a spectral bandwidth of 15
isolation during transmit mode is 25 dB with MHz. These dimensions are adequate to
a transmit insertion loss of 2 dB. The accommodate an experimental animal, such
insertion loss during receive mode is as low as a mouse, for imaging studies. The loaded
as 0.5 dB. This provided the necessary Q of the coil is maintained at 25. Q reduction
sensitivity in terms of excitation as well as in these resonators can be achieved by
desired bandwidth for imaging experi- overcoupling. In addition, a resistance in
ments. parallel (~3.3 kV) is also used to minimize
the dead time and also help reach the
Resonator desired spectral bandwidth. Pulses down
Several factors need to be considered in the to 30 ns give a power spectrum almost close
development of resonators for time-domain to the theoretical expectation. We have also,
EPR imaging methodology for in vivo in addition, investigated surface coil
applications. While in MRI, the frequency resonators for RF EPRI by connecting loops
bandwidth is about 20 kHz to obtain high- of the required diameter to a ½ length semi
resolution images of large-size objects, in rigid coaxial line to allow for seperation
EPRI the required bandwidth is at least 10 between the coil, and the tuning and
MHz, even for a resonator of 25-mm bore matching capacitors.
size. Desirable features of a resonator for
Signal Detection
such time-domain EPR applications are:
(1) ability to accept narrow and intense A mixer was used for phase-sensitive
pulses and convert to B1 with high efficiency; detection. The induction signals from the
(2) short resonator ring-down time; receiver arm are mixed with a local
(3) optimal Q-profiles with adequate spectral oscillator ~350 MHz. The intermediate, 50
bandwidth for imaging studies; (4) adequate MHz signal (IF) is isolated using a low pass
B1 field homogeneity; and (5) potential to filter and utilized for digitization and
be scaled up for larger objects. We have recovery of the FID. The first stage amplifier
designed parallel coil resonators for the RF in the receive arm is designed to avoid
FT EPR experiments to obtain: optimal, overload and also to facilitate fast recovery
homogeneous B1 field within the resonator to detect and amplify the weak FID signals.
for a given transmit power. Loops of After further amplification and filtering
conducting material spaced at equal followed by, rapid digitization and coherent
intervals, along the cylindrical axis are averaging provide adequate SNR.
338 Biomedical Magnetic Resonance: Proceedings of the International Workshop
strength. Even for spin probes with broad SPIN PROBES FOR EPRI
EPR signals, image resolution independent
Spectroscopy and imaging can have different
of line width can be achieved.15 Although
requirements; the former generally needs a
the pure phase encoding method produces
characteristic set of lines whereas the latter
high-resolution images with minimal
requires a signal as intense as possible to
artifacts, the SPI sequence is time inefficient
aid detection. Therefore a single spectral
because it requires N2 excitations for a 2D
line is preferable for imaging applications.
image of N2 pixels. Thus the 2D pure phase
But for in vivo applications, it is insufficient
encoding requires N times more phase
to simply select a probe which has a single
encode steps than a 2D frequency-phase
spectral line. The probe must have a chemical
image. Long data collection time of SPI may
significance to its application. A few
limit its use for in vivo imaging. But, efficient
desirable application-dependent properties
data acquisition system (DAS) may solve
of a useful spin probe for biological
this problem. Towards this direction, we
applications are:
have focussed further improvement the
i. Suitable solubility in the chosen
speed of our DAS. At present, we can collect
medium
2D SPI imagres (64 × 64) in 5 seconds.
ii. Narrow spectral lines.
iii. A simple EPR spectrum (in some types
of imaging or oximetry a single line is
desirable).
iv. Sharp lines with no unresolved
splittings, to provide maximum sensiti-
vity to detection,
v. Chemical, thermal and metabolic
stability.
vi. Non-toxic for in vivo applications.
vii. EPR parameters sensitive to physio-
logical conditions
Fig. 22.5: The schematics for data collection for single
The capability of EPR imaging has been
point (constant time) EPR imaging. Time-domain hampered by the lack of spin probes fulfilling
responses (FIDs) are collected in presence of the above criteria. In fact, the multiple lines
stationary gradients after applying a 90o pulse. The of nitroxyls cause hyperfine-based limita-
phase-encoding gradients are incremented in equal tions in image resolution and hyperfine-
intervals between ± Gmax. and applied in nested loops
based artifacts in the reconstructed image.
in a Cartesian raster. Although only a single time
point is needed for image reconstruction, the whole Solid state probes with a single-line ESR
FID (approx. 1500 points) is collected at 500 Ms/s spectrum have been tested. 25 The most
digitization rate. Each data point in the FID undergoes commonly used solid probes are lithium
a pseudo echo phase modulation (similar to the phthalocyanine (LiPc), fusinite a type of coal
gradient recalled echo imaging sequence in MRI). A and carbohydrate chars. The line broadening
one-dimensional gradient ramping and the
corresponding pseudo echo are shown on the right
of a single crystal of LiPc covers the range
hand side. Fourier transformation of each echo leads from 0.002 mT to 0.1 mT for 0 to 100 percent
to a one-dimensional image profile. oxygen respectively showing a linear
342 Biomedical Magnetic Resonance: Proceedings of the International Workshop
mouse were carried out using projection axis, the FOV of spatial window was
reconstruction (PR) and constant-time (CT) rescaled to 3.0 cm for all time points and
modalities using a CW EPR spectrometer/ interpolated to 128 points. The central 1.0
imager operating at 300 MHz frequency. G range of spectral axis was extracted and
Figure 22.6 illustrates this application with interpolated to 128 points after the FT along
phantom imaging. The phantom consists of the spectral axis. Finally, a spectral-spatial
three tubes of the Oxo63 solutions (5.0 mM) image represented by a 128 × 128 pixel-
saturated with air (21% O2), 10 percent O2 matrix is obtained.
and Ar (0% O2) gas at least for 1 hr at room Similarly, 3D spectral-spatial images
temperature respectively. The FOV of having 1.0 Gauss × 3.0 cm × 3.0 cm FOV
spectral and spatial window were selected were reconstructed upon 64 × 64 × 64 pixel
as 1.0 Gauss and 3.0 cm, respectively. A set matrix using 16 × 16 spectral data which
of 16 polar projections were obtained within obtained in the PR and CTSSI modality for
71.56° pseudo-viewing angle with constant the same phantom. From the 3D spectral-
angle increment in PR-SSI modality. The FOV spatial images reconstructed by FBP, the 2D
of spectral and spatial window were selected spatial distributions of the signal intensity
as 1.0 Gauss and 3.0 cm, respectively. A set (double integrated value) and the line width
of 16 polar projections were obtained within mapping were calculated and are shown in
71.56° pseudo-viewing angle with constant Figures 22.6A and B, Plate 13 respectively.
angle increment in PR-SSI modality. Sweep Figure 22.6C shows histogram of the line
width was changed from 1.00 to 3.16 Gauss width obtained from the area indicated on
depending on the projecting angle. Different the line width map (Fig. 22.6B, Plate 13). A
Filtered Back-Projection (FBP) programs suitable 2D spatial distribution (τ = 48) was
were employed to reconstruct the 2D chosen from the SPI data set (Fig. 22.6D,
spectral-spatial images. Plate 13). After the 2D spatial distribution
Although the principle of the CTSSI was masked by a threshold value, spectral
experiment is based on the time-domain shape was obtained by FFT of pseudo-time
EPR, EPR data in the frequency domain (CW domain only for the masked area. Then,
EPR) can easily be treated in the same the pixel-wise line width was calculated (Fig.
manner. The inverse FT of the acquired data 22.6E, Plate 13). Figure 22.6F, Plate 13 shows
with field or frequency sweep is the only the histogram of the line width obtained
extra step necessary to obtain the time from indicated area (dotted square) of
domain data. In the CTSSI modality, spectral Figure 22.6E, Plate 13. Both FBP and CTSSI
data were obtained under 16 linear steps of show similar good result although CTSSI
field gradient increment. All spectral data gives higher line width value than FBP in
were obtained at a constant sweep width this phantom study. The relatively low pixel
of 16.0 G with 1024 data points. The resolution of the spectral axis works as a
projections were Fourier transformed to smoothing filter for FBP image. FBP will
yield a 16 × 1024 pixel k-space matrix. 2D show a better range of line width when a
spectral-spatial images were reconstructed larger pixel matrix is employed to represent
in accordance with the principle of CTSSI a better pixel resolution. FBP shows
described above. Before final FT of spectral inhomogeneity of the line width distribution
344 Biomedical Magnetic Resonance: Proceedings of the International Workshop
between outer side and inner side of the tumor legs. The histogram from FBP image
image. On the other hand, fundamental shows that the values are varied widely.
spectral smoothness of CTSSI is not affected As described above, spatial resolution of
by pixel resolution. However, 3D CTSSI PR method is affected by line width. The
gives wider line width than 2D CTSSI. This clear 2D distribution of Oxo63 in mouse legs
is because each pixel has 4 neighboring was obtained by SPI slice. The line width
pixels leading to mutual broadening by mapping shows distinct difference between
overlap. In addition, CTSSI shows an appa- the normal and tumor legs. The in vivo
rent higher line width values at outer edges CTSSI shows wider line width than phantom
of the object. Nevertheless, CTSSI shows study, mainly due to the amplification effect
higher convergence for the line width values of T2* mentioned earlier. The wide spatial
compared with FBP and shows a greater distribution of Oxo63 in the mouse legs
promise of providing reliable line width generates broad blurred distribution at short
information with relatively noisier data. τ region of SPI data set. This low frequency
Figure 22.7, Plate 6 shows the application contribution on the pseudo-FID may cause
of these techniques for in vivo tumor an overall line width broadening. However,
oximetry. 2D spatial distributions and 2D the in vivo CTSSI study shows clear
line width mappings of normal and tumor differences in the line width between the
(1 cm size of SCC tumor) bearing mouse normal left hind leg and tumor-bearing right
legs of an anesthetized are presented in this hind leg. The line width mapping (Fig. 22.7F)
figure. Figure 22.7A shows a cartoon of the shows the distinctly hypoxic nature of the
positioning of mouse legs. Figures 22.7B and tumor leg. The histogram also clearly shows
22.7C show the 2D spatial distributions the difference between normal and tumor
(double integrated values) and the line legs (Fig. 22.7G).
width mapping respectively of Oxo63
injected in the tumor bearing mice obtained Oximetry by FT PR method
by FBP. Figure 22.7D shows histogram of In pulsed radio frequency (RF) EPR the spin
the line width obtained from indicated area probe is excited in the time domain with a
(dotted square) of Figure 22.7C. Figure short RF pulse and the resulting FID is of
22.7E and 7F show the 2D spatial the form, exp [-t/T2*] cos [(ω−ωo)t. Here,
distributions (SPI, τ = 24) and the line width T2* is a decay constant with contribution
mapping respectively of Oxo63 injected in from both natural T2 and magnetic field
the tumor bearing mice obtained by CTSSI inhomogeneties, and (ω−ωo) is the offset
modality. Figure 22.7G shows histogram of frequency. Fourier transformation of the FID
the line width obtained from indicated area gives the absorption spectrum from which
(dotted square) of Figure 22.7F. The 2D the line width can be measured after suitable
spatial distributions from FBP image are of phase correction. The FID is governed by a
lower intensity compared with SPI obtained first order decay constant, T 2*, which is
in CTSSI modality which shows a better related to the line width by,
contrast in the image. The line width Line width = (π T2*)-1 ...(8)
mapping from FBP image shows very little T2* is shortened by spin-spin interaction
difference between the normal and the between the spin probes. T2* can be further
EPR Imaging for Biomedical Applications 345
Figs 22.7A to G: 2D spatial distributions and 2D line width mappings of normal and tumor bearing mouse legs
of a C3H mouse with 1 cm size of SCC tumor on one of the legs. (A) A cartoon of the positioning of mouse legs.
(B) 2D spatial distributions of the double integration values and (C) the line width mappings calculated from
3D PR spectral-spatial image. (D) The histogram of the line width obtained from indicated area in C. (E) 2D
spatial distributions from SPI data and (F) the line width mappings calculated from 3D CT spectral-spatial
image. (G) The histogram of the line width obtained from indicated area in F. The differences in the line width
ranges between D and G arise from the differences in T2 and T2*
346 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 22.8: A schematic representation of T2* weighted spectroscopy. Calculated FIDs (top) of two signals at
50.5 and 52.5 MHz with the lower frequency signal corresponding to a larger line width. The initial time points
were progressively deleted (0, 100, 200 and 400 ns) to obtain the corresponding absorption mode spectra,
given in the bottom. The distinctly different slopes of intensity attenuation as a function of delay are also
indicated. The relatively slower attenuation of narrow line signal would indicate that hypoxic regions could be
better distinguished by the delayed processing scheme of ‘T2* weighted’ EPR imaging.
of single-point image depends on tp as given the thoracic, abdominal and pelvic regions
by Eq. (4). Therefore, a direct pixel-by-pixel are in the FOV. Projections corresponding
comparison cannot be done, because the to 2-D images were collected at 2.8, 9.0,
corresponding pixel represents different 13.0 and 16.9 minutes (B,C,D,E respectively)
spatial location. To correct for the change after spin probe administration using a
in field of view, images generated for gradient of 1.0 G/cm. The image at the first
different tp are resized. The resized images time point was interpreted as the spin probe
can be compared pixel by pixel, and slopes localization predominantly in the two
of the decay of pixel intensities can be kidneys with some intensity in other organs
computed to arrive at pO2, after calibration in the abdominal region such as liver, spleen,
with samples of known pO2. etc. Subsequent images suggest a
progressive redistribution of the spin probe
Pharmacokinetics from the circulation to the bladder as
Figure 22.10, Plate 14 shows a sequence of evidenced by the increasing image intensity
2-D CW EPR images of Oxo-63 distribution from the bladder. However, contours
in anesthetized mouse after intravenous corresponding to the two kidneys could be
administration.9 The mouse was positioned seen even in the presence of intense signal
(A) in the resonator in such a manner that from the bladder. The physical dimension
348 Biomedical Magnetic Resonance: Proceedings of the International Workshop
of the organs, obtained from the EPR images were collected for each 2D image which took
were in good agreement with the actual 108 seconds and a total of 8 images were
dimensions. Figure 22.10 F shows a graph collected separated temporally at 2-3 minute
of the decrease in the average spin count intervals. Sequential images obtained form
from the kidneys, and the increase in the the same phantom as a function of time show
spin count in the bladder as a function of that, while the intensity in tube A is
time. In this experiment the spin distribution invariant, there is a progressive loss in
in thoracic region could not be clearly intensity in tubes B, C and D, at rates
visualized, perhaps due to the low temporal proportional to the enzyme content in the
resolution of the CW EPRI modality. individual tubes. Reduction rates were
Figure 22.11, Plate 15 shows a sequence computed for individual voxels in the image
of 2D EPR images by SPI modality of a based on the time dependent intensities and
C3H mouse as a function of time after assuming a first order kinetics. Figure 22.12C
infusion of 100 μL of 100 mM Oxo63, shows a ‘redox-image’ where the intensity
administered via tail-vein cannulation. In the of each pixel is proportional to the first
first image, the spin probe is found to be order rate constant. In this image, the
predominantly localized in the thoracic and intensity corresponding to the tube A is
kidney regions. Subsequent images suggest within the image noise threshold, whereas
a progressive redistribution of the spin probe the image intensities corresponding to tubes
from the circulation to the bladder. B, C and D increase progressively reflecting
Improvement in the speed of data collection the enhanced rate of reduction. The
has enabled clear visulaisation of the spin histograms of the reduction rates and their
probe in the thoracic region. frequency of occurrence were computed
from each of the four tubes individually
Redox Mapping (Fig. 22.12D). The median reduction rate,
We have used 300 MHz, CW EPR imaging, derived from the histogram profiles of four
in vivo, to probe tissue heterogeneity and tubes correspond well with the XO
redox status in normal and RIF tumor concentration. This study illustrates that a
tissues in mice.30 Figure 22.12, Platge 15 time-series of 2D images measured from an
illustrates this application using a phantom object with spatially differing rates of spin-
imaging of four identical tubes containing loss can provide a redox map or a
nitroxides at identical concentration and pharmacokinetic image, which would be a
volume. Different rates of nitroxide reduc- reliable measure of the “metabolic” activity.
tion were established in each tube by
Redox Status in Tumors
adjusting the flux of O2- generated by the
aerobic reaction of 5 mM hypoxanthine (HX) EPR imaging experiments were also used to
at different levels of xanthine oxidase (XO). study the differences in nitroxide reduction
Tubes A, B, C and D contained 0.0, 0.4, 0.8 in normal muscle and implanted tumors.
and 1.6 U/ml of XO for nitroxide reduction Tumors were grown by implanting RIF-1
at different rates. EPR spectral projections cells in the hind leg of C3H mouse. The
were collected immediately after addition spin probe, 3-carbamoyl proxyl (3-CP) was
of the enzyme. A total of 12 projections administered by a tail vein cannulation.
EPR Imaging for Biomedical Applications 349
Sequence of data sets were collected to function of days after tumor implantation
compute pharmacokinetic image or the and the results shown in Figure 22.13B, Plate
redox map can be computed. Figure 22.13A, 16. As can be seen the reduction rates
Plate 16 shows representative spatial images increased in both the normal leg and the
obtained from such experiments on several tumor bearing leg as a function of time after
mice with tumors at different sizes (5 days, tumor implantation.
7 days, 10 days, 12 days, 14 days after tumor
implantation). Images for each mouse at FUTURE DIRECTIONS
different times after spin probe administ-
ration are shown with the corresponding Multimodality Approach
times indicated in the panel. The left hand In EPRI, the spatial as well as the spectral
side in each panel represents the image of information of the exogenously administered
spin probe distribution in normal leg and spin probes is obtained. Hence, EPR images
the right hand side that from the tumor present the in vivo visualization of the spin
bearing leg. The spatial images obtained probes and often do not provide anatomical
form such experiments show that the spin information. In contrast, MRI is a well-
probe accumulates in normal and tumor established modality that gives superior
muscle readily and the reduction of the anatomical information. Overhauser MRI
nitroxide proceeds rapidly as a function of (OMRI) is a double resonance technique that
time. The accumulation of the spin probe in couples the advantages of MRI with the
the tumor was found to be significantly sensitivity of EPR, by making use of the
altered as a function of tumor size. Over hauser effect. By saturating the EPR
The nitroxide reduction rates in the transition of a paramagnetic agent, the NMR
tumor bearing leg and normal leg were signal intensities of the coupled water
derived from the time dependent variation protons are enhanced by a significant factor
of nitroxide levels from each voxel. The by means of the Overhauser effect. The
image of the tumor leg showed a gradual Overhauser enhancement (OE) depends on
increase in nitroxide distribution area and the line width of the paramagnetic agent,
heterogeneity in the distribution of reduction which in turn depends on oxygen concen-
rates. Interestingly, the position of the lower tration, making it possible for coregistration
reduction rate was observed in the center of both the anatomical structures and the
of tumor on days 12 (size 118.44 ± 8.53 mm2 corresponding oxygen status. Using the
s.e.m) and 14 (size 132.43 ± 11.65 mm2 s.e.m) narrow-line trityl-based contrast agent, we
consistent with regions of necrosis in the have demonstrated the feasibility of
tumor core. This suggests that the image of obtaining morphological images of high
reduction rate may be related to the resolution, coregistered with oxygen
physiological reducing capacity, oxygen concentration in-formation.31 Based on these
concentration level (hypoxia) and necrosis. images, the pO 2 status of any specific
The differences in the reduction rates of location in the tumor can be interrogated
nitroxide in the region containing the normal accurately and repeatedly, thus providing a
and tumor legs were further examined by useful approach to evaluate the importance
making redox measurements calculated as a of oxygen status in tumor physiology.
350 Biomedical Magnetic Resonance: Proceedings of the International Workshop
A crucial aspect of EPRI concerning its Electron paramagnetic resonance for small
applicability to human studies is the animal imaging applications Ilar J 2001;42:209-
18.
powerdeposition caused by the EPR B1 field, 4. Krishna MC, Subramanian S, Kuppusamy P,
which may cause undesired heating. Mitchell JB. Magnetic resonance imaging for in
Developing new spin probes with narrower vivo assessment of tissue oxygen concentration,
line widths, compared with those currently Seminars in Radiation oncology, 2001;11:58-69.
used, will help in decreasing the RF power 5. Kuppusamy P, Afeworki M, Shankar RA,
Coffin D, Krishna MC, Hahn SM, Mitchell JB,
deposition. We have explored the application Zweier JL. In vivo electron paramagnetic
of correlation spectroscopy employing resonance imaging of tumor heterogeneity and
stochastic excitation and the Hadamard oxygenation in a murine model, Cancer
transform to time-domain FT-EPR spectros- Research, 1998;58:1562-68.
6. Krishna MC, Afeworki M, Devasahayam N,
copy in the RF band. Our results show
Cook J, Subramanian S, Mitchell JB. In vivo free
decrease in the total acquisition time, and radical detection and imaging by EPR: Non-
an improved FID SNR, compared to the invasive mapping of tissue oxygen status, Free
conventional coherent averaging approach. Radical Biology and Medicine, 1999;27:468
These techniques have the potential to Suppl. 1.
7. Van Dam GM, Afeworki M, Subramanian S,
reduce the RF pulse power to the levels
Krishna MC, Hoekstra HJ, Cook JA, Russo A,
used in CW EPR while retaining the Mitchell JB. A new functional in vivo imaging
advantage of time-domain EPR methods. technique of normal and ischemic liver using
These methods have the potential to electron paramagnetic resonance imaging
facilitate the progression to in vivo FT-EPR (EPRI), Gut, 2000;46:W51 Suppl. 2.
8. Halpern HJ, Chandramouli GVR, Yu C, Peric,
imaging of larger volumes. Barth, ME, Teicher BA, Grdona D. Pharmaco-
Recent development of narrow line spin logical compartment viscosity and polarity
probes for functional imaging and the measured with very low frequency EPR in
potential of the pulsed technique to provide tumors of living animals, Magnetic Resonance
better sensitivity and temporal resolution in Chemistry 1995;33:S147-53.
9. Yamada KI, Murugesan R, Devasahayam N,
for such narrow line spin probes, offer the Cook JA, Mitchell JB, Subramanian S, Krishna
attraction for the development of low MC. Evaluation and comparison of pulsed and
frequency pulsed EPR instrumentation. The continuous wave Radiofrequency electron
possibility of co-registering anatomic images paramagnetic resonance techniques for in vivo
(MRI) with functional images (EPRI)32 is detection and imaging of free radicals, J Magn
Reson, 2002;154(2):287-97.
likely to add an extra impetus to research 10. Murugesan R, Cook JA, Devasahayam N,
in this field. Afeworki M, Subramanian S, Tschudin R,
Larsen JA, Mitchell JB, Russo A, Krishna MC. In
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EPR Imaging for Biomedical Applications 351
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Murugesan R, Devasahayam N, Cook K, Matsumoto S, Utsumi H. Co-registration
JA, Golman K, Ardenkjaer-Larsen JH, image of free radical distribution and
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Studying Mobile Proteins and Peptides In vivo using MRI and MRS 353
23
Studying Mobile Proteins
and Peptides in vivo
using MRI and MRS
Jinyuan Zhou, Peter CM van Zijl
water pool is much larger than the saturated in situ WEX spectra (Figs 23.1A and B) at
solute proton pool, each exchanging solute 4.7 T show an initial increase (10-50 ms) in
proton is replaced by a nonsaturated water the resonance at 8.3 ppm, confirming its
proton. A few seconds of irradiation then exchange-related character. At longer tm,
leads to enhancement if the exchange rate signals at low frequency appear, which we
and saturation efficiency are sufficiently high. attribute to the intramolecular nuclear
Especially large enhancements in sensitivity Overhauser effect (NOE). It is important to
(up to 500,000,17) were demonstrated for stress that, when using the WEX approach,
amide protons on cationic polymers, such as only signals from more mobile macromole-
poly-lysine and dendrimers, which have a cules (proteins, peptides, some lipids) will
favorable exchange rate range (10-300 s-1) be visible, while those of small brain
under physiological conditions. These results metabolites are not.9 It is interesting to see
suggested that it should be possible to detect that the in vivo proton spectrum of endo-
the amide protons of tissue proteins and genous proteins and peptides at high
peptides in the water signal, via the amide frequency is very simple and resembles
proton resonance at 8.3 ppm. We recently protein spectra obtained in vitro in the sixties
demonstrated this capability, which was and seventies.7
called “amide proton transfer” or APT imag- The WEX data can also be used to assess
ing10,18 which should depend on endogenous the exchange properties of the amide
protein concentration, proton exchange rates, protons. Figure 23.2 shows the normalized
pH, temperature, and several other factors. signal integral values of the protons at about
8.3 ppm as a function of the mixing time,
tm. These spectra were used to study amide
DIRECT DETECTION OF THE proton exchange rates, which were
INTERACTION BETWEEN AMIDE determined to be 28.6 ± 7.4 s -1 for
PROTONS AND WATER: WATER normocapnia and 10.1 ± 2.6 s-1 (n = 5)
EXCHANGE (WEX) SPECTROSCOPY postmortem (10). Because no significant
change in amide intensity at tm = 300 ms
There are two approaches to study the
was seen in the first two hours postmortem,
interaction between water protons and amide it was concluded that this rate change is
protons. One is to magnetically label the due to the pH change upon death. To
water protons and study magnetization calibrate the exchange rate changes in terms
transfer (MT) to the proteins, the other is the of intracellular pH (pH i ) changes,
inverse pathway. The first approach has been phosphorus spectroscopy was also perfor-
demonstrated in vitro using so-called water med. The measured pHi values were 7.11 ±
exchange (WEX) spectroscopy,19,20 in which 0.13 at normocapnia and 6.66 ± 0.10 post-
water magnetization is selectively labeled mortem (n = 7). Using the fact that amide
and its transfer properties to the water- proton exchange is predominantly base-
catalyzed21,22 for pH > 5, it was deduced
exchangeable protons are monitored as a
that:
function of time after labeling (mixing time,
tm). Recently, WEX spectra were acquired k = kb [OH]= kb × 10pH – pK W
in situ in the rat brain9,10 to verify that the = 5.57 × 10pH–6.4 ...(1)
composite resonance at 8.3 ppm in vivo is in which pKw = 15.4 at 37oC.23
Using this
indeed due to proteins in tissue. The acquired result, an average pK value of 17.2 for the
Studying Mobile Proteins and Peptides In vivo using MRI and MRS 355
Figs 23.1A and B: Normocapnic (A) and postmortem (B) WEX spectra for rat brain as a function of mixing time,
tm Volume 16´12´4 mm3, SW 3000 Hz, 1024 points, 256 scans, TR 4 s, and TE 8 ms. (Reproduced, with
permission, from Zhou J, Payen J, Wilson DA, Traystman RJ, van Zijl PCM. Nature Med 2003;9:1085-1090)
amide groups contributing to the composite In order to selectively assess the APT effect,
resonance was calculated, in good agreement one can define an MT-ratio asymmetry
with literature values for amide protons. parameter:10,13 MTRasym = Ssat/S0 (negative
offset) - Ssat/S0 (positive offset). If the solid-
INDIRECT DETECTION OF AMIDE like MT effects were symmetric with respect
PROTONS THROUGH THE WATER to the water resonance, the additional APT
SIGNAL (APT MRI AND MRS) would give rise to a positive MT difference.
Interestingly, the rat brain asymmetry curves
In Situ Rat Brain APT Effects in (Fig. 23.3C) show a varying MT difference
the Water Signal that is initially slightly positive and then
Figures 23.3A and B, Plate 17 show in situ becomes negative. This result supports a
MT spectra of the rat brain during different report24 that the solid-like MT effect is asym-
physiological conditions. In these so-called metric with respect to the water resonance,
z-spectra, the water signal intensity with the MT center frequency in the aliphatic
reduction is acquired as a function of offset range. An additional series of experiments in
of the saturation pulse with respect to the which the irradiation range was extended
water resonance frequency. It is important (Fig. 23.3D, n = 3) shows that the MTRasym
to understand that the signal intensities in curve becomes constant and negative at about
these z-spectra reflect the effect of -3 percent for higher offsets. When subtract-
conventional MT based on solid-like tissue ing the normocapnic MTRasym plot from the
structure, direct water saturation, and APT. postmortem curve, the maximum signal
356 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 23.2: Fitting of proton exchange rates using the average amide proton integrals (n = 5) as a function of
mixing time. Integrals were normalized to the normocapnic value at tm = 300 ms. (Reproduced, with permission,
from Zhou J, Payen J, Wilson DA, Traystman RJ, van Zijl PCM. Nature Med 2003;9:1085-1090.)
change is found at an offset of 3.5 ppm peptides. APTR can be assessed under the
from water (Fig. 23.3E). Using 4.75 ppm for assumption that MTRasym (3.5 ppm) remains
the water resonance, this corresponds unaltered in the same experiment or by
exactly to the spectral frequency of the comparison with normal tissue.
composite amide proton resonance at 8.3 Using a two-site exchange model (a small
ppm (Fig. 23.1), indicating that this pool for amide protons and a large pool for
difference originates predominantly from bulk water protons) and assuming instanta-
the amide protons of the mobile proteins neous amide proton saturation, the APTR can
and peptides. Therefore, we have in zero be derived to be:25
order: k [amide proton] –R t
APTR = (1 – e 1W sat )
MTRasym (3.5 ppm) = [water proton] R1W
MTR’asym (3.5 ppm) + APTR (2) (3)
in which MTRasym (3.5 ppm) is the inherent in which k is the exchange rate for all amide
asymmetry of the solid-phase MT effect protons participating in the effect, R1W the
associated with immobile macromolecules average spin-lattice relaxation rate of water,
and membranes, and APTR is the proton and tsat is the saturation transfer time. The
transfer ratio for the amide protons water proton concentration [water proton]
associated with mobile cellular proteins and = 2 × 55 M × water content. In the
Studying Mobile Proteins and Peptides In vivo using MRI and MRS 357
postmortem study,10 which focused on the visible on the right side of both the pH-
first two hours after cardiac arrest, weighted and pH images is located in the
negligible changes in the combined effects caudate nucleus, a region commonly affected
for water content and R1W were found. The by infarction following MCA occlusion, as
amide proton content also remained confirmed by the diffusion-weighted image
constant in this early postmortem period and by histology, which acquired 8 hours
(Fig. 23.2). Thus, using ΔAPTR (-1.90%) and later. No infarct is visible on the T2-weighted
the measured exchanged rates at image
normocapnia and postmortem, the total One question that arises is whether it is
amide proton concentration from all proteins useful to make pH-weighted images if
and peptides could be calculated to be about diffusion images already show the initial
71.9 mM, leading to APTR values at the ischemia. Present hospital policy in advanced
3.5ppm offset of 2.94 percent at normocapnia academic centers for acute stroke treatment
and 1.04 percent postmortem. is to acquire MRI images and assess regions
of reduced diffusion constant (expected to
pH Imaging of the Ischemic Rat Brain go on to infarction) and zones of reduced
The APT approach for pH imaging has been flow (penumbra). If the reduced flow zone is
applied to brain ischemia following middle much larger than the diffusion-affected area
cerebral artery occlusion in rats.10,25 The (so-called perfusion/diffusion mismatch),
MTRasym spectra for the contralateral and treatment with clot lysing agents such as tPA
ipsilateral regions of interest (Fig. 23.4C, may commence. However, such treatment has
Plate 18) were found to compare very well a large risk of causing hemorrhage, while it
with those for normocapnic and postmortem is uncertain that the penumbra will progress
brain (Fig. 23.3C, Plate 17), respectively, to infarction. The thought is that a better point
indicating that ischemic brain has a similar of reference for treatment will be when
pH decrease to postmortem brain, typically oxidative metabolism becomes impaired. At
about 0.5 units.26 The pH-weighted images this point, pH changes will also occur and if
(i.e. the MTRasym (3.5 ppm) images) were this can be imaged in acute stroke, the
acquired using frequency-labeling offsets of treatment risk may be reduced by only
± 3.5 ppm (40 scans), in which B 0 applying treatment if a large part of the
inhomogeneity was corrected using field perfusion/diffusion mismatch shows reduced
maps. When combining Eqs. [1] and [3], and pH (pH/perfusion mismatch criterium).
using the experimental data that the amide
proton content does not change appreciably APT Imaging of Rat Brain Tumors
in the first hours post-occlusion, it can be APT contrast also reflects the tissue content
seen that APTR is a direct function of pH, of labile amide protons of mobile proteins
with a dependency on field strength and peptides and should thus be able to
(because of R1W). For the rat, using the post- provide an assessment of endogenous
mortem calibration data above, it was protein/peptide content. This possibility was
deduced that APTR = 5.73 × 10pH–9.4, which recently demonstrated18 in a 9L rat brain
was used to generated absolute pH images tumor model. The MTRasym curves in brain
(Fig. 23.5, Plate 18). The area of infarction areas with tumor and tumor-related edema
358 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 23.7: Comparison of several images for a 9L glioma (10 or 11 days) in a rat. The glioma (arrow) is visible
in all the MR images, as confirmed by histology, but it is much clearer in the APT-weighted image. Arrow head:
peritumoral tissue and open arrow: CSF. (Reproduced, with permission from Zhou J, Lal B, Wilson DA, Laterra
J, van Ziji PCM. Magn Reson Med 2003;50:1120-1126. Wiley-Liss, Inc)
types. Another is pH imaging. A method to time 1H NMR spectra of the rat brain. J Magn
image pH changes via endogenous contrast Reson 1999;141:104-20.
7. Wuthrich K. NMR of proteins and nucleic acids.
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New York: John Wiley and Sons; 1986.
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9. van Zijl PCM, Zhou J, Mori N, Payen J, Mori
and is eminently clinically translatable. S. Mechanism of magnetization transfer during
Future work will have to realize this potential on-resonance water saturation. A new approach
in the clinical setting. to detect mobile proteins, peptides, and lipids.
Magn Reson Med 2003;49:440-49.
10. Zhou J, Payen J, Wilson DA, Traystman RJ, van
ACKNOWLEDGMENTS
Zijl PCM. Using the amide proton signals of
The authors thank Drs Phillip Zhe Sun, Jean-Francois intracellular proteins and peptides to detect pH
Payen, David A. Wilson, Richard J. Traystman, effects in MRI. Nature Med 2003;9:1085-1090.
Bachchu Lal, and John Laterra for cooperation in 11. Chen W, Hu J. Mapping brain metabolites using
this project. This study was supported in part by a double echo-filter metabolite imaging
grants from NIH/NIBIB (EB02634 and EB02666) and (DEFMI) technique. J Magn Reson 1999;140:363-
the Whitaker Foundation. 70.
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362 Biomedical Magnetic Resonance: Proceedings of the International Workshop
24
Magnetic Resonance of
Biofluids and Tissues
Ian CP Smith, Tedros Bezabeh, Racquel Baert
been reported on human tissue ex vivo, times after removal from the host. One way
suggesting that the high information content to ensure this is to freeze the samples at
of the spectra should lead to effective clinical liquid N2 temperature within minutes of
use of NMR in vivo. excision. These frozen specimens are stable
Tissue differs drastically from biological for weeks. Likewise, a rigid protocol for
fluids in that it is macroscopically solid. This warming the specimens and obtaining their
leads to dificulty in optimizing the homo- spectra must be followed.
geneity of the applied magnetic field for NMR spectra contain a wealth of infor-
optimal spectral resolution. A technique has mation—some vital, some useful, some
been reported that minimizes this problem.27 useless. It is often dificult to decide by visual
Figure 24.2 shows the positioning of a piece inspection which regions of the spectra will
of human tissue within a capillary tube facili- be most useful in classifying the specimens.
tating the optimization of field homogeneity Ratios of peaks can be useful, but much time
and allowing an estimation of the volume must be spent before the most useful ratios
of the specimen. This method has been used are found. In our experience computerized
in most quantitative studies to date. methods to select the most discriminatory
Ex vivo tissue is not dead tissue; it has, region, and to perform the classification,
however, begun a progression toward death. are robust and accurate. The most exhaustive
In studies of human tissue ex vivo, great application of these methods is described in
care must be taken to ensure that the spectra Somorjai et al.28 Recently, a description of
of the many specimens are taken at similar the regional selection process has appea-
red. 29 A summary of applications and
methods is given in Lean et al.4
For spectral classification, multivariate
analysis has proved to be very effective. A
variety of methods, such as linear discri-
minant analysis, quadratic discriminant
analysis, neural networks, and genetic
algorithms may be used. Very often the
simplest method, linear discriminant ana-
lysis, is sufficient. In the most dificult cases,
a number of methods may be used and
finally entered into a meta classifier.28 The
combi-nation of methods is known as the
statistical classification strategy.4
It is crucial that the NMR spectra be
adequately prepared for the classification
process. They must be normalized for area,
adjusted for chemical shift, and the most
discriminatory regions determined. Data
must be divided into a training set, in which
the method is calibrated, and a test set, in
Fig. 24.2: Diagram of the positioning of a biopsy
specimen within a standard NMR tube of outside which the accuracy of the method is tested.
diameter 5 mm. (Reproduced by permission from Regrettably, this has been done very rarely
Kuesel et al.) in the literature to date, yielding promising
Magnetic Resonance of Biofluids and Tissues 365
results but with a method that is not robust. trated the ability to diagnose a number of
To enhance robustness, the leave-one-out metabolic disorders including histidinemia30,
method should be implemented. The citrullinemia,31 argininosuccinic acid lyase
procedure is challenged by leaving out one deficiency, 31,32 ornithine carbamoyl trans-
data set, classifying it by exposure to the ferase deficiency,31 cystinuria,33 oxalic aci-
existing classifier, leaving another out and duria,33 alkaptonuria,30 multiple acyl CoA
classifying it, until all data sets have been dehydrogenase deficiency (glutaric aciduria
thus treated. Finally, a sufficient number of type II),34 methylmalonic aciduria,35 propio-
data sets must be used if the classifier is to nic aciduria, 35 porphyria, 33 5-oxopro-
be accurate, specific, and robust. The linuria,33,36,37 homocysteinemia,38 trimethyl-
number required depends strongly on the
aminuria,39 and Fanconi syndrome.33
types of spectra compared.
More recently, both 1H-and 13 C-MRS
Using the procedures described above,
have been used to identify and quantify the
experimental results from which no obvious
presence of elevated levels of galactonate
discriminators can be seen, and in which
in the urine of 27 patients with uridyltrans-
there is a wide variation in spectra from a
ferase-deficient galactosemia. 40 750 MHz
given class, can be accurately classified. The 1
reader is referred to the articles.2-6 The H-MRS has also been used recently to
remainder of this article presents an examine the elevated levels of 2-hydro-
overview of the medical applications of high- xyglutaric acid in the urine of a patient with
resolution MRS ex vivo. Specifically, progress the rare condition of 2-hydroxyglutaric
has recently been made towards the aciduria.41 Maschke et al42 reported the use
diagnosis of inborn errors of metabolism, of 1H-MRS (300 MHz) for the analysis of
the measurement of renal injury, diagnosis trimethylamine (TMA) in the urine of a
of neurological disorders and cancer.2 patient with trimethylaminuria and of other
members of his family. In this study, 300
INBORN ERRORS OF METABOLISM MHz 1H-MRS was shown to have sufficient
sensitivity, resolution, and linearity to allow
High-field 1H spectroscopy of biofluids
the diagnosis of trimethylaminuria in a
offers great potential for furthering the
classical one-dimensional spectrum of the
under standing of disease processes in
urine of the patient (Figs 24.3A and B).
humans. Historically, one of the most
Moreover, this technique is easier than the
successful clinical applications of MRS has
established classical biochemical methods
been the detection of a large number of
that primarily use gas chromatography. The
metabolic disorders. In these disorders, the
increased TMA signal, and the decreased
reduction or absence of activity of an
enzyme or cofactor can have dramatic trimethy-lamine-N-oxide (TMAO), as com-
consequences for metabolism and its control. pared to a normal healthy subject, are clearly
Many inherited metabolic disorders result seen at 2.90 ppm and 3.27 ppm, respectively.
in the accumulation of large amounts of Although urine appears to be the
organic intermediates produced proximal to specimen of choice for the diagnosis of
the defective enzyme step, which eventually inborn errors of metabolism, the use of CSF
spill into the blood and urine. 1H-MRS has has also been reported.43,44 Derangements
been used to study the urinary excretion of of organic acid concentrations in CSF may
such compounds. Initial studies demons- occur independent of the systemic meta-
366 Biomedical Magnetic Resonance: Proceedings of the International Workshop
A C
Figs 24.4A and B: 1H-NMR spectra (600 MHz) of CSF from patients with: (A) isolated 3-methylcrotonyl-CoA-
carboxylase deficiency; (B) Canavan disease; and (C) histidinemia44
Magnetic Resonance of Biofluids and Tissues 367
and possibly of CSF, is capable of diagnosing markers of renal allograft rejection has been
a vast number of inborn errors of meta- studied extensively, 52,53 and found to be
bolism. MRS not only provides information relatively nonspecific. Foxall et al54 used 1H-
on endogenous biochemical processes, but MRS to investigate the pattern of metabolic
is also capable of rapid quantitation of changes associated with renal allograft
metabolites, using a small volume that dysfunction. As shown in Figures 24.5A to
requires little pretreatment. Furthermore, it E, the spectra of normal human urine
is not necessary to preselect the metabolites showed signals for creatinine, glycine,
of interest, as the technique allows simul- citrate, alanine, lactate, and N-methylated
taneous measurements of a range of compo- meta-bolites in the chemical shift range of
nents. There exists also, the possibility for 3.1 – 3.3 ppm. The spectra of patients’ urine
the detection of some novel markers of these collected following renal transplantation
diseases, or insights into the underlying were considerably different. Compared to
defects of these disorders. normal urine, the spectral pattern of urine
from patients with an immediate functioning
RENAL INJURY
graft, urinary tract infection, renal tubular
Early studies of 1H-MRS of urine demons- ischemia or with a nonfunctioning graft are
trated that in situations where renal damage widely different. Further studies associated
is present, the urinary profile of the low- an increased excretion of TMAO with
molecular-weight metabolites is altered.45,46 biopsy confirmed acute graft rejection.55 It
Furthermore, in studies of the effects of has also been shown that 1H-MRS of urine
region-specific nephrotoxins, the application can be used to predict rejection of a kidney
of 1H-MRS urinalysis indicates that abnormal much earlier than the usual chemical analysis
patterns of metabolites are associated with made in kidney biopsies.76 Thus, unlike the
different sites of nephrotoxic actions. Renal excretion of enzymes or proteins, 1H-MRS
proximal tubular toxins cause glycosuria, provides both diagnostic and prognostic
lactic aciduria, and amino aciduria; renal information.
papillary toxins produce abnormal excretion Since urine has been studied most
patterns; and renal papillary necrosis extensively, a large number of the reso-
produces an initial increase in TMAO and nances in the spectrum of normal human
dimethylamine (DMA) excretion followed urine have been assigned. A study was
by a subsequent increase in N,N-dimethyl-
performed to standardize the analytical con-
glycine, succinate, and acetate, and a decre-
ditions, to quantify the major metabolites
ase in TMAO and 2-oxoglutarate.47,48 This
present in urine of normal subjects, and to
information is useful when assessing renal
evaluate changes due to physiological condi-
disease progression or nephrotoxicity asso-
tions such as feeding. 56 In this study,
ciated with therapeutic agents. Subsequently, 1H-MRS, operating at 300 MHz, studied
1
H-MRS urinalysis has been used as a non-
invasive diagnostic technique for tubular and serial urine samples from 50 normal subjects.
papillary distortions in glomerulonephri- In all specimens, creatinine, lactate, alanine,
tis,49,50 and for the assessment of the degree citrate, DMA, TMAO, glycine, and hippurate
of chronic renal failure.51 were found to be present. All metabolites
The abnormal urinary excretion of were quantified on the basis of peak heights
specific enzymes and proteins as possible and were expressed as millimoles per mole
368 Biomedical Magnetic Resonance: Proceedings of the International Workshop
NEUROLOGICAL DISORDERS
The use of high-resolution 1H-MRS to evalu-
ate brain metabolism through the metabolic
profile of CSF constitutes a potentially
powerful strategy to aid the differential
diagnosis of neurological diseases. Although
the biochemical composition of CSF has been
well characterized by standard biochemical
techniques, a number of studies have been
done to evaluate the effectiveness of
1
H-NMR spectroscopy for examination of
CSF as an aid to the biochemical diagnosis
of central nervous system diseases. Early
studies reported the assignments of a
number of resonances. 13,43 Since then, a
number of pattern-recognition approaches
and discriminant analyses have been used
to separate samples into different classes,
and these used to differentiate between
normal controls and subjects with various
Figs 24.5A to E: 1H-NMR spectra (500 MHz) of (a) neurological disorders.57-59
normal human urine, and urine collected from four
Koschorek et al 58 reported that the
patients 3 days following renal transplantation
showing: (b) immediate functioning graft; (c) urinary spectra of CSF of normal controls and sub-
tract infection; (d) renal tubular ischemia; and (e) non- jects with tumors or multiple sclerosis (MS)
functioning graft. Abbreviations are as follows: Ac D can be perfectly separated, whereas those
acetate, Ala D alanine, Ch D choline, Cit D citrate, from subjects with disk herniations can be
DMA, GLC D glucose, Gly D glycine, Lac D lactate,
separated approximately 90 percent of the
myo-Inos D myo-inositol, Pep D peptides, P D
acetaminophen (paracetamol) metabolites, Suc D time using principal component analysis
succinate, TMAO, V D cyclosporin A drug metabolite54 (PCA).
Magnetic Resonance of Biofluids and Tissues 369
500 MHz 1H-MRS spectra of postmortem previously was not found in the CSF of any
CSF specimens from control subjects and of the patients with MS. Interestingly, Aasly
patients with Alzheimer’s disease were et al63 observed significantly lower levels
examined in a similar study.60 PCA achieved of lactate and glutamine in MS patients as
partial separation of the groups; more formal compared to controls. When the MS group
statistical analysis suggested that citrate by was divided into two sub-groups (chronic
itself was the best discriminator. progressive and relapsing–remitting forms
Spectra showing the metabolic brain of MS), no significant differences were
profile induced by degenerative dementia found in any of the parameters measured
are reported to be considerably different in the CSF, although there was a tendency
from those of control patients.61 An increase toward lower levels of lactate and glutamine
in many metabolites has been observed, and in the relapsing–remitting groups. What
for some (leucine–isoleucine) the concen- accounts for the different results in these
tration is increased three-fold. Furthermore, studies is unclear; it may simply reflect the
some abnormal and unassigned resonances lack of a standardized method for specimen
have been observed. These qualitative MRS prepara-tion and recording 1H-MRS spectra
from CSF. A standardized method for high-
abnormalities were not found to be corre-
resolution 1H-MRS of CSF needs to be
lated with any medical treatments.
adopted.44
In the CSF of patients with Huntington’s
chorea, 400 MHz 1H-MRS analysis demons-
CANCER
trated a significant increase (approximately
60%) in the pyruvate concentration as Increased research in the field of cancer
compared to controls. However, in serum diagnosis and treatment over the past
samples, no variation in this metabolite was several years included a search for an
detected.61 inexpensive, accurate, and noninvasive
In studies of CSF from patients with MS, screening test for early malignancy. It had
the findings among studies differ. Lynch et been hoped that a simple screening test of
al62 reported that there were no significant plasma, using 1 H-MRS, might reveal a
differences between the levels of most malignant condition. However, these investi-
metabolites, with the exception of acetate gations have been unsuccessful,64,65 and
and formate which were increased and attention has shifted toward the assessment
decreased respectively in patients with MS of various tissue specimens, and extracts of
as compared to controls. Moreover, in 93 tissue specimens, to determine whether
percent of patients with actively progressing 1H-MRS can differentiate between malignant
spectra was obtained, and the transverse from the specimen, resonances from these
relaxation time (T2) of the lipid resonance underlying tissues may confound the
at 1.3 ppm in the 1H spectrum was used as analysis. A definitive study was performed
an indicator of metastatic potential, whereas using well-defined specimens, and rigorous
the intensities of resonances due to choline- analytical methods, showing that 1H-NMR
containing compounds correlated with the can yield very high levels of sensitivity and
degree of invasion of the cancer (Dukes A, specificity for the detection of cancer.22 More
B, C, or D). Subsequent studies on cultured recently, this approach has been applied to
human cell lines of different degrees of the evaluation of other colonic disorders,
invasive capability showed that two- such as Crohn’s disease and inflammatory
dimensional COSY 1H spectra could be bowel disease.68 The chemical discriminatory
discriminatory67 (Fig. 24.6). power of 1H-NMR is expected to provide
More recent studies of colon tissue valuable insight into the progression of colon
revealed another critical element—the purity disease from dysplasia to malignancy, and
of the colon tissue. If care is not taken to to interpret the progression in terms of the
oncogenic genes expressed during the
exclude both muscle and subepithelial fat
progression.
Thyroid Cancer
A problem in thyroid cancer is the detection
of follicular malignancy. Regular histopatho-
logical procedures cannot yield a conclusion.
Only thyroidectomy, and analysis of the
entire gland, can yield a definitive diagnosis
of invasive disease. However, the patient
has already lost the thyroid gland. 1H-NMR
studies on thyroid biopsies by both surgical
and needle approaches demonstrated a high
level of sensitivity to thyroid cancer, and
provided a serious test of the multivariate
methods for spectral analysis.28 This appro-
ach is now used in Australia to determine
the therapy applied to neoplasms of the
thyroid (C.E. Mountford, personal communi-
Fig. 24.6: 1 H-NMR (360 MHz) COSY spectrum cation).
(magnitude mode, symmetrized) of excised tissue in
400 µL phosphate-buffered saline in D 2O, for a
specimen of human ovarian serous carcinoma, poorly
Brain Tumors
differentiated. Multiple cross-peaks are attributable A variety of human brain neoplasms have
to cell–surface fucosylation, namely Thr/Fuc I (1.33– been studied by this technique. Figure 24.7
4.27 ppm), which also contains a contribution from
shows the 1H-MR spectra of tissue from
the amino acid threonine, Fuc II (1.25 – 4.28 ppm)
Fuc IIb (1.19 – 4.20 ppm), and Fuc III (1.41 – 4.30 three different brain tumor types, and a
ppm), and correlated with tumor grade and loss of patient with epilepsy. Relatively simple data
cellular differentiation72 analysis allowed accurate classification of
Magnetic Resonance of Biofluids and Tissues 371
these cancers, and gave cause for hope for tumors have now been reported,71,77 and it
a method to determine their degree of is expected that 1H-MRS will be a valuable
aggressivity.29,69 The resonance at 1.3 ppm technique for the planning of treatment for
due to lipid was seen to be indicative of the brain tumors. For a good overview, see
degree of necrosis (dead tissue) in the Danielsen and Ross.77
tumor. 70 Many in vivo studies of brain
Ovarian Tumors
1
H-NMR spectra of ovary biopsy specimens
have shown good sensitivity to the presence
of malignant disease by both resonance
ratio72 and multivariate methods.73 In the
latter study, sensitivity of the tumor to drug
therapy was also detected by the method.
It is hoped that in vivo use of this method
will help detect ovarian cancer at an early
stage, and define its potential resistance to
therapy.
Prostate Cancer
Screening of prostate tissue is currently done
by transurethral or transcolonic biopsy. In
both methods, the sampling is not represen-
tative of the entire gland. 1 H-NMR of
prostate tissue has demonstrated remark-
able discriminatory power.74 Benign disease
can be distinguished from cancer with sensi-
tivity of 100 percent, and specificity of 95.5
percent. Furthermore, the method appears
to be capable of distinguishing the stromal
and glandular forms of benign prostatic
hypertrophy (Figs 24.8A and B). Transrectal
1H-MR spectra in vivo show similar detail,
CONCLUSION
The range of applications of 1H-NMR of
biological fluids is wide, and is growing
steadily. The study of the composition of
physiological fluids, and the changes thereof
in disease, are amenable to study by MRS.
Figs 24.10A and B: 360 MHz 1H MR spectra of head Although a greater availability of instru-
and neck tumors (A) control (B) failed case. ments, standardized methodology, a larger
Abbreviations: Chos, choline-containing compounds;
database of spectral changes correlated with
Cr, creatines; Gln, glutamine, Glu, glutamic acid; HOD,
deuteriated water; Lac, lactic acid; Leu, leucine; Lip, pathological conditions, and more NMR-
lipid; Tau, taurine; Val, valine. Assignments do not imply trained individuals in the clinical environ-
that these are the only substances contributing to a ment are necessary for routine use, the
particular peak9 discriminating power of MRS can and will
be exploited to attack difficult problems in
of screening is X-ray mammography, which
the clinic.
has a detection accuracy of approximately
The ex vivo 1H-NMR of tissue technique
70 percent. If lesions are detected, two has also enjoyed a remarkable degree of
approaches may then be followed; fine success in distinguishing cancer tissue from
needle biopsy or open biopsy, the former normal or otherwise abnormal tissue. The
being the less invasive. Diagnosis then methods thus developed are leading to
follows via conventional histology. extension of the studies to human subjects
1
H MRS of fine needle biopsies yields in vivo with a high degree of accuracy.
spectra which are significantly different for These studies are more time-consuming, but
normal and cancerous tissue. The ratios of it is expected that the savings in lives and
the areas of resonance due to choline and treatment cost will readily justify the effort.
creatine fall into two groups, with some With more automated methods for MRS or
overlap. Application of advanced statistical commercial MRI instruments, we should see
methods, including use of the genetic clinical MRS as a standard technique very
algorithm to choose the most discriminating soon.
regions of the spectra, and linear discri-
minant analysis, led to a specificity and ABBREVIATIONS AND ACRONYMS
sensitivity of 94 and 98 percent, with a high ADC Analog-to-digital Convertor
degree of robustness.79 An extra benefit COSY Correlated Spectroscopy
from this sophisticated analysis is the ability CSF Cerebrospinal Fluid
to determine with high accuracy, from only DMA Dimethylamine
a one pulse 1H MR spectrum of a biopsy of MRS Magnetic Resonance Spectroscopy
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378 Biomedical Magnetic Resonance: Proceedings of the International Workshop
25
MR Metabolomics: Studies
of Gene Function and
Novel Anticancer Drugs
JR Griffiths, YL Chung
considering the results that have been THE ROLE OF NUCLEAR MAGNETIC
produced with this approach, we should RESONANCE METHODS IN
review the problems and opportunities METABOLOMICS
presented by metabolomics. Most of the Metabolomics presents several methodologi-
published, systematic work is concerned cal challenges beyond those of the other -
with the metabolomes of plants2 or micro- omic sciences. Ideally, to specify the instan-
organisms such as yeast,3,4 and there has taneous state of the metabolome, we should
been little systematic work on animal like to know about the rates at which
metabolomes, still less in humans. However, metabolites flow through all the metabolic
the power of this approach in human disease pathways (“metabolic fluxes”). There is no
and particularly in drug development is way to measure pathway fluxes on a large
obvious, and studies on simplified systems scale at present, so it is necessary to measure
are already showing useful results. the steady-state concentrations of the
Like the transcriptome and proteome metabolites. This need for precise measure-
(but unlike the genome), the metabolome is ment of large numbers of metabolite
state-specific. Whereas an organism’s concentrations, ideally with an accuracy of
genome can be considered as an absolute, better than a few percent, is technically
the transcriptome in one of its cells will quite challenging. Many metabolites are
vary continuously according to its present at very low concentrations and some
physiological state, as different genes are are extremely labile. There is no current
expressed and the mRNA molecules are method that will simultaneously measure
spliced. There will be still more variation in the concentrations of thousands of
the proteome because of post-translational metabolites in a manner analogous to
modifications to the peptides translated from transcriptomics or even proteomics (although
the spliced mRNA molecules. The mass spectrometry methods may eventually
metabolome is yet more labile; metabolite do this), but by using NMR we can measure
concentrations can change within seconds sub-sets of the metabolome, termed
in response to nerve signals (e.g. the Ca2+ “metabolic profiles”.5 As studies of the entire
induced activation of glycogenolysis when metabolomes of microorganisms such as
a muscle receives a signal to contract) but yeast are completed, it will be possible to
in an unperturbed cell or tissue they usually correlate metabolic profiles of human cells
settle to a metabolic steady state. It is this or tissues with them. Even without such a
state-specificity that gives the “-omics” framework, useful information can be
methods their strengths in medicine: for obtained by interpreting the profiles in the
instance, one can consider the totality of light of conventional biochemical knowledge.
changes in gene expression or protein
content caused by a disease such as cancer NMR-Derived Metabolic Profiles
or even by a drug used to treat it. High-resolution NMR of cell or tissue ext-
Measuring the state of the metabolome will racts is currently a very attractive technique
enrich our understanding of the data from for obtaining metabolic profiles without the
the other –omics methods. need for elaborate sample preparation. A
380 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 25.1: 1H NMR spectrum of tumor cell extract. Peaks assigned as: sodium 3-trimethyl-silyl-2,2,3,
3-tetradeuteropropionate (1-included as a chemical shift reference and for quantification), isoleucine (2), valine
(3), lactate (4), alanine (5), acetate (6), glutamate (7), succinate (8), glutamine (9), creatine and phosphocreatine
(10), phosphocholine (11), scyllo-inositol (12), myo-inositol (13), glycine (14), creatine (15), phosphocreatine
(16), glucose (17), tyrosine (18), ATP and ADP (19), NADH (20), formate (21). For detailed peak assignments
see References 37,38
simple, one-dimensional NMR spectrum can 10 μM) in the cell will be detectable in a
quantify a substantial number (typically reasonable time by this rather insensitive
30-50) of metabolites, many of which show method. A metabolite must also have at least
variations in disease (Fig. 25.1). The main one resonance that can be resolved from its
constraint on NMR visibility is concentra- neighbors. In difficult cases, one can use
tion—only metabolites present at high methods such as the many types of 2-D NMR
concentrations (typically in the millimolar to resolve peaks. Shifting the pH of the
range, but with a lower limit of around sample is also often able to resolve a peak.
MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs 381
Brindle6 has pointed out two important on extracts made by grinding a sample with
reasons why these simple NMR-derived perchloric acid (for water-soluble
profiles can give useful insights into the metabolites) or organic solvent (for lipids).
metabolome. First, the metabolic pathways The extracted samples are then freeze-dried
within a cell interconnect so richly that each and reconstituted in deuterated solvents
metabolite is only a few removes from all prior to analysis. Extractions can be made
other metabolites. Thus, any metabolic of cultured cells or experimental tumors
perturbation, e.g. a gene knockout, or the grown from human cancer cells in rodents,
action of a drug on a single target protein, or even from frozen biopsies taken from
is likely to have an indirect effect on at patients.7,8 In all cases, it is important that
least one of the NMR-detectable metabolites. the sample should be frozen or the acid
Second, the fact that NMR measures nume- added as quickly as possible, since
rous metabolites simultaneously, in the same metabolism continues post-mortem and
sample, means that each was subject to an concentrations of some metabolites (e.g.
identical preparation and assay procedure, glucose and lactate, if the glycolytic pathway
and thus the ratios of their concentrations, is active) rapidly depart from the steady-
one to another, can be measured with great state values that originally existed in vivo.
precision. The pattern of metabolite signals A cell culture plate can be extracted by
in the profile is often very characteristic of, pouring off the culture medium and rapidly
for instance, a disease state, and this allows washing the cells with saline (to prevent
computer-based pattern recognition chemicals from the culture medium from
methods to give diagnostic information. In contaminating the profile), adding an aliquot
contrast, individual metabolite concentra- of perchloric acid and scraping the cells into
tions, obtained by conventional analytical the acid. Experimental tumors in animals
methods, are usually subject to several can be rapidly frozen by the freeze-clamp
percent error. Even if several replicates of method in which they are crushed between
each sample are analyzed the ratios between the metal jaws of a hand-held clamp that
the mean concentrations of metabolites are have been pre-cooled in liquid nitrogen.
known much less precisely than those in an Provided appropriate precautions are taken,
NMR metabolic profile. For this reason, even and the samples are then kept deep-frozen
though the number of metabolites used can until the extraction is performed, these
be greatly increased by incorporating mean methods give “metabolic snapshots” that
concentrations obtained from replicate reflect the steady-state metabolism of the
individual assays, the pattern recognition tissue concerned at the moment when the
process (e.g. in the FANCY method, see sample was taken.
below) is often less successful.
Magic Angle Spinning NMR
SAMPLE PREPARATION FOR By using Magic Angle Spinning (MAS) NMR,
METABOLIC PROFILES one can obtain well-resolved spectra from
solid tissue samples without having to
Extraction extract them (Fig. 25.2). This method avoids
Metabolic profiling studies of genes artefacts induced by the extraction process,
involved in cancer are typically performed but gives less well-resolved peaks than
382 Biomedical Magnetic Resonance: Proceedings of the International Workshop
agents on tumors in laboratory animals was Table 25.1: Phospholipid metabolite levels in mouse
in fact one of the first uses to be proposed carcinoma at 48 hours after vehicle or 5-FU treatment
for MRS in cancer.18 Most of the published Metabolite Control 5-FU treated
chemotherapeutic work by NMR has been PE 0.56 ± 0.11 0.95 ± 0.29*
performed on routinely-used drugs, but it PC 0.76 ± 0.11 0.61 ± 0.12
can also offer important insights into the GPE 0.25 ± 0.06 0.36 ± 0.10*
GPC 0.82 ± 0.24 1.82 ± 0.61*
actions of novel anticancer agents both in
the laboratory and in clinical trials on Data are expressed as Mean + S.E.M
patients, and thus, help to accelerate the Metabolite concentrations in μmol/g tissue.
drug development process. *Significantly different from control group.
With regard to clinical trials, there has
been a trend to move away from the tradi-
tional approach in which a Phase I trial was cytotoxic agents and on novel drugs that
solely concerned with establishing the were in the process of development.
toxicity and safe dose level of an anticancer
drug. Now-a-days, every effort is made to MRS Studies of a Conventional Cyto-
produce data on the efficacy of the drug toxic Agent – 5-Fluorouracil
and to verify its mechanism of action, even 5-Fluorouracil (5-FU) is a widely-used cyto-
in a Phase I trial. Many elaborate MRI and toxic drug in medical oncology. Its PK and
PET protocols and even tissue biopsies are metabolism have been extensively studied
used to maximise the data obtained at the in vivo in animals and patients by 19 F
Phase I/II stage, and MRS methods could MRS19,20 and its PD has been monitored by
have a role here. Understanding whether a 31
P MRS. Street et al,21 used in vivo and in
candidate drug is acting on its intended vitro 31 P MRS to examine the
target is made more difficult because many pharmacodynamic effect of 5-FU on a mouse
of the anticancer agents currently under mammary carcinoma model. An increase in
development are not intended to cause NTP/Pi and PCr/Pi ratios was observed in
tumor shrinkage when used as single agents vivo 48 hours after 5-FU treatment. The in
(as they usually are in Phase I or Phase II vivo PE to PC ratio was also elevated relative
clinical trials). This means that conventional to controls and this was found to be due to
clinical indications of response or even an increase in PE (Table 25.1; as confirmed
sophisticated imaging methods will be by in vitro 31P MRS). In addition, increases
inadequate. Noninvasive MRI methods in GPC and GPE were also observed 48
(particularly, in the case of antivascular and hours after 5-FU treatment.21
antiangiogenic agents, dynamic contrast-
enhanced MRI) are therefore being adopted
MRS Studies of a Cyclin-Dependent
as surrogate pharmacodynamic (PD)
Kinase Inhibitor-R-
biomarkers. MRS could provide an
Roscovitine (CYC202)
alternative PD method for trials of such
drugs. CYC202 (R-roscovitine, Cyclacel Ltd,
The following sections will provide some Dundee, Scotland) inhibits the cyclin-
examples of PD studies performed by MRS dependent kinases 1, 2 and 7 and thus,
and metabolic profiling both on conventional blocks cell cycle progression. In vivo 31P MRS
386 Biomedical Magnetic Resonance: Proceedings of the International Workshop
was used to examine xenografts treated with analogue was used as a control. In all the
CYC202, to gain insights into the biochemical cell lines studied, there were significant
changes that are associated with cell cycle increases in phosphocholine and glycero-
disruption.22 phosphocholine levels following 17AAG
CYC202 treatment for 4 days caused treatment.
inhibition of tumor growth. 31P MRS of the In vivo, after 4 days of 17AAG treatment
tumors in vivo showed a significant decrease the HT29 tumors showed significant growth
in the intracellular pH and also in NTP/ delay and an increased in ratios of phos-
TotP (total phosphorus signal) ratio. A phomonoester/phosphodiester (PME/PDE),
significant increase in the Pi (inorganic PME/total phosphorus (TotP) and PME/β-
phosphate)/TotP and the Pi/NTP ratios nucleoside triphosphate (β-NTP), as well as
were also observed post-treatment, whereas a decrease in the β-NTP/TotP ratio (Fig.
no significant changes were seen in the 25.3). The elevated PME observed in vivo
vehicle group. As well as reducing cell was due to increased levels of phos-
proliferation, CYC202 impaired tumor phoethanolamine and phosphocholine as
bioenergetics and decreased tissue pH. If shown in vitro by 31P MRS of tumor extracts
CYC202 comes to clinical trial, these 31P MRS (Fig. 25.3). A significant inverse correlation
effects may provide a non-invasive marker was found between the percentage of
for assessing tumor response in patients. change in PME/PDE ratio and the
percentage of change in tumor size following
MRS Studies on 17AAG, 17AAG treatment. These 31P MRS changes
an Inhibitor of HSP90 suggested that it would be possible to
The heat-shock protein HSP90 is of interest monitor tumors in patients during the Phase
as an anticancer target because it helps 1 clinical trials of 17AAG, and such a study
maintain the shape of many oncogenic is currently under way.
proteins. Inhibiting single oncogenes with
“magic bullet” drugs has proved somewhat Pharmacokinetics and Metabolism
disappointing as the cancers often become MRS also has the unique ability, in some
resistant; a drug that acts indirectly on a cases, to monitor the changing concentration
large number of potentially oncogenic of the anticancer drug itself (PK) as well as
proteins may be able to overcome this its metabolism and detoxification. The first
problem. The HSP90 inhibitor 17AAG was MRS studies 19 on an anticancer drug,
developed with this strategy in mind and is 5-fluorouracil (5-FU), used 19F MRS on rat
currently under trial as an anticancer agent. tumors to monitor 5-FU PK, activation to
A surrogate marker of tumor response was toxic fluoronucleotides and the appearance
needed for the clinical trial, and noninvasive of detoxification products from the liver.
MRS methods were therefore investigated. There have been many other animal studies
The actions of 17AAG on several cultured on 5-FU and its derivatives (for a review,
human colon cancer cell lines and on the see Reference 20) and this widely used drug
HT29 human colon tumor xenograft in nude has also been monitored by 19F MRS in
mice were studied in vivo and in vitro using patients.24,25 A recent clinical study26 has
31 P MRS. 23 Treatment with an inactive
shown that the detoxification products of
MR Metabolomics: Studies of Gene Function and Novel Anticancer Drugs 387
Figs 25.3A and B: Expanded in vitro 31P MR spectra of extracts from (A) a control and (B) a 17AAG-treated
HT29 tumour. C and D: In vivo 31P MR spectra of an HT29 tumour (C) before and (D) after 17AAG treatment.
Peak assigned as: phosphomonoester (PME), comprised mainly of phosphocholine (PC) and
phosphoethanolamine (PE); inorganic phosphate (Pi); phosphodiester (PDE), comprised of
glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE); phosphocreatine (PCr), α-, β- and
γ-nucleoside triphosphates (α-, β-, γ-NTP).
Note that the ppm scale on A and B is referenced to methylenediphosphonic acid, whereas the ppm scale
for the in vivo spectra in C and D is referenced to PCr. The box drawn on C indicates the region of the in vivo
spectrum corresponding to the in vitro spectra shown in A and B
5-FU are conjugated and excreted via the species were also found in the apparently
bile duct after storage in the gall bladder. normal livers of two patients who failed to
In another recent study it proved possible develop liver metastases.27
to monitor the toxic fluoronucleotides in MRS studies using other nuclei to monitor
tumors in patients using a conventional 1.5 T anticancer drugs have also been performed
MRI instrument; surprisingly, these toxic on tumors in vivo. 1H MRS has been used to
388 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Table 25.2: Rate constants (k) of 5′DFUR, capecitabine and its intermediary metabolites
Fig. 25.4: A: In vivo 19F MR spectra of a tumour 52-72 minutes after an injection of capecitabine. B: Time
course of the accumulation and breakdown of 5′DFCR/5′DFUR. Spectral peaks assigned as: 5′-deoxy-
5-fluorocytidine (5′DFCR); 5′-deoxy-5-fluorouridine (5′DFUR); 5-fluorouracil (5-FU); α-fluoro-β-alanine (FBAL).
11. Allen J, Davey HM, Broadhurst D, Heald JR, The effects of CYC202 on tumors monitored
Rowland JJ, Oliver SG, Kell DB. High- by magnetic resonance spectroscopy.
throughput classification of yeast mutants for Proceeding of AACR, 1664 2002.
functional genomics using metabolic 23. Chung Y-L, Troy H, Banerji U, Jackson LE,
footprinting. Nat Biotechnol. 2003;6:692-696. Walton MI, Stubbs M, Griffiths JR, Judson I R,
12. King RD, Whelan KE, Jones FM, Reiser PG, Leach MO, Workman P, Ronen SM. Magnetic
Bryant CH, Muggleton SH, Kell DB, Oliver SG. Resonance Spectroscopic Pharmacodynamic
Functional genomic hypothesis generation and Markers of the Hsp90 inhibitor 17-allylamino,17-
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26
Metallopharmaceuticals as
Exogenous Contrast Agents
RK Sharma, R Mathur, AK Mishra
Gd-DTPA 10
Gd-EDTA 0.3
Gd-CI3 0.4
Meglucamine diatrizoate 18
Common X-ray contrast agent
Half life in urine = 21.0 min and in blood = 19.6 min after intravenous injection
Metallopharmaceuticals as Exogenous Contrast Agents 397
Fig. 26.2A: Gadolinium complexes with macrocyclic Fig. 26.2B: Acyclic chelating agents with different
chelating agents used as contrast agents functional groups as Gd-based MR contrast agents
selectivity of different ligands towards Gd3+ lable for interaction with Gd3+ complexes.
as compared to Zn3+, Cu3+, Ca3+. Iron (III) The main competitor to Gd3+ was found to
was not considered because it is tightly be Zn2+, and the most important thermo-
bound in vivo by the storage proteins ferritin dynamic criterion of toxicity is the selecti-
and hemosiderin and is essentially unavai- vity of the ligand for Gd 3+ over other
398 Biomedical Magnetic Resonance: Proceedings of the International Workshop
endogenous metal ions. Zinc trans bis (amide),30 silyl lewis X mimetic31 with
metallation was found to be the most likely the aim to obtain better relaxation and hence
mode of both acute and sub chronic toxicity a better contrast.
in experiments on rats. Complexes whose
structure make in vivo trans metallation Iron Oxide Nanocompounds
reactions much slower than renal excretion Nanoparticles are typically defined as the
rates have significantly improved toxicities particles in the nanometer size range
than would be predicted by thermo- (ranging typically from 10–200 nm) with
dynamics. Slower clearance from the body colloidal properties so that the particles do
is likely to significantly increase the toxicity not settle out of the solution. The size,
of any Gd3+ complex shows other ligands charge, hydrophilicity and composition of
often used in gadolinium studies. the nanoparticles can be manipulated to
Gd3+ has been shown to inhibit Ca2+ achieve optimal uptake within a tumor and
binding to mammalian cardiac sarcoplasmic specific targeting ligands (i.e., peptides,
reticulum. The mechanism of toxicity could monoclonal antibodies, small molecules) can
involve hemodynamic disruption. Potential be attached to the surface for specificity.
improvement also lies in the idea of The iron oxide nanoparticles tend to be
covalently coupling the ligand to protein to magnetic or superparamagnetic in nature
generate tissue-specific contrast agents.27 with the particle approximately 20 nm being
Ca2+ plays a very important role in signal the later. For the purpose of acting as MRI
transduction and its presence or absence is contrast agent the iron oxides can be
seen to be affecting the relaxivity of the classified as MION (mononcrystalline iron
DOPTA-Gd complex.28 oxides) and CLIO (cross linked iron oxides)
Understandably, search for new potential They are basically the primary iron oxide
ligands for Gd3+ complexation is still hot nanoparticles (the core of the iron oxide
area of investigation. The complexes are crystal consists of and iron oxide crystal
evaluated against Gd-DTPA, the only coated with a polymer. This class of
approved compound for human use so far. compounds normally consists of dextran
Criteria include thermodynamic stability, coated iron oxide preparations. The CLIO
rates of excretion, toxicity, lipophilicity, particles represent a stabilized dextran
biodistribution, percent change in MR signal coated iron oxide nanoparticle in which the
intensity. Some of the complexes are slightly iron oxide core is caged in the cross-linked
better than Gd-DTPA in particular tests, dextran coating. These compounds consist
others are a little worse. However, neither of nonstoichiometric microcrystalline
of them got as much attention as Gd-DTPA magnetite cores, which are coated with
itself.29 This complex is far not be the best dextrans (in ferumoxides) or siloxanes (in
choice as of today, but it is relatively well- ferumoxils). In case of SPIO (superpara-
known choice, widely used in many MRI magnetic iron oxide) the nanoparticle have
facilities on a daily basis. The Gd-DTPA a size greater than 50 nm (coating included)
complexes have been over the period been where as cases where the size is smaller
improvised chemically by using different than 50 nm the nanoparticles are termed as
substituents and derivatives like the DTPA- USPIO (Ultra small superparamagentic iron
Metallopharmaceuticals as Exogenous Contrast Agents 399
oxides). Lumirem and Endorem are (<10 nm) magnetic domains, which undergo
examples of SPIO where the former contains superparamagnetic relaxation or collective
silica coated particles 300 nm in diameter magnetic excitation on the Mossbauer time
and the later contains magnetite particles scale.33 There is no evidence of covalent
with 150 nm diamter. They are being used bonding between the iron surface and the
for the gastrointestinal tract imaging and surrounding dextran.35
for the liver and spleen disease detection. The SPIO compounds are promising
Sinerem is an example of USPIO and is contrast agents since their properties may
composed of magnetite particle with 30 nm be fine tuned for the specific application.
diameter and is currently being used for They are non-toxic and rapidly cleared from
tumor detection. The particle size greatly the organism. Experiments have been
affects the physicochemical and the successful in receptor-specific SPIO deli-
pharmacokinetic properties. SPIO agents are very.36
much more effective in MR relaxation than Porphyrins have been known for decades
their paramagnetic counterparts. Since they as indicators of various metabolic disorders
are nonstoichiometric, there was and still is and disease states.37,38 They are used in
much interest in studying these compounds photodynamic therapy of tumors. 39 Low
with all the vast array of modern physical- toxicity of metalloporphyrins and their
chemical methods: single crystal X-ray selective retention in tumors have led
diffraction, powder X-ray diffraction, recently to their study as an MRI contrast
Mossbauer spectroscopy, transmission media. 40-45 Theoretical treatment of
electron microscopy, dynamic light relaxivity of some of the metalloporphy-
scattering, atomic adsorption spectroscopy, rinshas been studied by Mercier. The
spectrophotometry, electron microscopy, proposed mechanism via which porphyrins
superconducting quantum interference accumulate in tumors is through selective
devices, etc.32-35 uptake by benzodiazepine receptors
The compositions and physiochemical abundant in tumor cells but not in normal
properties of nonstoichiometric magnetite’s cells.46 Mangafodipir trisodium (MnDPDP)
are continuously variable between those of was shown to be useful in establishing the
Fe3O4 and Fe2O3. Conceptually, these cation- diagnosis of acinar cell carcinoma, a rare
deficient, inverse-spinel phases are formed pancreatic exocrine neoplasm.47 MnDPDP,
by partial oxidation of Fe(II) in stoichio- which specifically labels liver tumors over
metric magnetite 24. The Fe2+ content is healthy liver tissue, helps determine if
typically 8-15 mol percent. The lattice surgery on hepatic tumors is a viable
parameters of these colloids also fall between option. 48-50 This is a fairly new area of
those of Fe3O4 and Fe2O3. development, but metalloporphyrins of
The particles are usually of varying sizes Mn(III) and Fe(III) show favorable pro-
from several to several hundred nanometers. perties as MRI contrast agents for tumor
They are irregular in shape and highly light- detection.
absorbing. They have no magnetic hysteresis
at ambient temperatures, which is charac- Gastrointestinal Contrast Agents
teristic of superparamagnetic materials. The gastrointestinal tract cannot be reliably
Mossbauer spectra are characteristic of small studied by MRI without the use of contrast
400 Biomedical Magnetic Resonance: Proceedings of the International Workshop
agents. Oral contrast agents may drama- heavily T1 weighted images. This effect is
tically improve utility of MRI for gastro- successfully utilized in contrast enhanced
diagnostics. The only clinically approved MR angiography.62,63 Various iron oxide
agents for that purpose are soluble iron nanoparticles are suitable contrast agents for
compounds. (ferrous gluconate, ferric MRI angiography.64,65 Superparamagnetic
ammonium citrate) and Gd-DTPA. There is iron oxide particles have been shown to
a problem with dosage of iron salts, which accumulate in human atherosclerotic plaques
may not exceed the levels above those when which indicates potential for noninvasive
iron supplementation is used. There are no assessment of active atherosclerotic
particulate agents approved for oral use yet. disease.66
Gadolinium(III) DTPA has been used to
depict the lumen of the digestive organs in Other Contrast Agents
rats.51 It is effective in the primary diagnosis
Examples of contrast agents other than
of Crohn’s disease and in differentiating
paramagnetic involve rather mechanical
nonactive from active forms of the
action than changing the characteristics of
disease.52,53 Gadolinium(III) DOTA has been
the surrounding tissue. They are thus, similar
used to examine the movement of material
to agents used in X-ray methods. One recent
exiting the stomach and for diagnosis of
example successfully utilized vege-table oil
delayed gastric emptying 54,55 zeolites
for rectal MRI applications. 67 Other
containing gadolinium have been shown to
possibilities include insufflation of air to
be an inexpensive, non-toxic alternative to
achieve better contrast of intestinal walls.68
X-rays and barium salts for imaging of the
GI tract.56,57 Further, hectorite clay that has
undergone ion exchange with gadolinium CONCLUSION
ions has been used as an oral contrast agent We have summarized the various exogenous
for GI tract imaging,58 superparamagnetic contrast agents available for MRI in this
iron oxide agents have been used to image chapter. Contrast agent enhanced MRI is an
the GI tract and assess ulcerative colitis.59,61 invaluable tool to the diagnosis of cancer
and many other diseases, and recently in
Contrast Agents For Angiography molecular imaging of experimental animals.
MR angiography usually requires a rapid With improved targeted delivery of these
bolus injection of a paramagnetic ECF agent agents, the ability to diagnose diseases will
since these agents rapidly redistributes from become more sensitive, accurate, and
the vascular space into the interstitium. ultimately simplified. The activatable agents
Following such a bolus injection the initial that respond to biological phenomena by
gadolinium concentration in blood can easily altering the intensity of signal enhancement
be ten times higher than the steady state in a conditional fashion are steps toward
concentration. The benefit of such a high unraveling the complex connectivity of
gadolinium concentration is that the developmental biological systems. In order
resulting T1 of blood is extremely short to further the usefulness of contrast agents,
typically less than 100 ms thus giving rise chemistry needs to be used to develop
to a very large blood background contrast agents that are responsive to biological
Metallopharmaceuticals as Exogenous Contrast Agents 401
phenomena and directed to specific regions. 17. Friebolin H. Basic One- and two-dimensional
These two improvements are closely related NMR spectroscopy, VCH, Germany, 1993.
18. Runge VM, Clanton JA, Herzer WA et al.
in that many biologically significant
Radiology 1984;153:171.
molecules are located in specific parts of 19. Mendonca-Dias MH, Gaggelli E, Lauterbur PC.
the body. Further, these agents may Semin Nucl Med 1983.
represent the prelude to complete, non- 20. Vlaardingerbroek MT, Boer, den JA. Magn
invasive, medical examinations that are safe, Reson Imaging. Theory and Practice 1996;
fast, and accurate. 242-43
21. V Runge VM, Clanton JA, Lukehart CM et al.
Am J Roentgend 1983;141:1209.
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27
High-resolution MR Spectroscopy
in Clinical Medicine
GA Nagana Gowda, Ashish Gupta, M Bhandari, CL Khetrapal
bacterial strains: P. aeruginosa (ATCC-25922, day before and at every 24 hrs after the
NCTC-10662); E. coli NCTC-10418; ATCC- transplant for a period of 7 days. Blood
25923, K. pneumonia NCTC-9633; ATCC- samples were centrifuged at 5000 rpm,
13883, Enterobacter ATCC-13048; Acinetobacter 20oC and the supernatant was decanted to
ATCC-19606; Proteus mirabilis ATCC-49565; obtain the serum. These body fluids samples
Citrobacter ATCC-8090; Enterococcus faecalis were subjected to one dimensional MR
ATCC-19433; Streptococcus gp B ATCC-13813; experiments with water suppression by
and Staphylococcus aureus ATCC-12600. presaturation. Two-dimensional experiments
Bacterial growth media were first prepared were performed for the confirmation of the
by addition of nicotinic acid (1.1 mg/ml) in assignments made using one-dimensional
sterile urine and bacteria of about 3 × 107 spectra. Assessment of the liver graft was
cfu. The media were divided into two parts based on biochemical parameters obtained
of 1.0 ml each. One part was used for from conventional method.
counting the bacterial cells by growing them
on Mac Conkey agar medium on culture RESULTS AND DISCUSSION
plates and counting the colonies grown after
incubation. The other part was kept for Malabsorption Syndrome
incubation at 37oC with shaking at a speed Typical spectra of urine sample before and
of 150 rpm, for 6 hours. Subsequently, the after xylose-test are shown in Figure 27.1.
bacterial suspensions were removed and the Quantity of D-xylose estimated in same urine
supernatant parts were subjected to MR
specimens was higher with MR than
experiments. MR experiments were also
colorimetric method in patients without
performed on the bacterial media of
malabsorption syndrome [median 1.58 (0.72
P aeruginosa varying from about 103 to 107
to 3.20) versus 1.03 (0.32 to 1.89) g, p <0.0001,
cfu/ml in multiple of 10. Control samples in
triplicate (without bacteria) were also made Wilcoxon’s signed rank test). Xylose
under similar conditions and subjected to estimated by colorimetry from standard
MR experiments. Experiments on the urine additions showed a maximum error of 20
samples from 30 patients of P. aeruginosa percent, while that estimated by MR
infection were performed. Nicotinic acid (1.0 showed 7 percent. The quantity estimated
mg/ml) was added to the urine, incubated by MR in the presence of glucose showed
at 37oC and the supernatant was taken for 8 percent error. MR is less prone to error as
the MR experiments as explained above in compared with colorimetry in estimating
the case of standard bacterial strains. For D-xylose. Quantification of D-xylose using
unambiguous assignment of the metabolic colorimetry and its potential limitations are
product of the nicotinic acid induced by P. reported in detail in literature. 3,4 It is
aeruginosa, homonuclear and heteronuclear reported that the concentrations of the
two-dimensional experiments were standard xylose samples estimated using
performed. identical procedure show widely different
results. These varied results have been
Assessment of Liver Graft Function attributed to temperature, time and light
The blood and urine samples of the patients dependence of the resultant colored
undergoing liver graft were collected one complex. 3 The results presented here
406 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 27.1: One dimensional 1H MR spectra of typical (a) pretest and (b) postxylose urine specimen of a patient
with suspected malabsorption syndrome. D - xylose signals of both the isomers (α and β) are clearly seen in
the postxylose urine (figure 1b) which are absent in the pretest urine (figure 1a). The assignments of signals
of α and β isomers are made in figure 1b. The structures of α- and β-D - xylose are shown with the numbering
of all the nonlabile protons.
isomers, overlap with the glucose signals. Therefore, addition of small quantity
Further, proton 1 of β−xylose cannot be used (about 1 mg/ml) of nicotinic acid to the
for quantification for the reason of partial urine specimen containing the micro-
saturation during water suppression and organism P. aeruginosa yields 6-OHNA after
hence, in such cases, proton 1 of the α− incubation for about 6 hours.
xylose at 5.2 ppm is the only signal left for Analyses of the MR spectra of the
the quantitative estimation of the total bacterial media with variable cell count of
xylose. Therefore, glucose was estimated P. aeruginosa strain showed that the intensity
using the ratio of the isomers as 1 : 1.9 of nicotinic acid signals gradually decreased,
which requires area of only proton 1 of the while, the intensity of the signals of the
α isomer. There was no significant difference 6-OHNA increased with increasing number
between the xylose estimated in the presence of bacterial cells. This clearly establishes that
of glucose using the ratio (maximum error the 6-OHNA produced depends on the
8%) and that estimated using the average number of bacteria.
area of the signals (maximum error 7%). On In the spectra of E. coli NCTC-10418;
the other hand, in the colorimetric method ATCC-25923, K. pneumonia NCTC-9633;
the presence of glucose is shown to interfere ATCC-13883, Enterobacter ATCC-13048;
with the quantification of the xylose Acinetobacter ATCC-19606; Proteus mirabilis
concentration leading to misleading results.20 ATCC-49565; Citrobacter ATCC-8090; Enter-
Thus colorimetric method may pose a serious ococcus faecalis ATCC-19433; Streptococcus gp
problem in patients with uncontrolled B ATCC-13813; and Staphylococcus aureus
diabetics mellitus having glycosuria. ATCC-12600 showed that they do not
metabolize nicotinic acid. This suggests that
Bacterial Infection only P. aeruginosa metabolises nicotinic acid
among the bacteria causing UTI and thus
Figure 27.2 shows one dimensional 1 H
nicotinic acid metabolism is specific to P.
spectra of incubated bacterial media of
aeruginosa.
E. coli K. pneumonia, and P. aeruginosa. In
29 out of 30 patients of UTI showed
the spectra of P. aeruginosa media, Nicotinic
metabolism of nicotinic acid to 6-OHNA as
acid signals were totally absent and, simul- observed by the reduction of the intensity
taneously, new set of signals were seen. of nicotinic acid signals with concomitant
Using two-dimensional experiments, DQF- appearance of 6-OHNA signals. However,
COSY and HMBC, the new set of signals in in one of the samples, 6-OHNA could not
the media of P. aeruginosa were assigned to be detected under similar conditions owing
6-hydroxynicotinic acid (6-OHNA). to its less concentration. The concentrations
Pseudomonas group of bacteria metabolize of 6-OHNA determined from the MR
NA giving rise to 6-OHNA as follows: spectra for 29 patients were in good agree-
COOH COOH
ment with that obtained from conventional
H2O
H2
method. Thus from a single MR experiment,
N HO N the identity of the bacteria as well as the
Nicotinic acid 6-Hydroxynicotinic acid viable bacterial count could be obtained.
408 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 27.2: Parts of 1H MR spectra highlighting the signals of substrate (nicotinic acid, marked as NA) and its
metabolite 6-OHNA. The spectra shown are of sterile urine [marked as “medium”], growth medium [sterile
urine with 1.1 mg/ml nicotinic acid, marked as “medium +NA”] and the remaining ones are in the growth media
after incubation for 6 hours in presence of 107 cfu/ml of E. coli (ATCC-25923, NTCC-10418), K. pneumonia
(ATCC-13883, NCTC-9633) and P. aeruginosa (ATCC-25922, NCTC-10662)
High-resolution MR Spectroscopy in Clinical Medicine 409
Fig. 27.3: Part of the 1H MR spectra of serum, showing levels of glutamine ( within dotted lines) in mg/dL on
different days: (a) 1 hour before transplantation, (b) 5 hours after transplantation on day 1, (c-f) from day 2 to
day 5 post transplantation, respectively. The numbers on the extreme left denote the corresponding INR, ALT,
AST (in international units) and Lactate (LAC, mg/dL) as observed by routine biochemical methods. ND: not
detected
410 Biomedical Magnetic Resonance: Proceedings of the International Workshop
urine culture to predict significant bacteriuria. CL. A new dimension of 1H NMR spectroscopy
J Clin Microbiol 1986;23:160-62. in assessment of liver graft dysfunction. NMR
13. Gupta A, Dwivedi M, Manocha H, Nagana Biomed 2003;16:185-88.
Gowda GA, Ayyagari A, Bhandari M, Khetrapal 18. Holbrook IB. Gastrointestinal tract function
CL. 1H NMR Spectroscopy in the Diagnosis of tests. In the Varley’s practical clinical biochemis-
Pseudomonas aeruginosa induced Urinary Tract try, 6th edition, Gowenlock Ah et al. (eds).
Infection. NMR Biomed (communicated). Heinemann Professional, Oxford 1988;706.
14. Kuntz E, Kuntz HD. Hepatology Principles and 19. Benesi AJ, Falzone CJ, Banerjee S, Farber GK.
Practice: Springer-Verlag, Heidelberg 2002; NMR assignements for the aldopentoses.
79:48-49. Carbohydr Res 1994;258:27-33.
15. Waniewski RA. Physiological levels of ammonia
20. Kohout P. Small bowel permeability in dia-
regulate glutamine synthesis from extracellular
gnosis of celiac disease and monitoring of
glutamate in astrocyte cultures. J Neurochem
1992;58:167-74. compliance of a gluten-free diet (gut perme-
16. Suarez I, Bodega G, Fernandez B. Glutamine ability in celiac disease). Acta Medica (Hradec
synthetase in brain: effect of ammonia. Neuro- Kralove). 2001;44:101-04.
chem International. 2002;41:123-42. 21. Choi CG, Yoo HW. Localized proton MR
17. Singh, HK, Yachha SK, Saxena R, Gupta A, spectroscopy in infants with urea cycle defects.
Nagana Gowda GA, Bhandari M, Khetrapal Am J Neuroradiol 2001;22:834-37.
28
Clinical Assessment of
Breast Cancer using 1H MRS at
High Magnetic Field
M Garwood, A McIntosh, PJ Bolan, S Meisamy, CJ Snyder,
A Stycznski, L DelaBarre, J Ellermann, E Gulbahce, TM Tutte,
LI Everson, TH Emory, MT Nelson, D Yee, JT Vaughan
disease. It is often used when there is a similar to those of normal and benign breast
mammographic abnormality, a palpable tissues. Thus, reliable detection of breast
lesion, and during pregnancy. This modality cancer by MRI requires intravenous injection
avoids the exposure to ionizing radiation. of a paramagnetic reagent (Gd-DTPA) that
The specificity and sensitivity ranges from enhances image contrast. 21 Due to the
80 to 93 and 33 to 50 percent, respectively, greater blood volume, leakiness of the lesion
in high risk women.6,7 vasculature, and extracellular space, Gd-
The imperfect specificity and sensitivity DTPA accumulates and thus, shortens T1
of these modalities leads to missed cancers more in the lesions than in normal (fibro-
and benign biopsies. Magnetic resonance glandular) tissue.
imaging (MRI) has been proposed as an Many studies have shown that contrast-
alternative diagnostic to improve screening enhanced MRI offers nearly 100 percent
performance in high-risk women.9 Addi- sensitivity for detecting breast cancer, but
tionally MRI may be used to follow response unfortunately its ability to differentiate
to neoadjuvant chemotherapy,10-15 to detect benign from malignant breast lesions appears
local recurrences16 and occult primaries,17 less impressive, with published values for
and to improve preoperative staging.18,19 specificity varying in the 30 to 90 percent
Importantly, MRI scanning is noninvasive range. 22 Low specificity is a significant
and exposure to a strong static magnetic drawback because ~70 percent of breast
field in the range typically used for magnetic lesions found in the general population are
resonance scanning (~1-10 Tesla) results in benign and their detection results in
no known long-term biologic effects. In unnecessary surgical procedures, costs, and
addition to providing high-resolution images patient anxiety. Although recent reports
of soft tissue anatomy, MRI offers the support the use of MRI for screening women
possibility to measure multiple physiologic with a hereditary predisposition to breast
and functional properties of tissue. The use cancer (e.g., see9,23), the variable specificity
of MRI in detecting breast cancer was first of contrast-enhanced MRI limits its utility
reported in 1985.20 for screening and diagnosing breast cancer
Commonly exploited sources of image in the general population, despite its
contrast in MRI rely on the existence of impressive sensitivity.7
differences in the water relaxation rates A related technique, magnetic resonance
described by the longitudinal and transverse spectroscopy (MRS), offers a noninvasive
relaxation rate constants, T 1 and T 2 . way to measure a metabolic profile of the
Differentiation based on the uniqueness of tumor, providing a “fingerprint” of cancer.
T1 or T2 in different tissues is often sufficient The ability to detect metabolic alterations
to allow good separation of normal from noninvasively in cancer cells can be exploited
malignant tissues in many parts of the body. to enhance our understanding of their
However, standard imaging methods (or aberrant metabolic and biochemical pro-
“pulse sequences”) designed to detect perties. Furthermore, the metabolic profile
differences in the relaxation rates usually that can be measured by MRS may provide
perform poorly in breast because the T1 and information that can be used to improve
T2 values of malignant lesions are often very the accuracy of diagnosis and may provide
414 Biomedical Magnetic Resonance: Proceedings of the International Workshop
tCho signal intensity between malignant and the boot-strap method.48 In the author’s
benign lesions would need to be a factor of experience, sometimes the tCho signal is not
>100 for the qualitative approach to be detectable or has low SNR due to small
reliable for diagnosis, but the difference in lesion size, deep location, large adipose
tCho signal is not typically this large. tissue content, and/or low cellularity. In
Therefore, a procedure to calibrate the tCho such cases, the MRS measurement may be
signal is required to yield a tCho concen- indeterminate, in which case the error
tration, [tCho]. assessment must make this known to the
The two basic elements of a quantitative reader. When using MRS for breast cancer
MRS methodology are the referencing diagnosis, for example, it is important to
strategy and the spectral fitting technique. know when the [tCho] measurement is
Our preferred referencing strategy uses indeterminate since in this case it should
water as an internal reference signal.44 This not be a factor in the decision-making
approach models the breast as a two- process. The error estimation should provide
compartment tissue (i.e., lipid and aqueous an indication of whether a weak or absent
fractions), with the assumption that tCho tCho signal is truly reflecting low [tCho] in
molecules reside only in the aqueous the lesion, in which case the lesion is
fraction. Accordingly, the water referencing probably benign, or is caused by a technical
approach compensates for the partial volume limitation, such as inadequate SNR because
of adipose tissue in the voxel and naturally the lesion is too small and/or has a low
leads to a molal concentration (mmol/kg) aqueous fraction, for example. To achieve
for the water-soluble metabolite, tCho. The this, a minimum detectable level (MDL) of
fitting technique is based on the TDFD [tCho] can be calculated.44 In any measure-
approach,46 which allows flexible lineshape ment in which the MDL is greater than the
definition using a time domain model and threshold [tCho] value that is indicative of
has excellent frequency-selection properties malignancy (>1 mmol/kg water), it is
since the residuals are evaluated and obvious that the MRS measurement is
minimized in the frequency domain. The indeterminate and as such the information
ability to select a narrow frequency range should not be used in making the diagnosis.
is crucial for fitting weak signals in the Besides the procedures described above,
presence of very large ones, as is the case research has revealed additional technical
in breast spectra containing large lipid requirements for performing high quality
signals. Previous studies using this method breast MRS, beyond those commonly
have shown that breast malignancies encountered in brain MRS. Firstly, it was
typically present with [tCho] >1 mmol/kg.44 discovered that metabolite peaks such as
The ability to estimate the error of the tCho are sometimes obscured by the
[tCho] measurement is another advantage abundance of mobile lipids which produce
of the quantitative approach. A common spurious sidebands that interfere with
method to estimate the error in quantitative metabolite signals. To solve this problem,
MRS is based on the Cramer-Rao minimum echo-time averaging was found to be
variance bound,47 although other approaches necessary. 49 Secondly, large shot-to-shot
may be better suited to breast MRS, such as frequency shifts associated with respiratory
Clinical Assessment of Breast Cancer using 1H MRS at High Magnetic Field 417
motion of tissues in the chest and abdomen determined by the wash-out phase: pixels
occur at high magnetic fields. If not showing decreased signal are colored red,
corrected, these frequency shifts significantly those with little or no change are colored
reduce spectral resolution and increase the green, and those with increased signal are
error in assessing the tCho signal.50 Finally, colored blue.
to obtain high quality spectra of breast, the
RF detector coil must be specifically BREAST CANCER TREATMENT
designed and optimized for the breast.
With these tools in hand, high quality Treatment of breast cancer has changed
1
H spectra of breast lesions can be acquired dramatically in the last 40 years, due to
fairly routinely. Representative examples are advances in surgical technique, chemo-
shown here for two types of benign lesions, therapy, radiation therapy, and radiographic
detection. Throughout the last century,
cyst and fibroadenoma, and an IDC. In the
breast cancer treatment was mainly surgical,
cyst (Fig. 28.1, Plate 19), the tCho level was
and was based on local control of the
too low to be quantified. The simulated peak
disease. With this local control in mind, most
(dashed line) provides an estimate of the
surgeons performed radical mastectomies,
tCho signal that would have been necessary
as popularized by Halsted.52 However, it
to obtain a reasonably accurate measure-
became apparent that smaller surgeries could
ment (i.e., the MDL), with an error of ±0.4
also achieve excellent local control, and
mmol/kg in this case. In the fibroadenoma several large studies showed no survival
and IDC (Figs 28.2, Plate 19 and 28.3, Plate benefit to radical surgical excision when
20), tCho was measurable, and the fit of the compared to procedures that left the breast
tCho resonance and the residual are shown intact.53,54 Local control remains important
above and below each full spectrum. In each as recurrences were substantially reduced
case, the tissue location giving rise to the with whole breast irradiation after lumpec-
spectrum shown is indicated by the box tomy when compared to women who
drawn on the image. The anatomical images received no radiation treatment.55
depict a slice from the high-resolution 3D Once it was understood that initial
image dataset acquired approximately 5 surgical technique did not affect overall
minutes after Gd-DTPA injection. The Gd- survival, additional systemic strategies were
DTPA enhancement kinetics are para- necessary to reduce the risk of recurrence
meterized using a color overlaid on the in distant organs. Both chemotherapy and
anatomical images. These color-coded maps hormone therapy had proven to be effective
are based on the three-time point (3TP) in women with advanced breast cancer, so
algorithm described by Degani et al.51 This these treatments were applied to women
method uses images acquired at three time with respectable disease. Both types of
points–precontrast, immediately post- systemic therapy were effective at reducing
contrast, and delayed postcontrast–to recurrence and death from breast cancer56,57
characterize the “wash-in” and “wash-out” highlighting the need to provide appropriate
phases of the Gd-DTPA uptake curve. The systemic treatment, even in apparently early
brightness of each image pixel is determined stage breast cancer.
by the amount of enhancement during the Systemic therapy, either hormonal or
wash-in phase. The color of each pixel is chemotherapy, can be given prior to surgery
418 Biomedical Magnetic Resonance: Proceedings of the International Workshop
in women with large tumors.58,59 This type protocol without technical problems, the
of “neoadjuvant” therapy can reduce tumor change in [tCho] between baseline and 24
size prior to surgery. While neoadjuvant hours after the first dose of PST showed
chemotherapy provides no survival significant positive correlation with the
advantage over post-surgical adjuvant change in lesion size measured at the end
chemotherapy,58 it does allow clinicians to of four cycles of PST (R = 0.79, p = 0.001).
assess the response of tumor to a particular The change in [tCho] within 24 hours was
drug and increases the number of women significantly different between responders
who can receive breast conserving therapy. and nonresponders (p = 0.007), based on
Archer et al showed that tumor apoptosis RECIST criteria.62 These results suggest that
can be detected almost immediately after the change in [tCho] within 24 hours after
the administration of chemotherapy.60 Thus, the first dose of PST can serve as an early
response to specific chemotherapeutic agents indicator for predicting clinical response to
could be identified in patients treated with doxorubicin-based chemotherapy in locally-
neoadjuvant chemotherapy. Armed with the advanced breast cancer. A representative
knowledge that malignant and benign set of MR data from an objective responder
lesions have differing biochemical profiles, is shown here (Fig. 28.4a, Plate 20 and 28.4B
and that these changes can be detected quite and C, Plate 21 and 28.4D, Plate 22).
early, we hypothesized that quantitative 1H
MRS can detect early specific changes in
FUTURE PROSPECTS
breast lesions that directly correlate with
apoptosis and subsequent tumor response With the increasing availability of high field
to neoadjuvant treatment. (>3 Tesla) systems and improved
methodology, spectral quality can be
Predicting Response to Neoadjuvant significantly better than that of previous
Chemotherapy using 1H MRS years. It is clear that high field MR scanners
will have an increasing role in the clinical
Primary systemic therapy (PST), also known assessment of cancer in the near future.
as neoadjuvant chemotherapy, given prior From our experience, we feel that now is
to surgery offers several advantages over the appropriate time to distribute the latest
standard post-operative chemotherapy. The technology and to begin multicenter trials
ability to immediately detect response to a to evaluate the use of MRS as an adjunct to
specific choice of therapeutic regimen would MRI in diagnosing breast cancer and for
be ideal, since it would allow for optimal assessing response to primary systemic
individualization of the regimen with therapy in locally advanced breast cancer.
improved likelihood of achieving a
pathologic complete response. With the ACKNOWLEDGMENTS
above MRS tools in hand, we performed a
study designed to determine whether early This research was supported by the DOD Breast
Cancer Research Program DAMD 17-01-1-0331, NIH
changes in [tCho] could provide a biomarker grants RR08079, CA92004, CA94002, RR00400, PHS
of clinical response as soon as 24 hours after Cancer Center Support Grant P30 CA77398, the
the first dose of doxorubicin-based PST for Tickle Family Land Grant Endowment in Breast
locally advanced breast cancer.61 Of the first Cancer Research, and the Lillian Quist-Joyce Henline
13 patients who successfully completed the Chair in Biomedical Research.
Clinical Assessment of Breast Cancer using 1H MRS at High Magnetic Field 419
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MR Applications in Experimental and Animal Model Systems 423
29
MR Applications in Experimental
and Animal Model Systems
Kurt V Schenker
Figures 29.2A to C show images of the 6-month-old wild-type (control) mouse (A),
mouse brain acquired with a DWI pulse a 6-month-old transgenic mouse APP23 (B),
sequence. The echo time was TE = 50 ms, a 25-month-old wild-type mouse (C) and a
the repetition time TR = 1325 ms, the 25-month-old transgenic mouse APP23 (D).
resolution was 128 x 64 pixels with a field- The color red encodes high diffusivity, blue
of-view of 25.6 x 25.6 mm2, i.e. an in-plane encodes a reduced diffusivity. The images
resolution of 200 μm × 400 μm, which is reveal a substantial ADC decline in the
typically sufficient for DWI applications and medial and dorso-lateral cortex of the aged
also allows to keep the scan time short. The APP23 mice compared to the age-matched
total acquisition time for the five b values control mouse or young APP23 and control
measured to produce the ADC maps animals. In the dorso-lateral cortex of the
represented in Figure 29.3, Plate 22 was 6-month-old APP23 mouse few areas with
7 minutes. The slice thickness was 1 mm, reduced ADC are visible.
the inter-slice gap 0.6 mm. The diffusion One day after the DWI experiments mice
encoding gradient was the applied along were sacrificed and brains were prepared
the slice direction, i.e. the experiment is for immunohistochemistry. APP23 mice
sensitive to the incoherent motion revealed robust compact amyloid-β plaques
component of water perpendicular to the in the neocortex but only few and diffuse
image plane. Animals were anesthetized amyloid in the caudate putamen. No
with isoflurane, intubated and artificially amyloid deposition was detected in
ventilated. This helps to avoid motion 6-month-old APP23 mice and wild-type
artefacts to which DWI sequences are rather control animals (Fig. 29.4, Plate 23).
sensitive. For further data processing Observations suggest that the ADC
regions-of-interest (ROI) were defined: ROI reduction in the neocortex of the aged APP23
1 for the lateral and medial caudate mice is related to the amyloid deposition in
putamen, ROI 2 for the neocortex, the brain of these animals. However, ADC
additionally subdivided into medial cortex values were not reduced in all cortical areas
(2a), dorso-lateral cortex (2b), and ventro- containing amyloid. For example, the ventro-
lateral cortex (2c). lateral part of the neocortex did not show
The color-coded ADC maps in Figure a reduction in ADC although this region
29.3, Plate 22 show each a single slice of a exhibited a significant amyloid load.
Figs 29.2A to C: Sections through the mouse brain with regions-of-interest (ROI) selected for quantitative
data analysis indicated (see text). Courtesy M. Rudin, Novartis Inst. for Biomedical Research, copied from.4
MR Applications in Experimental and Animal Model Systems 427
Interestingly, this brain region develops we can express the activation induced
more diffuse amyloid compared to the percentage change in CBV as
medial and dorso-lateral parts of the ΔCBV%(t) = ln {S(t)/S0}/ln{S0/Spre} × 100
neocortex. Thus, one may argue that not
Animals were anesthetized with isoflu-
only the amount of amyloid deposition but
rane and actively ventilated. As a contrast
also its nature (compact versus diffuse)
agent 50 mg/kg ENDOREM™ (ferumoxides,
determines diffusivity within cerebral tissue.
11.2 mg Fe/ml; Laboratoire GUERBET,
Roissy, France) was injected intravenously.
AGE-DEPENDENT IMPAIRMENT OF Twenty minutes after injection the plasma
SOMATOSENSORY RESPONSE IN concentration of ENDOREM has reached a
APP23 MICE5 steady state, the plasma half-life was
Functional MR imaging (fMRI) methods are measured to be 3.5 hours. A single slice
suited to measure neuronal function non- including the somatosensory cortices was
invasively. fMRI probes secondary changes recorded in the fMRI studies using a fast
associated with neuronal activation such as spin-echo sequence (see Figs 29.5A and B).
regional changes in cerebral blood volume The sequence parameters were: TR = 1135
(CBV), or cerebral blood flow (CBF), or a ms, inter-echo time TE = 6.7 ms, effective
change in the ratio of oxygenated versus echo time TEeff = 80 ms, echo collection factor
de-oxygenated hemoglobin (blood was 32. The field-of-view was 25.6 × 25.6
oxygenation level dependent contrast, mm2 with a resolution was 128 × 128 pixels,
BOLD contrast). The goal of these the slice thickness was 1 mm. With four
experiments was to assess the functional averages the acquisition time per image
response in distinct areas in the amounted to 21 s. Moreover, high-resolution
somatosensory cortex of transgenic APP23 anatomical MR images were acquired onto
mice at different ages by using the CBV- which the colored fMRI activation maps
fMRI method upon electrical stimulation of were superimposed (see Figs 29.6A to C,
the animal’s paw. Plate 23).
Intravascular paramagnetic contrast For somatosensory stimulation pairs of
agents that maintain a steady-state blood small needle electrodes were implanted
concentration permit to measure relative subcutaneously in each hind paw. Electrical
CBV values with a good temporal stimulation was performed by using constant
resolution. Neuronal activity leads to an current pulses with 0.5 ms pulse length at a
increase in CBV, i.e. a larger amount of frequency of 1.5 Hz. The stimulation
paramagnetic contrast agent in the paradigm consisted of a resting period of
respective brain area. The relevant effect of 105 s followed by a stimulation period of
the paramagnetic contrast agent is to reduces 105 s. This basic module was repeated three
the T2 relaxation time. Hence a T2 sensitive times with progressive current strength of
pulse sequence will lead to a decrease of 0.5 mA, 1.0 mA and 2.0 mA.
the signal intensity. If Spre, S0 and S(t) denote Figure 29.5 shows statistically derived
the signal intensity before and after ROIs overlaid on the spin-echo images
administration of the contrast agent and applying a correlation threshold of r>0.3 and
during the fMRI experiment, respectively, a minimum cluster size of 10 pixels. The
428 Biomedical Magnetic Resonance: Proceedings of the International Workshop
processes, in particular loss of synaptic horizontal scout image acquired for each
connectivities, and/or severe cerebral animal (see Fig. 29.8). Great care was taken
amyloid angiopathy (CAA) compromising to reposition the voxel always at the same
the vascular reserve capacity. location fully within the brain, and to avoid
the ventricles. The size of the voxel was 1.7
ALTERED METABOLIC PROFILE IN × 1.7 × 3.4 mm3, the sensitive volume was
THE FRONTAL CORTEX OF PS2APP 10 μl.
TRANSGENIC MICE6 A STEAM pulse sequence with the
Metabolic profiling in human Alzheimer’s VAPOR water suppression scheme was
disease patients by MR spectroscopy (MRS) employed to acquire the localized 1H spectra.
revealed a decrease of N-acetyl-aspartate The sequence parameters were: TE = 8 ms,
(NAA), indicating a neuronal loss. An TM = 7 ms, TR = 2000 ms, number of
increase of the inositol MRS signal has also averages 1024, leading to a total scan time
been reported for patients with probable of 35 minutes. Spectra shown in Fig. 29.9
Alzheimer’s disease and a transient recovery have been acquired in a 20-month-old
of NAA in patients treated with donepezil PS2APP mouse and an age-matched control
has been described. Based on these findings mouse. The major metabolites NAA, creatine
in humans 1 H MRS experiments with (Cr), choline (Cho), taurine (Tau) and inositol
PS2APP transgenic mice as an Alzheimer’s (Ins) can be readily identified. The typical
disease model were performed. Animals line width of all spectra was 18 Hz (0.09
were anesthetized with isoflurane and ppm).
positioned on a cradle with their heads In view of the limited signal-to-noise
immobilized in a stereotaxic holder. A ratio of MR spectroscopy, it is important to
72 mm diameter birdcage coil was used for assess the reproducibility of the measure-
RF excitation, and an actively decoupled ment method. Therefore, 9 animals approxi-
surface coil positioned on the head of the mately 22 months old were subjected to two
animal for MR signal reception. measurements each about one month apart.
The sensitive voxel for localized 1 H The NAA concentration measured in
spectroscopy was selected in the frontal individual animals is shown in Figure 29.10.
cortex and was positioned on a sagittal and A large variability between individual
Figs 29.8A and B: Voxel position for localized 1H MR spectroscopy in frontal cortex,
defined in sagittal and horizontal image planes. Voxel size is 1.7 x 1.7 x 3.4 mm3, voxel
volume is 10 µl. Courtesy M. von Kienlin, Hoffmann-La Roche Ltd, copied from.6
MR Applications in Experimental and Animal Model Systems 431
Figs 29.9A and B: Localized 1H spectra from a 10 µl voxel as defined in Fig. 29.8 of 20-month-old mice: (A)
from a control wild-type mouse, (B) from a transgenic PS2APP mouse. The main metabolites are: N-acetyl-
aspartate (NAA, 2.0 ppm), creatine (Cr, 3.0 ppm), choline (Cho, 3.2 ppm), taurine (Tau, 3.4 ppm), inositol (Ins,
3.8 ppm), glutamate and glutamine (Glu and Gln, 2.0-2.5 ppm), lactate (Lac, 1.3 ppm) and lipids (Lip, 0.8-1.8
ppm). Courtesy M. von Kienlin, Hoffmann-La Roche Ltd, copied from.6
animals is evident, with NAA 29.11 illustrate the findings for four major
concentrations, which ranged from 6 to 12 cerebral metabolites in 24-month-old mice.
mM. Within the measurement interval of In the PS2APP mice, there is a statistically
one month the value measured for NAA in significant decrease of both the NAA/Cr
each animal remained very stable. The four (P<0.01) and Glu/Cr (P<0.002) ratios com-
animals with the highest NAA concen- pared to control animals. No significant
trations were controls, the five with the difference was detected for any of the other
lowest NAA concentrations were PS2APP. metabolites.
The main question to be addressed was For the analysis of the cortical plaque
whether some significant difference could load, thioflavin-S staining was performed
be detected in the metabolic profile of the in the 24-month-old PS2APP mice directly
animal in the end stage of the pathophysio- after the last MRS session. The same areas
logical process. The box charts in Figure of the frontal cortex as those investigated
432 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 29.11: Box-plot of the main metabolites measured in 24-month-old PS2APP (hashed, n=12) and control
mice (open squares, n=11). Both NAA and Glu are significantly lower in the PS2APP mice, no significant
differences were found for Ins or Gln. Courtesy M. von Kienlin, Hoffmann-La Roche Ltd, copied from.6
by MRS were stained and documented. The neurons in Alzheimer’s disease. Figures
12 PS2APP brains analyzed revealed a high 29.12A and B shows the NAA/Cr and Glu/
inter-individual variability resulting in Cr ratios throughout the life-span of
significant differences in amyloid plaque PS2APP and control mice from age 4 to 24
load. Also the MRS analysis showed months. In control animals the NAA/Cr
astonishingly high variability of metabolite ratio is stable around a mean value of 0.96
levels within the PS2APP mouse group. In (see Fig. 29.12 A). In the PS2APP group,
order to investigate whether these variations however, there is a trend towards reduced
are predictive for the terminal plaque NAA starting at 16 months. This trend
pathology, the metabolic profiles were reaches significance at 20 months, and
correlated with the histological data. The continues further at 24 months. Linear
markers inositol, glutamine, taurine and regression of the evolution starting at
choline did not show any trend or 12 months yields a reduction of NAA/Cr
correlation with plaque load. The neuronal of –17% per year in the PS2APP mice,
marker NAA and glutamate showed a compared to –4.3% in controls. The
negative correlation with plaque-covered evolution of glutamate shows a similar
area, the former reaching significance pattern: In the control group the Glu/Cr
(P<0.05). remains more or less stable with an average
The main observation of this study is value of 1.42, while there is a more
the reduction of NAA and glutamate in pronounced decline in the PS2APP mice at
PS2APP mice. This finding is consistent with older age. Computation of the slope of linear
the general view of a loss of functional decline starting from age 12 months yields
MR Applications in Experimental and Animal Model Systems 433
Table 29.2: Quantification of signal integrals obtained from 13C MR spectroscopy of adipose fat in rats.
vivo. The aims of this study were to 1) rendering a chemical shift for the EMCL
establish an animal model in rats for IMCL methylene protons of 1.5 ppm. If the
detection, and 2) systematically investigate magnetic field B0 is oriented perpendicular
the influence of age, gender, muscle type, to the muscle fibers this chemical shift would
and rat strain on IMCL levels. be 1.0 ppm (see Fig. 29.19)
The most relevant spectroscopic signals
of muscle tissue stem from total creatine
(tCr) at 3.05 ppm and from the methylene
and methyl resonances of fatty acyl chain
protons at 0.8-1.7 ppm (see Fig. 29.18), one
from (IMCL) and one from extramyocellular
lipids (EMCL) which are separated by a
frequency shift of 0.25 ppm. This frequency
shift has been explained by magnetic
susceptibility differences between these two
compartments, and the anisotropic spatial
arrangements of muscle fibers.
The chemical shift of the EMCL resonance
has been shown to be orientation-dependent,
which is most likely due to the arrangement
of adipocytes along the muscle fibers. IMCL,
on the other hand, is stored in the cytoplasm
of muscle cells within spherical droplets,
resulting in no spatial dependence of their
chemical shift on the magnetic field
direction. This orientation dependence is Fig. 29.18: 1H MR spectrum in rat muscle soleus
essential for the separation of EMCL and (muscle fiber oriented parallel to the magnetic field
IMCL by MR spectroscopy. The best B0) from a voxel of (A) 18 mm3 and (B) 8 mm3. In (A)
signals due to IMCL and EMCL are present, while in
separation between the two signals is
(B) the EMCL signals are no longer detectable.
achieved when the investigated muscle lies Courtesy C. Neumann-Haefelin, Aventis Pharma Ltd,
roughly parallel to the magnetic field B0, copied from.8
MR Applications in Experimental and Animal Model Systems 439
Fig. 29.19: A schematic representation of muscle fibers with the isotropic intramyocellular lipid (IMCL) droplets
and the anisotropic extramyocellular lipid (EMCL) within adipocytes. The methylene protons of EMCL resonate
at 1.5 ppm when the B0 field is aligned in parallel to the muscle fibers or at 1.0 ppm when the orientation is
perpendicular where as the IMCL methylene protons resonate at 1.3 ppm. Measurements in this study were
all done with the magnetic field oriented parallel to the muscle fibers.
440 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Figs 29.20A and B: Typical voxel localization in muscle soleus (1) and muscle tibialis anterior (2) shown in a
sagittal (A) and transverse (B) slice of the rat hind leg in a gradient echo image. The box in the sagittal image
(A) indicates the slice position shown in image (B). The rat is lying on its side on the surface coil with the inner
aspect of the hinge facing upward. Courtesy C. Neumann-Haefelin, Aventis Pharma Ltd, copied from.8
were found between muscle types: IMCL body parts would result. An obvious
levels in muscle soleus were significantly solution to the problem is to make the
influenced by gender and rat strain, while acquisition faster. This has been successfully
no such influence was detected in muscle implemented on newer MRI hardware.
tibialis [Figs 29.21 and 29.22 (Plate 24)]. In Scan times from 20 seconds down to
female rats, the age related decline of IMCL 20 milliseconds are nowadays available,
levels overlaps with effects related to sexual sufficiently short for a breath-hold scans or
maturity. In conclusion, this study shows even to freeze out cardiac motion.
that before investigating diseases such as Nevertheless, fast imaging techniques are
insulin resistance and diabetes as well as often compromising the image quality and/
the effects of drugs on these diseases, normal or resolution.
IMCL levels for a chosen rat strain and An image data acquisition (again we look
gender at a given age must be determined. at a 256 x 256 matrix) is usually composed
of 256 individual phase-encoding steps,
STUDIES ON THE MOUSE during each 256 points are collected, which
AND RAT HEART9,10 last only a few milliseconds. The individual
phase-encoding steps are separated by the
MRI is inherently a rather slow imaging TR time, which is in the order of a second
technique. For the acquisition of a 2D image or so. If we now can synchronize the phase-
matrix of say 256 x 256 points a typical scan encoding steps with the periodic cardiac
time in the minute range is required. Of and/or respiration motion we may get
course this is very long compared to the unblurred images. A combination of fast
timeframe of a beating heart or respiration imaging techniques and synchronization may
motion and thus blurred images of these further improve the situation.
MR Applications in Experimental and Animal Model Systems 441
Figs 29.21A and B: Typical 1H spectra observed in muscle soleus in male (A) and female (B) Wistar rats at age
4, 6, 10, and 17 weeks. A clear age dependence of the IMCL level is detected. Courtesy C. Neumann-
Haefelin, Aventis Pharma Ltd, copied from.8
Clinical MRI techniques for cardiac These models allow e.g. quantification
applications have now been implemented of ventricular volumes and mass, assessment
most successfully and MRI is seriously of global and regional wall motion,
challenging the gold standards in cardiology measurement of myocardial perfusion and
like X-ray DSA or ultrasound techniques. In regional blood volume, assessment of flow
rodent applications things are complicated and tissue velocities, and detailed visuali-
by the fact that rat hearts beat at a rate of zation of cardiac morphology. Figure 29.24
around 300 per minute (bpm), mouse hearts gives an example of a quantitative evaluation
even around 600 bpm and it may be higher of the myocardial motion, comparing
in diseased animals. Figure 29.23, Plate 24 infarcted and healthy control mice.10
proves that one still can get cardiac images
with an astonishingly high resolution from UNDERSTANDING THE
juvenile mice weighing between 20 g and 2 g MONKEY BRAIN11-17
only.9 These images are frames from a
movie, i.e. the data acquisition was In the early 1990s brain functional MRI
synchronized in such a way that the cardiac (fMRI) has become a popular and significant
cycle can be resolved into individual images tool for studying the operational organi-
with a time resolution of approximately 4 zation of the (human) brain.11,12 The basic
milliseconds and renders spatial an in-plane concept behind brain fMRI is that neuronal
resolution of 100 μm. This is obviously a activity will lead to an increase in glucose
sound technical basis to be used in cardiac use and blood supply at the site of
rodent models. activation. These changes are much greater
442 Biomedical Magnetic Resonance: Proceedings of the International Workshop
Fig. 29.24: Infarcted (bottom) and control (top) mice were scanned with magnitude and phase contrast
imaging techniques rendering vector maps. The infarcted myocardium can clearly be recognized. The vectors
depicted in the diagrams represent the myocardial motion. These images were acquired with a FLASH
gradient echo sequence with velocity compensation in all three gradient directions. In order to accomplish
additional motion encoding, the spin phase was prepared using bipolar gradient pulses which resulted in a
linear dependence between the voxel velocity and the spin phase. This method provides accurate quantification
of the velocity magnitude and direction of the murine myocardium at a spatial resolution of around 200 µm
and a temporal resolution of 10 ms. The data were acquired at 7 Tesla on a BRUKER BIOSPEC 70/20 system.
Courtesy J. Ruff, Axel Haase, University of Wuerzburg.
MR Applications in Experimental and Animal Model Systems 443
than those in oxygen consumption, thus changes, leaving many questions regarding
resulting in an increase of the oxygen level its relation to the actual neuronal activation
in those areas of activation. MRI is capable unanswered. In order to get a better
to map hemodynamic variations, such as understanding of the underlying mecha-
alterations in cerebral blood volume (CBV), nisms experiments with great spatial and
cerebral blood flow (CBF), and blood temporal resolution and simultaneous
oxygenation level dependant (BOLD) measurements of intracordical field poten-
response with a relatively high temporal and tials proved to be extremely valuable.
spatial resolution. The BOLD contrast relies Furthermore, high magnetic field strength
on a change in concentration of deoxy and a very careful implementation of the
hemoglobin (dHb) which is paramagnetic experiments is pivotal to the outcome of
and acts as an endogenous contrast agent. the experiments.14,15 (See Figs 29.26, Plate
The local increased in oxygen supply results 25, 29.27, Plate 26 and 29.28, Plate 27)
in an equilibrium shift form dHb to oxy- Diffusion tensor imaging (DTI) is another
hemoglobin, i.e. a lower dHb concentration technique that has attracted much attention
rendering an increase in MRI signal. and there is no doubt that it can greatly
Mainly BOLD fMRI has generated much contribute to the understanding of the
interest as a tool for mapping brain functional architecture of the brain.16 DTI
activation and as a means of studying has the ability to visualize anatomical
dynamics of neuronal networks. 13 For a connections between different parts of the
number of reasons the visual cortex became brain, noninvasively and on an individual
one of the main application areas of brain (rather than a statistical) basis. Diffusion
fMRI research. In Figure 29.25, Plate 25 the weighted imaging is based on the concept
concept of such an experiment is outlined. that the signal of spins, i.e. water molecules,
The stimuli used here to generate neuronal will dephase as they randomly move along
activity in the visual cortex of monkeys were the diffusion gradient direction, resulting
polar-transformed checkerboard patterns, in a signal attenuation. This signal
rotating at different speeds, changes of attenuation quantitatively depends on the
rotation direction at different rates and diffusion coefficient and the gradient
covering smaller or larger field of vision. strength (also see section 2.1 and Fig. 29.1).
To study the stimulus-induced activation in In an ideal isotropic environment without
the visual cortex a multi-slice T2* sensitive any restriction for the diffusion motion the
gradient-recalled echo-planar imaging (GE- signal strength can be described by the
EPI) sequence to image the entire brain was simple equation given in section 2.1. In
used. The EPI data representing the BOLD surroundings like brain white matter,
contrast signal changes are superimposed diffusion is highly anisotropic reflecting the
on a high resolution 3D anatomical image organization of white matter in bundles of
rendering the functional map. parallel fibers. As a consequence the signal
So far brain fMRI can be rather straight- attenuation will be more pronounced when
forward. However, BOLD responses can the diffusion gradient is aligned with the
only measure hemodynamic changes or fiber orientation as compared to a
intravascular magnetic susceptibility perpendicular orientation where diffusion
444 Biomedical Magnetic Resonance: Proceedings of the International Workshop
motion is hindered by the tissue structure. (Ca2+), which can enter cells through calcium
The diffusion tensor D is a description of pathways such as voltage gated calcium
the diffusion properties for each individual channels and is confined to the intracellular
voxel. Equivalent to the isotropic case the compartment. In MRI experiments injections
signal attenuation in an anisotropic medium of MnCl 2 allow the determination of
may be described by the equation projections and connectivities in the brain.
S(bij)/S0 = exp{-∑ bij × Dij} Mn2+ is a paramagnetic contrast agent. It is
D is a 3 x 3 matrix with diagonal elements actively transported along fibers and axons,
(Dxx, Dyy, Dzz) that describe the mobility in e.g. the optic nerve and tract with a speed
perpendicular directions and off-diagonal of a few mm per hour. The concentration of
elements describing the coupling between Mn2+ required for these investigations was
different directions (Dxy, Dxz, Dyz). So the found not to have toxic effects. Figure 29.31,
task is to determine D for every voxel. This Plate 28 gives an example of MRI studies
is achieved by repeating DWI acquisitions with MnCl2 injections. Such paramagnetic
for a set of diffusion gradient strengths and tracer studies may also be used to validate
orientations. At least six different diffusion and further develop noninvasive fiber
gradient directions must be chosen and at tracking techniques, such as diffusion tensor
least one experiment with very low diffusion
MRI. In contrast to histological validation
weighting is necessary as a reference. In
methods, Mn 2+ enhanced tracts can be
fact, the choice of an optimal set of diffusion
readily acquired under identical conditions,
gradient directions is still a matter of
and in exact spatial co-registration with DTI
contention. The representation of DTI data
is not straightforward. An image ought to where as histological methods are prone to
convey information about diffusivity and tissue distortions or destruction during the
the degree anisotropy as well as its spatial procedures. The Mn2+ loaded tracts show a
orientation (See Figure 29.29, Plate 27). signal enhancement in T1 weighted images.
Figure 29.30B, Plate 28 shows one attempt This signal enhancement can be observed
to achieve this. It is important to note that over a period of approximately two to three
diffusion imaging is truly quantitative days.
method that gives direct insight into the
ACKNOWLEDGMENTS
voxel-averaged microscopic physical
properties of tissue. The author thanks all the research groups that have
generously provided images and data for this article
A very different way of “fiber tracking”
and workshop lecture. These are in particular (in the
can be done with MnCl 2 . 17 The axonal order of appearance in the article):
transport of the radioactive isotope 54Mn2+ • Novartis Institute for Biomedical Research, Basel,
was first studied using histological methods. Switzerland; Markus Rudin, Nicolau Beckmann,
Martin Rausch, Thomas Mueggler and co-
Although these studies were carried out
authors.
with the goal of understanding the regional • MR Imaging and Spectroscopy Group, F.
specificity of Mn 2+ distribution, they Hoffmann-La Roche Pharmaceuticals Ltd, Basel,
indicated the usefulness of Mn2+ as an Switzerland; Markus von Kienlin, Basil
anterograde neuronal tract tracer. The Kuennecke, and co-authors.
• LO-MRI lab, Aventis Pharma Ltd, Frankfurt,
divalent manganese (Mn2+) has an ionic Germany; Hans-Paul Juretschke, Claudia
radius similar to that of the calcium ion Neumann-Haefelin, and co-authors.
MR Applications in Experimental and Animal Model Systems 445
• Biophysics Group, University Wuerzburg, analysis in awake mice and rats by magnetic
Germany; Axel Haase, Joerg Streif, and co- resonance relaxometry. Obesity Res in press.
authors. 8. Neumann-Haefelin C, Kuhlmann J, Belz U,
• Max Plank Institute for Biological Cybernetics, Kalisch J, Quint M, Gerl M, Juretschke H-P.
Tuebingen, Germany; Nikos Logothetis and co- Determinants of intramyocellular lipid
authors. concentrations in rat hindleg muscle. Magn
• Bruker BioSpin MRI Ltd, Ettlingen, Germany; Reson Med 2003;50:242-48.
Martin IIg and his applications team. 9. Ruff J, Wiesmann F, Hiller K-H, Voll S, von
Kienlin M, Bauer W, et al. Magnetic resonance
microimaging for noninvasive quantification
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Index
expanded from 2.9 - 4.7 ppm, at the level of the basal Chemical shift 90
of perchloric acid extract ganglia 133 Chemical shift displacement 81
of skeletal muscle tissue Chemical shift imaging 89, 415
321 B Chemical shift selection artefact 81
4-D Fourier transform 80 Bacterial infection 407 CHESS (chemical shift selective
Acquired signal 89 Bacterial infection MR spectro- saturation) pulses 88
Acquiring ADC trace maps 146 scopy 404 Chicken embryo model 150
Acquisition of two-dimensional Balanced fast field echo Choline peaks in MRS of breast
raw data for imaging 36 (balanced FFE, Philips) 63 carcinoma 100
Acute disseminated Balanced gradients 63 Classical view of NMR
encephalomyelitis 161 Baseline 1H MRS 144 spectroscopy 12
Acyclic chelating agents with Classification and regression
Basic gradient-echo imaging
different functional groups trees (CART) 100
sequence 35
as Gd-based MR contrast Coding to create ‘spatial
Biological applications of NMR 3
agents 397 frequencies’ and ‘spatial
Blood oxygenation level
ADC values 145 phases’ 30
dependent (BOLD) contrast
ADC changes 238 Coherence transfer echoes 90
effect 142
ADC histograms in response to Coherence transfer pulse 91
Body MR imaging 257
taxol 236 Coherence-transfer-echo 91
Bohr relation 2
ADC images 144 Coherent complex signal
Breast cancer treatment 417
ADC maps 143, 238 addition 83
Burst imaging 65
ADC trace maps 145 Combined exposure to LPS and
b-values 237
ADC values 235, 238 hypoxia 150
Advantage of L-SECSY over L- Comparing key characteristics
COSY 94 C of animal imaging
Advantage, 2D spectroscopy 20 Cabinet sequence 91 techniques 423
Ambient FiO2 level 147 Catheterization 313 Concentration of metabolites
Antecedents of MRI methods 9 Cerebral 1H spectra estimated in the muscle tissue
chemical applications 9 Cerebral lactate/creatine ratios extracts, neuromuscular dise-
echoes 10 before, during and after ases 323
the first NMR “image” 9 hypoxia for normal growth Continuous wave (CW)
Apparent diffusion coefficient restricted chick embryos 148 saturation 95
(ADC) 142 Cerebral lactate/creatine ratios Contrast 52
Applications in drug plotted against time for Contrast agents for angio-
development, NMR 27 chick embryos that had graphy 400
Applications in physics and received hypoxia + LPS (n = Contrast and relaxation 43
chemistry NMR 3 9) and hypoxia alone 149 Contrastenhanced MRI for
Applications of echoes 10 Cerebral trace ADC values plot- detecting breast cancer 413
APT imaging 354, 359 ted against time for chick Control system 76
APT imaging of rat brain embryos that had received COSY sequence 87
tumors 357 hypoxia 149 COSY spectra 87, 100
Assessment of liver graft Change in cerebral T 2* values Critical experiments in
function 405, 410 plotted against time for chick biomolecular studies 20
Automated linear combination embryos that had received HMQC 20
modeling method 143 hypoxia + LPS (n = 9) and J-correlated spectroscopy
Axial MRI of a 42 year old hypoxia alone (n = 10) 150 (COSY) 20
healthy breast with a voxel Changes in ADC and TGD in NOESY 20
of 2D L-COSY 101 response to taxotere 236 ROESY 20
Axial T1-weighted images with Chemical exchange saturation TOCSY 20
individual activation clusters transfer (CEST) 353 Cross peak imaging (CPI) 102
Index 449
PX-478 using both diffusion MRI Signal frequency 29 Spectra from healthy prostate
237 Signal phase 29 tissue 183
Signal strength or amplitude 29 Spectra of fine-needle aspirates
Q Signal-to-noise ratio 63, 162 of breast tissue 100
Quantitation of short TE 1H clinical Simple pulse sequence for NMR Spectral editing techniques 86
spectra 84 excitation and detection 13 Spectroscopic imaging (MRSI)
Simulated echo acquisition mode 415
R 88 Spectroscopic imaging (SI) 89
Simultaneous acquisition of spa- Spherically symmetric Hamming
Radio-frequency FAST 62 tial harmonics (SMASH) 66 k-space filter 81
Ratio of resonances at 1.7 ppm Single volume localizing MRS Spin echo 33, 78, 89, 91, 143
and 0.9 ppm for normal sequence 91 Spin phase 31
thyroid tissue 181 Single-array head coil 83 Spin-echo sequence 79
Reciprocal space concept in Single-quantum (SQ) resonance Spin-echo SVS sequence 79
crystal physics 39 of water protons 94
Recovery time 89 Spoiled gradient echo 62
Single-voxel PRESS spectra Spoiled GRASS 62
Redox inhibitor of thioredoxin 240 recorded at 3.0 tesla from a
Refrigerator system 74 Spoiling gradients 79
phantom containing N-acetyl SQ coherences of metabolite
Relaxation properties 52 aspartate (NAA) using a 6-
Repetition time 100, 143, 163 protons 94
channel head 162 Standard diffusion weighting
Rephased echoes 33 Single-voxel spectroscopy 414
Resonances in one dimensional protocol 235
Skeletal muscle metabolism 314
1H MR spectra 176 Steady-state imaging physics 58
amino acids metabolism 316
RF coil 71 STEAM (stimulated echo
fatty acid metabolism 315
RF pulses 78, 80, 87, 91, 92, 95, acquisition mode) 78
glucose metabolism 314
218 STEAM sequence 80, 87
krebs cycle 315
RF shielding 75 Stern’s molecular beam technique
Slice center 81
RF system 74 2
Slice selection gradient 81
RF-FAST, Picker international 62 Slice selection profile 80 Stimulated echo 10, 78
ROC analysis 237 Slice selection pulses 81 Stimulated-echo acquisition
Slice-selective RF-pulse 80 mode (STEAM) 184
S Small MW contrast reagents Studies of body fluids, tissues
(SMWCR) 240 and cells 23
Sagittal MR image that is T1 Studies on spermatozoa 24
weighted, contrast Specific absorption rate (SAR) 56
Specific Nuclei 219 biochemical changes during
dependent on relaxation 44 cell maturation 26
5-fluorouracil 222
Sagittal short tau inversion effect of Argon on activity of
carbon-13, 277
recovery (STIR) fastspin spermatozoa 27
fluorine-19 219
echo MR image 185 identification of small mole-
future prospects 227
Sagittal spine MRI 38 cular weight compounds 24
hyperpolarized gases—3He
Sample preparation for metabolic metabolism in cells 25
and 129Xe 226
profiles 381 lithium-7 222 Summary of classifiers and spec-
extraction 381 nitrogen-15 227 tral regions using SCS 175
magic angle spinning NMR other nuclei 227 Summary table of breast in vivo
381 10
B 227 data 186
MRS profiling in vivo 382 133Cs 227 Sum-of-squares normalization 83
Sampling time 217 17O 227 Supportive system, MR scanner 76
SECSY 87 195Pt 227
additional console 76
Select a particular J-coupled 27Al 227
camera 76
metabolite 86 2H 227
chiller 76
SENSE-MRSI scan 165 87
Rb 227 injector 76
Sensitivity encoding (SENSE) 66 sodium-23 226 intercom 76
Index 455