Horizons in Neuroscience Research, Volume 24

HORIZONS IN NEUROSCIENCE RESEARCH
HORIZONS IN
NEUROSCIENCE RESEARCH
VOLUME 24
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HORIZONS IN
NEUROSCIENCE RESEARCH
VOLUME 24
ANDRES COSTA
AND
EUGENIO VILLALBA
EDITORS
New York
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CONTENTS
Preface vii
Chapter 1 Multifactorial Influences of Electroacupuncture on
Non-Painful and Painful Sensory Pathways in the Spinal Cord:
An Option for the Treatment of Neuropathic Pain 1
Salvador Quiroz-González, Erika Elizabeth Rodríguez-Torres
and Ismael Jiménez-Estrada
Chapter 2 What Are the Evidence about Electrical Stimulation for Pressure
Ulcer Prevention and Treatment in Spinal Cord Injury? –
A Systematic Review and Meta-Analysis 21
Liang Q. Liu, Julie Moody, Rachel Deegan and Angela Gall
Chapter 3 Structure and Function of the Entorhinal Cortex with Special
Reference to Neurodegenerative Disease 59
Richard A. Armstrong
Chapter 4 Ginsenosides as Brain Signaling Molecules and Potential Cures
for Neurological and Neurodegenerative Diseases 83
Ze-Jun Wang and Thomas Heinbockel
Chapter 5 Neuroplasticity and Neurogenesis: Two Paths, One End 101
Rosmari Puerta Huertas
Chapter 6 Cross-Modal Plasticity: Pre and Post Cochlear Implant Study
in Deaf-Blindness 113
L. E. Charroó-Ruíz, F. Rivero Martínez, A. Calzada Reyes,
M. C. Pérez-Abalo, M. C. Hernández, S. Bermejo Guerra,
B. Bermejo Guerra, B. Álvarez Rivero, A. S. Paz Cordovés,
M. E. Sevila Salas, M Estévez Báez, L. Galán García
and A. Álvarez Amador
Chapter 7 Dexamethasone Actions on Brain Vascular Endothelium in
Health and Disease 125
Malgorzata Burek
vi Contents
Chapter 8 Dexamethasone Suppresses Neurosteroid Biosynthesis in Human

Glial Cells via Cross-Talk with Vitamins A and D 137
Hiroomi Tamura
Chapter 9 Vitamin B12 Importance for the Proper Body Condition 149
José Luis Cabrerizo-García and Begoña Zalba-Etayo
Chapter 10 Sex-Steroid Hormones and Neuropsychiatric Disorders:
Pathophysiology, Symptomology and Treatment 157
Anna Schroeder, Michael Notaras and Rachel A. Hill
Index 203
PREFACE
This book provides readers with the latest developments in neuroscience research. Topics
covered include the multiple effects of electroacupuncture on the synaptic efficacy of
neuronal ensembles in the spinal cord of experimental animal models of neuropathic pain and
their neuromodulation by neuropeptide hormones and cytokines; electrical stimulation for
pressure ulcer prevention and treatment of spinal cord injury; the structure and function on the
entorhinal cortex with special reference to neurodegenerative disease; recent advances in the
understanding of the effects of different ginsenosides on CNS targets and how ginsenosides
can contribute to cures for some of the most devastating neurological disorders and
neurodegenerative diseases; neuroplasticity and neurogenesis; neuroplastic changes in
subjects with deaf-blindness using the topographic distribution maps of the somatosensory
evoked potential by stimulation of the median nerve (SEP-N20) pre-CI versus post-CI;
vascular functions of dexamethasone, a synthetic GC with a focus on dexamethasone action
on the blood-brain-barrier (BBB); the relationship between the effects of GCs on neurosteroid
biosynthesis and on cognitive behaviors and hippocampal neural activity; a review of the real
case of a patient with subacute combined spinal cord degeneration and pancytopenia
secondary to severe and sustained vitamin B12 deficiency; and the role of sex-steroid
hormones in anxiety, affective, eating and psychotic disorders.
Chapter 1 - Acupuncture is an ancient therapeutic modality that emerged from Traditional
Chinese Medicine. The efficacy of acupuncture for the treatment of pain has been evaluated
in numerous clinical trials, but its action has not been experimentally supported due to some
conceptual and methodological limitations. Recently, neuronal electrical stimulation therapies
have gained popularity for the treatment of pain because the stimulus parameters, such as
frequency and strength, can be standardized in an appropriate and relatively easy manner.
Electrophysiological studies have shown that direct electrical stimulation of Aβ and Aδ fibers
in nerves may depress the nociceptive activation of spinal dorsal horn neurons for short or
long periods of time. Electroacupuncture stimulation (EA) is a relatively novel procedure for
the peripheral activation of sensory pathways by the application of transdermal electrical
current pulses at acupuncture points. Such acupoints are located at specific skin areas with
abundant sensory cutaneous receptors, and their stimulation by EA induces alterations in the
activity of neuronal ensembles located at different spinal and brain levels during normal or
pathological conditions, such as neuropathic pain. Experimental studies performed in animal
models have shown that the development of neuropathic pain involves not only neuronal
pathways but also neuroglial cells and the immune system. In addition, several lines of
viii Andres Costa and Eugenio Villalba
evidence have hypothesized that non-painful sensory pathways play a major role in the
pathogenesis of neuropathic pain. In concordance, EA effects are not restricted to nociceptive
inputs, extending to non-painful sensory inputs and the glial, immune and endocrine systems.
In this chapter, the authors will review the multiple effects of electroacupuncture on the
synaptic efficacy of neuronal ensembles in the spinal cord of experimental animal models of
neuropathic pain and their neuromodulation by neuropeptide hormones and cytokines. In
addition, the authors will analyze the action of EA stimulation on low-threshold non-painful
spinal sensory pathways and the probable mechanisms involved in such effects.
Chapter 2 - Context: Electrical stimulation (ES) can confer benefit to pressure ulcer (PU)
prevention and treatment in spinal cord injuries (SCIs). However, the clinical guidelines on
ES for PU prevention in SCI are limited. New NPUAP/EPUAP clinical guideline 2014
recommends the use of ES to facilitate healing in recalcitrant stage II, any stage III and IV
PU. Yet the effectiveness of different type and mode of ES for PU healing in SCI is unclear.
Objectives: To critically appraise and synthesize the research evidence on ES for PU
prevention and treatment in SCI.
Method: Any types of interventional studies published from 1985 to June 2015 were
included. Target population included adults with SCI. Interventions of any type of ES were
accepted. Any outcome measuring effectiveness of PU prevention and treatment was
included. Methodological quality was evaluated using established instruments. Pooled
analyses were performed to calculate the mean difference for continuous data, odds ratio for
dichotomous data.
Results: Thirty studies were included, 17/30 studies were preventive studies, 13/30 trials
were therapeutic trials. Two types of ES modalities were identified in therapeutic studies
(surface electrodes, anal probe). Four types of modalities in preventive studies (surface
electrodes, ES shorts, sacral anterior nerve root implant, neuromuscular ES implant). ES
enhanced PU healing in all 13 therapeutic studies. Pooled analyses of eight unique controlled
therapeutic trials showed ES significantly improved weekly healing rate (WHR) (mean
difference (MD by 22.5 (95% CI 5.27-15.73, p < 0.0001). Pooled subgroup analysis of
therapeutic trials showed that pulsed current ES significantly improved WHR compared with
no ES 13.1, 95% CI 5.70-20.4, p = 0.0005, I2 = 94%). Subgroup analyses of two therapeutic
trials showed significantly higher numbers of ulcers healed (odds ratio (OR) 2.95, 95% CI
1.69-5.17, p = 0.0002, I2 = 0%) with ES treatment. Subgroup analysis of three therapeutic
trials showed that patients with ES treatment reported significant less number of PU worsened
than control group. (OR 0.30, 95% CI 0.10–0.89, p = 0.03, I2 = 9%).
Conclusion: The methodological quality of the studies was poor, in particular for
prevention studies. The findings of ES for PU prevention in SCI are inconsistent across
studies. The great variability in ES parameters, stimulating locations, and outcome measure
prevent a formal meta-analysis on ES for PU prevention. ES appears to increase WHR in SCI.
Pulsed current ES seems to confer better benefit on PU healing than direct current. Future
research is suggested to improve the design of ES devices, standardize ES parameters
alongside outcomes measures and address device-related adverse events for PU prevention
studies. To confirm the beneficial effect on the enhancement of PU healing in SCI, more
rigorous preclinical studies and clinical trials on determining the optimal stimulation
parameters and electrodes placement are warranted.
Chapter 3 - The entorhinal cortex (EC) comprises the anterior portion of the
parahippocampal gyrus (PHG) and constitutes an important part of the medial temporal lobe
Preface ix
(MTL) of the brain. The EC is involved in a variety of brain functions including the analysis
of olfactory information, various types of memory, and the final integration of sensory data
before being transmitted to the hippocampus (HC) via the perforant path. Significant
pathology occurs in the EC and HC in neurodegenerative disease and therefore, these regions
may be involved in the development of the cognitive deficits characteristics of these
disorders.
Based on the severity of pathological change in the EC, neurodegenerative disorders can
be divided into three groups: (1) those in which high densities of neuronal cytoplasmic
inclusions (NCI) and/or extracellular protein deposits occur in the EC, e.g., Alzheimer’s
disease (AD), Down’s syndrome (DS), and sporadic Creutzfeldt-Jakob disease (sCJD), (2)
those with moderate densities of NCI and/or extracellular protein deposits in the EC, e.g.,
argyrophilic grain disease (AGD), dementia with Lewy bodies (DLB), progressive
supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD), neuronal
intermediate filament inclusion disease (NIFID), and variant CJD (vCJD), and (3) those in
which there is relatively little pathology in the EC, e.g., Parkinson’s disease dementia (PD-
Dem), frontotemporal lobar degeneration with TDP-43-immunoreactive inclusions (FTLD-
TDP), and multiple system atrophy (MSA). Hence, EC pathology varies significantly among
disorders which could contribute to differences in the development of memory deficits among
dementias.
Pathological differences among disorders could reflect either differential vulnerability of
the EC to specific molecular pathologies or variation in the degree of spread of pathological
proteins among regions of the MTL.
Chapter 4 - Ginseng has been used as a traditional medicine in Asia for thousands of
years and is a popular natural medicine throughout the world. Ingredients of ginseng that have
biological activity include more than 30 different compounds known as ginsenosides. Recent
studies have reported that ginsenosides display beneficial effects on central nervous system
(CNS) processes and disorders such as aging, deficit of memory and learning capabilities, and
neurodegenerative diseases. Ginsenosides can be classified into four types of aglycones:
protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid types.
This review will discuss recent advances in the authors’ understanding of the effects of
different ginsenosides on CNS targets and how ginsenosides can contribute to cures for some
of the most devastating neurological disorders and neurodegenerative diseases. The authors
focus in more detail on ocotillol as a derivate of pseudoginsenoside-F11, which is an
ocotillol-type ginsenoside found in American ginseng.
The authors describe the effect of ocotillol on neuronal activity in individual cells and
circuits and on locomotor behavior.
Chapter 5 - Neuroplasticity is the ability of the authors’ NS to respond structurally and
functionally to intrinsic or extrinsic stimuli. The mechanisms of response and functional
adaptation to stimuli express the onset and continuity of constant neuromodulation involved
in the process of neuroplasticity. The discovery of neurogenesis in the adult brain, generating
new neurons, demonstrated in the hippocampus and olfactory system, together with the
process of neuroplasticity in light of changes, guarantee homeostasis in the authors’ NS.
We only understand a small percentage of the brain's potential, which we still do not fully
comprehend. As our science advances each day with new lines of research in this discovery,
we adopt action strategies to favor this complex phenomenon in the authors’ NS, which starts
x Andres Costa and Eugenio Villalba
during our embryonic development and facilitates our survival in the adult years, by
reorganizing damaged circuits as a spontaneous response of natural plasticity.
Therapeutic intervention programs are based on brain plasticity. The authors can assert
what occurs on a molecular level in the authors’ complex brain microanatomy to be able to
understand this ability, but the authors have one question: how can the authors use these
mechanisms in a targeted way to be able to achieve the authors’ desired therapeutic
objectives?
Chapter 6 - Introduction: Significant neuroplastic changes occur in deaf and blind
subjects as a result of the sensory impairment that affect them. However, the changes that
occur in deaf-blind subjects, after the auditory (re)habilitation post Cochlear Implant (CI) are
not well studied.
Objective: To identify the neuroplastic changes in subjects with deaf-blindness using the
topographic distribution maps of the somatosensory evoked potential by stimulation of the
median nerve (SEP-N20) pre-CI versus post-CI.
Methods: SEP-N20 was studied in seven deaf-blind children, after 5 years of auditory
(re)habilitation post-CI. Topographic distribution maps of SEP-N20 post-CI were compared
with the SEP-N20 pre-CI and the SEP-N20 maps of a control group. In addition, the authors
obtained Cortical Auditory Evoked Potential (CAEP). Results: The author’s study provides
evidences of the cross-modal plasticity. The deaf-blind children implanted before age 7
therefore, with less than 7 years of auditory and visual deprivation, did not show changes in
the topographic distribution maps of SEP-N20 pre-CI when compared with the control group.
Similar result was found after the auditory (re)habilitation post-CI. On the contrary, in deaf-
blind children implanted after age 7 (with 7 or more years of auditory and visual deprivation)
changes occur in the SEP-N20 topography, both pre-CI and post-CI. With the responses of
the CAEP it was possible to reveal the auditory area activation by the sensory input through
the CI.
Conclusions: In deaf-blind children, with 7 or more years of auditory and visual
deprivation before the CI, changes occur in the topographic distribution maps of SEP-N20
pre-CI versus post-CI. It is evidence of cross-modal plasticity, which may be an expression of
how important in these subjects the somesthetic information is, probably due to the
relationship with tactile language, as well as the functional interaction of auditory and
somesthetic information during the auditory (re)habilitation post-CI.
Chapter 7 - Due to their anti-inflammatory and immunosuppressive properties,
glucocorticoids (GCs) are the most often prescribed medicine with manifold physiological
effects in the human body. GC can function through genomic and non-genomic pathways.
Activation of glucocorticoid receptor (GR) by GCs leads to transcriptional activation of GC-
target genes. A lot of work has been done to identify the GC-target genes as well as factors
influencing the expression and the activity of the GR. This chapter will give an overview
about vascular functions of dexamethasone, a synthetic GC with a focus on dexamethasone
action on the blood-brain-barrier (BBB). BBB maintains homeostasis of the central nervous
system and is disrupted in many pathological disorders, e.g., multiple sclerosis or ischemia.
The results of in vitro and in vivo studies showed that GC-treatment improved BBB-
properties. Several direct targets of GCs have been found among the endothelial cell junction
proteins. GCs have been shown to stabilize their expression under inflammatory and ischemic
conditions. Better knowledge of the mechanisms of action of this fascinating group of
Preface xi
endogenous and synthetic molecules will allow better future treatment and therapy in the
clinics.
Chapter 8 - Emerging evidence indicates that stress hormone glucocorticoids (GC) are an
important modulator of brain development and function. To investigate whether GCs
modulate neurosteroid biosynthesis in neural cells, the authors studied the effects of GCs on
steroidogenic gene expression in human glioma GI-1 cells. The GC dexamethasone (Dex)
reduced StAR, CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression in a dose-
and GC receptor-dependent manner. In addition, Dex reduced de novo synthesis of
progesterone (PROG). Furthermore, Dex inhibited all-trans retinoic acid (ATRA) and vitamin
D3 (VD)-induced steroidogenic gene expression and PROG production. Metabolism of GCs
is carried out by the enzymes 11β-hydroxysteroid dehydrogenases (HSD11B1 and
HSD11B2). These enzymes control the intracellular concentration of active GCs which in
turn are related to brain functions. The authors found that Dex inhibits the ATRA/VD-
induced HSD11B2 gene expression.The author’s results suggest that GC regulates
neurosteroid synthesis in neural cells via cross-talk with the two fat-soluble vitamins, A and
D. The relationship between the effects of GCs on neurosteroid biosynthesis and on cognitive
behaviors and hippocampal neural activity is also discussed.
Chapter 9 - Vitamin B12 or cobalamin is produced by bacteria in the large bowel of
humans and by external bacteria and fungi. However, cobalamin from the former source is
not absorbed, and humans need to introduce it solely from the diet. The major sources of
cobalamin are animal proteins, mainly meats and eggs. Vitamin B12 deficiency causes a wide
range of hematological, gastrointestinal, psychiatric and neurological disorders. However, the
clinical picture does not always accompany the analytical variations. Hematological
presentation of cobalamin deficiency ranges from the incidental increase of mean corpuscular
volume to pancytopenia or symptoms due to severe anemia. Neuropsychiatric symptoms may
precede hematologic signs and are represented by myelopathy, neuropathy, dementia and, less
often, optic nerve atrophy. The spinal cord manifestation, subacute combined degeneration, is
characterized by symmetric dysesthesia, disturbance of position sense and spastic paraparesis
or tetraparesis.
In this chapter, the authors present a review of the real case of a patient with subacute
combined spinal cord degeneration and pancytopenia secondary to severe and sustained
vitamin B12 deficiency. Such cases are rare nowadays and have potentially fatal
consequences. Correction of the deficit is important, especially in severe cases, and
neurological sequelae may be irreversible if treatment is not started in good time.
Chapter 10 - Neuropsychiatric disorders, as a category of illness, arise from complex
interactions that comprise both biological and environmental factors. While progress has been
made in understanding the pathophysiology of various disorders and the molecular genetic
architecture of risk, comparatively speaking, little progress has been made in understanding
the mechanisms underlying sex differences within these disorders. While sex-steroid
hormones are clearly associated with reproductive health and pathology, emerging evidence
reveals that these hormones also have a role in the pathophysiology, symptomology and
treatment of a range of psychiatric illnesses. This chapter will address the role of sex-steroid
hormones in anxiety, affective, eating and psychotic disorders. The molecular biology of
these hormones, with a particular focus on actions of 17ß-estradiol, and their impact on the
clinical phenotype, as well as risks, will be discussed in detail.
xii Andres Costa and Eugenio Villalba
In this regard, selective estrogen receptor modulators, which have estrogen-like effects in
the brain, but lower risks for side effects in the periphery, will be evaluated as possible
adjunctive therapeutic treatments of these illnesses, where relevant.
In: Horizons in Neuroscience Research. Volume 24 ISBN: 978-1-63484-325-6
Editors: Andres Costa and Eugenio Villalba © 2016 Nova Science Publishers, Inc.
Chapter 1
MULTIFACTORIAL INFLUENCES OF
ELECTROACUPUNCTURE ON NON-PAINFUL AND
PAINFUL SENSORY PATHWAYS IN THE SPINAL CORD:
AN OPTION FOR THE TREATMENT
OF NEUROPATHIC PAIN
Salvador Quiroz-González1, Erika Elizabeth Rodríguez-Torres2

and Ismael Jiménez-Estrada3
1
Department of Acupuncture and Rehabilitation,
State University of Ecatepec Valley, Ecatepec State of Mexico, Mexico
2
Center for Research in Mathematics, Autonomous University of Hidalgo,
Hidalgo State, Mexico
3
Department of Physiology, Biophysics and Neurosciences,
Center for Research and Advanced Studies,
National Polytechnic Institute, Mexico City, Mexico
ABSTRACT
Acupuncture is an ancient therapeutic modality that emerged from Traditional
Chinese Medicine. The efficacy of acupuncture for the treatment of pain has been
evaluated in numerous clinical trials, but its action has not been experimentally supported
due to some conceptual and methodological limitations. Recently, neuronal electrical
stimulation therapies have gained popularity for the treatment of pain because the
stimulus parameters, such as frequency and strength, can be standardized in an
appropriate and relatively easy manner. Electrophysiological studies have shown that
direct electrical stimulation of Aβ and Aδ fibers in nerves may depress the nociceptive
activation of spinal dorsal horn neurons for short or long periods of time.
Electroacupuncture stimulation (EA) is a relatively novel procedure for the peripheral
activation of sensory pathways by the application of transdermal electrical current pulses
at acupuncture points. Such acupoints are located at specific skin areas with abundant
sensory cutaneous receptors, and their stimulation by EA induces alterations in the
activity of neuronal ensembles located at different spinal and brain levels during normal
2 S. Quiroz-González, E. Elizabeth Rodriguez-Torres and I. Jiménez-Estrada
or pathological conditions, such as neuropathic pain. Experimental studies performed in

animal models have shown that the development of neuropathic pain involves not only
neuronal pathways but also neuroglial cells and the immune system. In addition, several
lines of evidence have hypothesized that non-painful sensory pathways play a major role
in the pathogenesis of neuropathic pain. In concordance, EA effects are not restricted to
nociceptive inputs, extending to non-painful sensory inputs and the glial, immune and
endocrine systems. In this chapter, we will review the multiple effects of
electroacupuncture on the synaptic efficacy of neuronal ensembles in the spinal cord of
experimental animal models of neuropathic pain and their neuromodulation by
neuropeptide hormones and cytokines. In addition, we will analyze the action of EA
stimulation on low-threshold non-painful spinal sensory pathways and the probable
mechanisms involved in such effects.
Keywords: electroacupuncture, neuropathic pain, cytokines, spinal cord, non-painful sensory

input
INTRODUCTION
The International Association for the Study of Pain (IASP) defines pain as an unpleasant
sensory and emotional experience associated with actual or potential tissue damage, or
described in terms of such damage (Bonica, 1979). Woolf (2010) classified pain as
nociceptive, inflammatory and neuropathic pain. The first type is the result of stimuli that
exceeds a specific harmful intensity, and it is divided into visceral and somatic pain. The
second type is associated with tissue damage and infiltration of immune cells, and the third
type is evoked by direct damage of the nervous system.
A patient who suffers neuropathic pain frequently refers to a spontaneous electrical,
burning or stabbing pain. Clinically, it is characterized by allodynia, in which a non-
nociceptive stimuli that normally do not produced any pain begin to do so, and hyperalgesia,
in which noxious stimuli evoke a large and prolonged sensation of pain at the damaged site
and normal adjacent areas (Paice et al., 2003; Woolf et al., 2010).
The mechanisms underlying neuropathic pain are complex and appear to involve several
peripheral and central components in the nervous system. For example, changes in the
excitability of peripheral sensory structures, such as a reduction in the threshold of cutaneous
nociceptors (peripheral sensitization), could be considered as a peripheral component,
whereas increased excitability of nociceptive neurons in the central nervous system (central
sensitization) is a central component (Woolf and Salter, 2000). At the cellular level, it has
been shown that peripheral and central alterations are mediated by alterations in the release of
neurotransmitters, expression of membrane receptors and/or the properties of ion channels,
which are modified as a result of distortions in the permanent peripheral afferent input,
altering the normal input-response relationship of the pain sensory pathway.
Current pharmacological treatments for neuropathic pain include the use of opioids,
nonsteroidal anti-inflammatory drugs (NSAIDs), tricyclic antidepressants, and anticonvulsant
agents. Unfortunately, these therapeutic options provide a modest reduction in pain in
approximately 50% of patients, and their effects are variable in magnitude and include several
side effects (Martin and Eisenach, 2001; Namaka et al., 2004). Due to the latter, the search
Multifactorial Influences of Electroacupuncture … 3
and development of traditional and non-traditional therapeutic strategies for the treatment of
neuropathic pain have been extensively promoted.
Acupuncture is part of Traditional Chinese Medicine (TCM), which has been used for the
treatment of very diverse disease modalities. Although the World Health Organization
recommends its use (Zhang et al., 2014) and it is currently endorsed by the National Institutes
of Health and the National Center for Complementary and Integrative Health (Eskinazi and
Jobst, 1996; Yin et al., 2010), its real efficacy has been questioned by the inconsistency of
acupuncture results, as assessed by several systematic reviews and meta-analyses (WHO,
2001). Such inconsistency is probably due to variables that remain uncontrolled in
acupuncture research, such as the standardization and/or individualization of the sites to be
stimulated, selection of credible control procedures, duration of the effect, length of
stimulating sessions and time interval between treatments (Napadow et al., 2008). In addition,
the training of acupuncturists is not uniform, and there is a large diversity of clinical practices
related to acupuncture.
Electroacupuncture (EA) is a relatively recent acupuncture technique in which an
electrical stimulator delivers small current pulses through needles inserted in skin acupoints.
In this procedure, the intensity of the electrical current is gradually increased during
treatment. EA offers several advantages over traditional acupuncture, namely, manual
acupuncture stimulation. EA stimulation is simple to control and is accurately reproducible
because some stimulus parameters, such as frequency, intensity and width of pulses, can be
easily adjusted to induce discernible effects such as analgesia. The analgesic effects of EA on
different types of acute and persistent inflammatory pain have been explored in rodents and
humans (Silverio-Lopes, 2007; Dai et al., 2001; Hwang et al., 2002; Kim et al., 2004).
BEHAVIORAL RESPONSES AND EA IN EXPERIMENTAL

ANIMAL MODELS
To study the effect of EA on neuropathic pain, several animal models of injury have been
developed, particularly in rodents (Dai et al., 2001; Kim et al., 2004). Neuropathic pain is
provoked in animals by diverse experimental procedures, including resection of the caudal
end of the S1 and S2 spinal nerves, sciatic nerve chronic constriction injury (CCI), or L5
spinal nerve ligation (SNL).
These animal models develop mechanical hyperalgesia, heat hyperalgesia and mechanical
allodynia after the evoked injury (Hoke, 2012). Such behavioral responses are similar to those
observed in humans, and they are experimentally used to determinate the efficacy of EA for
the treatment of pain (Zhao, 2008).
In the sciatic nerve CCI model, it has been shown that EA significantly decreases the
expression of neuropathic pain behaviors, such as mechanical allodynia and heat hyperalgesia
in one study (Dai et al., 2001) and mechanical allodynia in the rat tail resection in another
study (Hwang et al., 2002). Similar observations have been reported in spinal nerve ligation
models (Huang et al., 2004). Several researchers considered that low frequency EA
stimulation (2-15 Hz) produces robust and longer-lasting effects than 100 Hz on neuropathic
pain behaviors (Kim et al., 2004) and longer inhibitory effects on dorsal horn-nociceptive
neurons in the rat spinal cord (Kim et al., 1997). Altogether, these results suggest that low-
frequency EA is suitable for the treatment of neuropathic pain, and it must to be properly
considered in clinical practice.
SPINAL MECHANISMS OF ELECTROACUPUNCTURE RELATED TO

NEUROPATHIC PAIN TREATMENT
The spinal cord is the first structure in the central nervous system that receives primary
afferent connections from somatic receptors. It is proposed that plastic changes in spinal
nociceptive synapses after nerve injury may contribute to the development of central
sensitization in the spinal dorsal horn and ultimately lead to the generation of neuropathic
pain (Navarro et al., 2007). Experimental studies in animal models have shown that the
development of central sensitization involves not only neuronal pathways but also
interactions between neuroglial cells and the immune system (Scholz and Woolf, 2007).
Several lines of evidence have shown that EA treatment is characterized by evoking
actions on multiple neuronal systems (Quiroz-Gonzalez et al., 2014a). At the spinal cord
level, EA activates serotonergic, adrenergic, opioid and cholinergic pathways (Yan et al.,
2011; Kim et al., 2005; Park et al., 2010), but its effects are not restricted to neuronal
pathways, extending to neuroglial cells and the immune system (Cha et al., 2012). In addition,
it has been proposed that EA exerts its main role at the neuroinmunomodulatory level. For
example, EA applied at both ST36 Zusanli and PC6 Neiguan acupoints decreases the plasma
levels of inflammatory cytokines (TNF and IL-1β); increases systemic anti-inflammatory
cytokines, such as IL-10; reduces iNOS and NF-κB activity; and mitigates an acute kidney
injury in endotoxemic rats (Gu et al., 2011). Other studies have also shown that acupuncture
at the LI4 Hegu acupoint or direct stimulation of the vagus nerve inhibits systemic serum
TNF levels in endotoxemic animals through a mechanism mediated by the spleen (Song et al.,
2012).
In the neuropathic pain induced by a peripheral nerve injury model, EA stimulation
provokes a noticeable decrease in the expression levels of IL-1β, IL-6, and TNF-α found in
peripheral nerves and dorsal root ganglia (DRG) (Cha et al., 2012).
In addition, the increased expression of proteins and receptors evoked in neuropathic pain
models is restored to normal expression levels after EA treatment. In contrast, proteins or
receptors showing down-regulation increased their expression after EA treatment (Zhang et
al., 2014; Kim et al., 2013).
SYNAPTIC TRANSMISSION
Several lines of evidence have shown that plastic changes in spinal dorsal horn
nociceptive neurons after nerve injury may contribute to the development of central
sensitization leading to neuropathic pain. Such changes include hyperexcitability at the spinal
nociceptive synaptic level, reduction in the threshold for evoking spinal long-term
potentiation (LTP) and a significant decrease in the threshold of spinal C-fiber field
potentials, concomitant to an increment in the amplitude of such potentials (Woolf and Salter,
2000). Accordingly to the latter, LTP of C-fiber potentials is considered an index of enhanced
synaptic transmission between afferent C-fibers and spinal dorsal horn neurons, and it is
proposed as the central mechanism of neuropathic pain (Buesa et al., 2008).
The convergence of impulses originating from skin pain receptors and acupoints occurs
in the spinal dorsal horn, where the neuronal nociceptive responses appear to be depressed by
both pre- and post-synaptic inhibitory mechanisms during EA stimulation (Zhao, 2008; Li et
al., 1993). In addition, an important superimposition of low-threshold sensory afferents and
acupoint inputs in the dorsal horn at several segments in the spinal cord of the rat has been
demonstrated (Figure 1. Quiroz-Gonzalez et al., 2014b). Such evidence may indicate that
sensory inputs generated by non-painful and painful peripheral skin receptors and EA
acupoints probably share spinal pathways that can interact at the spinal cord level.
In the SNL model, EA at 2 Hz applied to the acupoints ST 36 and SP 6 over 30 min
depressed the amplitude of evoked C-fiber field potentials in the spinal dorsal horn for more
than 3 h (Xing et al., 2007). This experimental evidence indicates that 2 Hz EA stimulation
induces a long-term depression (LTD) on the C-fiber evoked potentials. Interestingly, 100 Hz
EA induced LTP instead of LTD in the spinal dorsal horn in SNL rats (Xing et al., 2007).
Long-term potentiation (LTP) and LTD are considered electrophysiological correlates of
plastic, long-lasting changes in the efficacy of spinal synaptic transmission (Randic, 1996;
Sandkuhler, 2000). In this way, EA stimulation could induce a modulatory action, in a
frequency-dependent manner, on the transmission of nociceptive information in the spinal
cord.
As a marker of neuron activation, cFos expression has been widely used to monitor
changes in neuronal activity evoked by peripheral inputs (Terayama et al., 2015). Increments
in the number of cFos-labeled neurons activated by a noxious stimulus in both the superficial
and deep laminae of the spinal dorsal horn have been reported in CCI rats.
Modified from Quiroz-Gonzalez et al., 2014b.
Figure 1. Cord dorsum potentials (CDPs) produced by SU nerve and EA stimulation at the ST36 and
SP6 acupoints. A. Schematic representation of the experimental protocol and the first negative
component of the cord dorsum potential (N1-CDPs) provoked by SU nerve and EA stimulation
recorded at the L4 to S1 spinal cord segments. B. Plot illustrating the longitudinal distribution of N1-
CDP amplitudes evoked by SU nerve and EA stimulation at several spinal segments (L4 to S1).
EA stimulation (at 100 Hz) applied at the Zusanli acupoint suppresses the increment in
the number of cFos-labeled cells in the spinal dorsal horn induced by neuropathic pain and
significantly reduces mechanical and heat hyperalgesia; however, mechanical allodynia is not
affected (Day et al., 2001).
It has been shown that P2X3 receptor subunits contribute to the transmission of
nociceptive signals in inflammatory and neuropathic states (Liu and Salter, 2005). P2X
purinoreceptors are a family of ligand-gated ion channels that open in response to the binding
of extracellular adenosine 5'-triphosphate (ATP). Seven P2X subtypes (P2X1 to 7) have been
identified (Burnstock, 2007); among them, the P2X3 receptor subunit is selectively localized
in small- and medium-diameter DRG neurons (Xiang et al., 2008) and in the spinal dorsal
horn (Burnstock, 2013). Recently, Wang and coworkers (2014) showed that EA stimulation
within the frequency range of 2 to 100 Hz applied at the ST-36 and GB-34 acupoints
depressed the fast ionic currents activated in dorsal root ganglion neurons by ATP and
purinergic 2X3 receptor in CCI rats (Wang et al., 2014). EA stimulation was more efficient in
reducing mechanical allodynia and thermal hyperalgesia when combined with A-317491 (a
blocking agent of the P2X3 receptor). These results may indicate that EA inhibits the primary
afferent transmission of neuropathic pain associated with the activation of P2X3 receptors. In
addition, it could be proposed that EA stimulation combined with the application of A-
317491 might have an additive effect in inhibiting the transmission of pain mediated by the
P2X3 receptor.
NEUROTRANSMITTERS AND THEIR RECEPTORS

It has been suggested that EA stimulation applied at the GB30 and BL40 acupoints
alleviates neuropathic pain by reducing the release of excitatory amino acids and increasing
the release of inhibitory amino acid neurotransmitters in the spinal cord. This suggestion
indicates a possible role of excitatory and inhibitory substances in the spinal cord
participating in the regulation of neuropathic pain, among other mechanisms.
EXCITATORY NEUROTRANSMITTERS
A possible role of excitatory neurotransmitters in the effects provoked by EA stimulation
on neuropathic pain has been proposed in several studies. EA stimulation (2 Hz) applied to
the GB 30 and BL 40 acupoints significantly reduces the induced high concentration of
glutamate and aspartate in the lumbar spinal cord during neuropathic pain and decreases the
mechanical and thermal pain thresholds in the CCI animal model (Yan et al., 2011). In
addition, EA-induced LTD in CCI animals is blocked by pretreatment with MK-801 (an
NMDA receptor antagonist) or naloxone (an opioid receptor antagonist) but not by picrotoxin
(a GABA-A receptor antagonist; Xing et al., 2007). This evidence suggests that the analgesic
effects of 2 Hz EA depends on the induction of NMDA receptor-dependent LTD via
activation of the endogenous opioid peptidergic system (Xing et al., 2007).
In the chemically induced model of neuropathic pain (by the injection of paclitaxel, an
antitumoral drug or oxaliplatin), EA stimulation significantly attenuates the evoked
mechanical allodynia and thermal hyperalgesia (Moon et al., 2014; Meng et al., 2011).
Intrathecal pretreatment with naloxone (an opioid receptor antagonist), idazoxan (an α2-
adrenoceptor antagonist) or propranolol (a β-adrenoceptor antagonist), but not prazosin (an

α1-adrenoceptor antagonist) reduced the antinociceptive effect of EA stimulation (Choi et al.,
2015; Kim et al., 2005). However, EA markedly suppressed the PTX-enhanced
phosphorylation of the NMDA receptor-NR2B subunit in the spinal dorsal horn (Choi et al.,
2015), and intrathecal pretreatment of naloxone, idazoxan (IDA) or propranolol exerts a
blocking action on the effects provoked by EA (Choi et al., 2015). In conclusion, EA
stimulation at the ST36 acupoint significantly diminished PTX-induced neuropathic pain via
the activation of spinal opioid receptors and α2- and β-adrenoceptors in mice.
OPIOIDS
In a review by Han in 2003, it was demonstrated that the frequency of EA stimulation
determines the type of opioid peptide released in the central nervous system. EA at 2 Hz
induces endomorphin and enkephalin release, whereas EA at 100 Hz induces the release of
dynorphin and EA at 15 Hz induces the release of all of the three opioids. In the tail
neuropathic pain model, EA stimulation applied to the ST36 acupoint reduce the behavioral
signs of mechanical allodynia. In this animal model, EA stimulation at 2 Hz induced longer
and robust effects than EA at 100 Hz (Kim et al., 2004).
It is well known that endomorphin, enkephalin, and dynorphin are the endogenous
ligands of the μ, δ and κ opioid receptors in the spinal cord, respectively (Obara et al., 2009),
and opioid receptors are localized in primary afferent fibers of the spinal cord (Martin-Schild
et al., 1998). According to Cheng and collaborators (1992), the antinociceptive effects
induced by EA (2 Hz) are mediated by met-enkephalin via μ and δ receptors.
Intrathecal injection of the μ opioid antagonist, b-FNA or naltrindole, a δ opioid
antagonist, significantly blocks the depressing effects exerted by EA stimulation on
neuropathic pain, whereas nor-binaltorphimine dihydrochloride (BNI, a κ opioid antagonist)
does not block such EA effects (Meng et al., 2011). In the paclitaxel-induced neuropathic
pain model, antagonists of the three opioid receptors reduce the anti-allodynia and anti-
hyperalgesia effects exerted by EA stimulation at 10 Hz, indicating that all three opioid
receptor subtypes are involved in EA analgesia (Meng et al., 2011).
Agonists of μ and δ opioid receptors, such as D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-
ThrNH2 (CTOP) and naltrindole hydrochloride (NTI), reduce the depolarization-induced
Ca2+ currents in single small dorsal root ganglion neurons and inhibit the synaptic actions of
afferent C-fibers in the dorsal spinal cord of the rat, but BNI does not produce any effect
(Wang et al., 2010). These results suggest that μ and δ, but not κ, opioid receptors, play a
relevant role in the mechanical allodynia-relief effects of EA (2 Hz) in the spinal cord.
Because EA significantly depresses the induced allodynia/hyperalgesia symptoms through the
activation of spinal opioid receptors, it could be proposed that EA stimulation (10 Hz) in
combination with opioid agonists is a useful clinical treatment for neuropathic pain.
INHIBITORY NEUROTRANSMITTERS
The pharmacologically isolated GABAA receptor-mediated inhibitory postsynaptic
currents (IPSCs) recorded in spinal neurons appear decreased in magnitude and frequency of
apparition in CCI and SNI animal models of neuropathic pain (Moore et al., 2002). The latter
is suggestive of a noticeable reduction of GABA content and, in consequence, is indicative of
a decreased GABAergic transmission in the spinal dorsal horn of neuropathic pain in animal
models.
Electroacupuncture stimulation (2 Hz) applied to the GB 30 and BL 40 acupoints up-
regulates the expression of GABA and taurine receptors in the lumbar spinal cord and
substantially reduces the threshold of mechanical and thermal pain responses in CCI rats (Yan
et al., 2011). In the tail neuropathic pain model, EA stimulation (2 Hz) at the ST36 acupoint
significantly inhibits the cold allodynia symptom (Park et al., 2010). Intrathecal
administration of gabazine or baclofen provokes a reduction in the pain-relief effect exerted
by EA stimulation on cold allodynia (Park et al., 2010). These results may suggest that spinal
GABAA and GABAB receptors mediate the antinociceptive effect of low frequency EA and
implies that EA-induced anti-allodynia is at least partially mediated by the activation of spinal
inhibitory mechanisms, in which GABA and its receptors have a relevant role (Park et al.,
2010).
NITRIC OXIDE
Nitric oxide, a free radical gas, acts as a messenger molecule in the nervous system and is
involved in the sensitization of peripheral and sensory neurons after noxious stimulation
(Zimmerman, 2001). A series of studies have provided evidence that nitric oxide acts as a
modulator of spinal dorsal horn nociceptive pathways, enhancing the release of pro-
nociceptive neurotransmitters such as glutamate and tachykinin (a molecule participating in
the synthesis of substance P; Garthwaite et al., 1995). EA stimulation at 1 Hz, applied to
ST36 (Zusanli) and SP9 (Yinlingquan) acupoints, induced a notable decrease in the nitric
oxide synthase immunoreactivity in superficial regions of the spinal cord (Cha et al., 2010).
This observation indicates that EA stimulation induces a reduction in the spinal content of
pro-nociceptive neurotransmitters by modulating the expression of nitric oxide synthase in
spinal neurons (Cha et al., 2010).
NEUROGLIA AND NEUROIMMUNE MODULATION

Several lines of evidence have shown that glial cells in the spinal cord are involved in the
manifestation of pathological painful states. Activated microglia and astrocytic glial cells are
important for the maintenance of pain (Cao et al., 2008). In this sense, matrix
metalloproteinases (MMP) are implicated in neuroinflammatory processes and in glial
activation (Kawasaki et al., 2008): MMP-9 induces neuropathic pain and microglia activation
during the early stages of neuroinflammation, whereas MMP-2 maintains neuropathic pain
and astrocyte activation during the establishment of the inflammation process (Kawasaki et
al., 2008).
In the tail neuropathic pain model, EA stimulation (2 Hz) applied at the ST36 acupoint
reduced allodynia and MMP-9 or MMP-2 activity (Gim et al., 2011). As a result of injury,
microglia and astrocytes released pro-nociceptive substances that enhance pain transmission,
such as prostaglandins, pro-inflammatory cytokines, ATP, excitatory amino acids and nitric
oxide (Watkins et al., 2001). In such conditions, EA stimulation reduces the activities of pro-
inflammatory cytokines, tumor necrosis factor (TNF)-a, interleukins (IL)-1β, and IL-6 and
upregulates serum IgG concentrations. In cultured macrophages, IgG inhibits the production
of MMP-9 (Shapiro et al., 2002). According to the latter, it is proposed that EA stimulation
suppresses MMP-9 levels by upregulating serum IgG (Gim et al., 2011). Meanwhile, in the
neuropathic pain induced in a peripheral nerve injury model, EA stimulation provokes a
noticeable decrease in the expression levels of IL-1β, IL-6, and TNF-α found in peripheral
nerves and dorsal root ganglia (DRG). These results suggest that EA stimulation induces a
significant reduction of the increased pro-inflammatory cytokines levels generated by the
nerve injury (Cha et al., 2012).
Several membrane receptors are expressed in glial cells of the spinal cord. Among these
receptors, the P2X4, TLR2/4 and NMDA receptors participate in the modulation of neuronal
activity (Vallejo et al., 2010). Thus, EA stimulation inhibits glial activation via TLR4 and
NMDA receptors (Cha et al., 2012).
In diverse acute and chronic pain animal models, p-p38 MAPK (a stress-activated protein
kinase) is expressed in the spinal cord microglia. It has been shown that p-p38 MAPK is
activated through a double phosphorylation of threonine and tyrosine residues by cellular
stress and pro-inflammatory cytokines (Jin et al., 2003) and participates in the initiation and
maintenance of pain hypersensitivity after tissue and nerve injury (Svensson et al., 2005;
Katsura et al., 2006). In the SNL animal model of neuropathic pain, application of EA
stimulation at 2 or 100 Hz at Zusanli (ST36) and Kunlun (BL60) acupoints diminishes the
expression of p-p38 MAPK and subsequently reduces the activation of OX-42 (a marker of
microglia) (Liang et al., 2005). The manifestation of neuropathic pain after SNL is associated
with the up-regulation of COX-2 expression in the spinal dorsal horn, particularly at laminae
II of the spinal white matter. In the model of neuropathic pain produced by SNL, EA
stimulation depressed the spinal COX-2 immunoreactivity, ipsilateral to the injured nerve.
Such an effect is associated with alleviation of neuropathic hypersensitivity (Lau et al., 2008).
It has been shown that Ephrin-B/EphB signaling complexes modulate the synaptic
efficacy of the spinal cord, contributing to sensory abnormalities in persistent pain conditions
(Ruan et al., 2010). Activation of the spinal Ephrin-B1–2/EphB1 signaling complex plays a
critical role in the development and maintenance of chronic pain after peripheral nerve injury
(Liu et al., 2011; Ruan et al., 2010). Activation of the Ephrin-B2/EphB1 signaling complex in
the spinal dorsal horn and primary sensory neurons activates astrocytes and microglial cells
and up-regulates the phosphorylation of NR1 and NR2B receptors and the phosphorylation of
Src within the N-methyl-D-aspartate receptor complex (Kobayashi et al., 2007). All these
processes contribute to the occurrence of pain behaviors (Liu et al., 2011).
EA stimulation induces a reduction in the high expression of ephrin-B1 mRNA and
enhances the ephrin-B3 and ephrin-B3/B6 mRNA expression in the dorsal horn of
neuropathic pain animal models.
In addition, EA stimulation up-regulates ephrin-B3 protein levels in the rat spinal dorsal
horn. This evidence indicates that acupuncture activates ephrin-B/EphB signaling complexes
in experimental animals with neuropathic pain and improves their neurological function (Ju et
al., 2013).
In summary, several lines of evidence have shown that the expression of pro-
inflammatory cytokines is dramatically decreased after EA stimulation in injured peripheral
nerves, DRG and the spinal cord of neuropathic rats, and the analgesic effect of EA may be
partially mediated via inhibition of inflammation and glial activation. EA stimulation has
been proposed as a useful clinical procedure for pain attenuation.
NEUROTROPHIC FACTORS
Experimental evidence has indicated that the glial cell line-derived neurotrophic factor
(GDNF) and its receptors, GFRa-1, in the spinal cord could exert potent analgesic effects on
evoked neuropathic pain of experimental rats (Boucher et al., 2000; Obata et al., 2006). There
are several other mechanisms underlying the analgesic action of GDNF; for example, it
prevents the sprouting of low threshold afferent fibers into the lamina II (Bennet et al., 1998),
depresses spontaneous ectopic discharges in large-diameter myelinated afferent fibers and
promotes the release of endogenous somatostatin (an endogenous analgesic non-opioid
neuropeptide) in adult primary sensory neurons (Malcangio et al., 2003). It also suppresses
the production of neuropeptide Y provoked by nerve injury (Wang et al., 2003). Taken
together, the evidence suggests that GDNF plays an important role in the modulation of
nociceptive signals during neuropathic pain.
It has been demonstrated that EA stimulation modified the expression of GDNF and its
receptors in dorsal root ganglions and the spinal dorsal horn in the rat (Dong et al., 2005). In
the CCI model, EA stimulation applied in alternating strings of dense sparse frequencies (60
Hz and 2 Hz) to the GB-30 and GB34 acupoints induces an enhanced expression of GDNF
and GFRa-1 in DRG cells and the spinal dorsal horn. These observations indicate that EA
activates endogenous GDNF and GFRa-1 systems in rats suffering neuropathic pain, and this
might partially underlie the effectiveness of EA stimulation for the treatment of neuropathic
pain (Dong et al., 2005).
SPINAL ADRENERGIC AND SEROTONERGIC SYSTEMS

It has been reported that spinal 5-HT and their receptors, 5HT1 and 5-HT3, have
antinociceptive effects (Millan et al., 2002). In the tail neuropathic pain model, the pain
relieving effects of EA stimulation (2 Hz) on cold allodynia were suppressed by the
application of yohimbine (an α2-adrenoceptor antagonist), NAN-190 (a 5-HT1A receptor
antagonist) and MDL 72222 (a 5-HT3 receptor antagonist), but not by prazosin (an α1-
adrenoceptor antagonist) or ketanserin (a 5-HT2A receptor antagonist) (Kim et al., 2005).
This suggests that spinal α2-adrenergic, 5-HT1A and 5-HT3 receptors, but not α1-adrenergic
or 5-HT2A receptors, are involved in the analgesic effect of EA stimulation (2 Hz) on cold
allodynia in neuropathic rats (Kim et al., 2005).
CHOLINERGIC RECEPTORS
Cholinergic receptors are present in the superficial and deep dorsal horn of the spinal
cord, and activation of spinal nicotinic or muscarinic acetylcholine receptors produces
analgesia (Millan, 2002). In the tail neuropathic pain model, 2 Hz of EA stimulation applied
at the ST36 acupoint reduces cold and warm allodynia (Park et al., 2009). Intrathecal
administration of atropine (a nonselective muscarinic antagonist), but not mecamylamine (a
non-selective nicotinic antagonist), eliminates the pain-relieving effects of EA stimulation. In
addition, pirenzepine (an M1 muscarinic antagonist) attenuates the anti-allodynic effects of
EA stimulation, whereas methoctramine (an M2 antagonist) and 4-DAMP (an M3 antagonist)
had no effects. These results could suggest that spinal muscarinic receptors, especially the M1
subtype, mediate the EA-induced anti-allodynia in neuropathic rats (Park et al., 2009).
OTHER PROTEINS
By means of a proteomic analysis, it was demonstrated that EA stimulation restored to
normal the expression of several proteins upregulated during neuropathic pain (Sung et al.,
2004). The proteins showing such restorative effect of EA stimulation include the α-enolase
and the vitamin D binding precursor, both of which play an important role in the immune
response signal transduction process and a relevant role in neuropathic pain development
(Sung et al., 2004). In addition, EA treatment induces a noticeable reduction in the enhanced
up-regulation of proteins 14-3-3c and α-adrenergic receptor kinase evoked by neuropathic
pain. Extracellular signal-regulated kinases 1 and 2 (ERKs 1 and 2) are mitogen-activated
protein (MAP) kinases that mediate several cellular responses during the mitogenic and
differentiation processes (Lewis et al., 1998). It has been shown that β-adrenergic receptor
stimulation activates ERKs (Mikhailov and Rusanova, 1993) and contributes to hyperalgesia
as a result of nociceptor sensitization by inflammatory mediators (Aley et al., 2001). In
addition, the α-adrenergic receptor kinase 1 protein also has an important role in hyperalgesia
via the increased expression of ERKs 1 and 2 (Aley et al., 2001). Therefore, it is likely that
the down-regulation of ERKs 1 and 2 could be involved in the analgesic effect of EA
stimulation in neuropathic pain diseases (Sung et al., 2004).
Several interesting genes, such as the MAP kinase, zinc finger protein, and tyrosine
phosphatase genes, have been identified in rodents under neuropathic pain. The expression of
such genes decreased approximately 40-60% in the neuropathic pain model but after one day
of EA treatment, their expression levels were restored to normal values. It is known that such
genes play an important role in signal transduction pathways and gene expression. Therefore,
multiple signaling pathways, including opioid receptor pathways and MAP kinase-mediated
pathways, as well as other gene expressions, might be involved in pain development and are
likely involved in the analgesic effects of EA stimulation (Ko et al., 2002).
NON-PAINFUL SENSORY INPUTS IN THE DEVELOPMENT OF

NEUROPATHIC PAIN
Several lines of evidence have hypothesized that large-diameter sensory fibers play a
major role in the pathogenesis of neuropathic pain (Devor 2009; Campero et al., 1998; Ochoa
1994). Devor (2009) showed that dorsal root ganglion Aβ afferents, which normally sense
touch and vibration signals, changed their electrical and synaptic properties when they are
axotomized. This condition seems to switch the sensory input of Aβ afferents from non-
painful to painful signals (“phenotypic switching”) and changes the triggering and
maintenance of central sensitization. The transmission of non-nociceptive and nociceptive
information through primary afferents is modulated by means of pre- and/or post-synaptic
mechanisms in the spinal cord (for example, pre- and post-synaptic inhibition; Le Bars, 2002;
Rudomin and Schmidt, 1999; De LaTorre et al., 2009; Rudomin and Hernandez, 2008; Eccles
et al., 1963). Electrical stimulation of Aβ fibers may depress nociceptive activation of spinal
dorsal horn neurons (Chung et al., 1984). Electrical stimulation of afferent C-fibers and
prolonged high-frequency stimulation of the sciatic nerve at Aδ fiber stimulus-strength
produce long-term depression (LTD) on C-fiber evoked field potentials (Liu et al., 1998). In
addition, LTD of synaptic transmission in substantia gelatinosa neurons can be induced by
low-frequency stimulation of primary Aδ-afferent fibers (Sandkuhler et al., 1997). All of
these observations indicate that activation of primary afferent fibers effectively modulates the
synaptic efficacy of the same and/or other afferent fibers at the spinal cord level (De LaTorre
et al., 2009; Rudomin and Hernandez, 2008; Eccles et al., 1963).
EA stimulation also evokes significant changes in the synaptic transmission in a similar
way to peripheral nerve stimulation. EA stimulation depresses the enhanced evoked responses
of the deep dorsal horn (lamina IV–VII) neurons as well as after discharges developed in
ankle sprained rats (Kim et al. 2011) and induces LTD in the C-fiber-evoked field potentials
recorded within the spinal dorsal horn of rats with neuropathic pain. In contrast, 100 Hz EA
induced LTP but not LTD in control rats (Xing et al. 2007). Recently, we have shown that
conditioning low and high frequency EA stimulation at the ST36 and SP6 acupoints
depressed the spinal dorsal neurons that receive non-painful sensory input from SU nerve
(Quiroz-Gonzalez et al., 2014b).
The depressive effect of low-frequency EA stimulation occurred at the time interval
between 5 and 90 ms, and it is partially reverted by picrotoxin (an antagonist of the GABAA
receptor). The pharmacological and temporal characteristics of the depression evoked by EA
stimulation (Figure 2) resembled those measured during the presynaptic depolarization of
afferent fibers and presynaptic inhibition (Rudomin and Schmidt, 1999).
Because EA stimulation exerts analgesic and antinociceptive effects by modulating the
activity of spinal dorsal horn neurons and depressing the cord dorsum responses evoked by
low-threshold non-painful sensory pathways, it could be proposed that presynaptic
mechanisms could participate in the depressive action of acupuncture on non-painful and
probably on painful pathways provoking neuropathic pain in the spinal cord of the rat.
However, further studies are necessary to explore this possibility by analyzing the effect of
EA on low-threshold sensory pathways in an animal model of neuropathic pain.
Modified from Quiroz-Gonzalez et al., 2014b.
Figure 2. Inhibition of the first negative component in the cord dorsum potential (N1-CDP) produced
by sural nerve stimulation (SU-CDP) and recorded in the L6 spinal segment before EA stimulation (A)
during 2 Hz EA stimulation applied on the ST36 and SP6 acupoints, 70 ms prior to the test stimulus
applied to SU nerve (B), 40 ms (C), 20 ms (D), and 30 ms after SU-evoked N1-CDP (E). Plot
illustrating percent reduction values of the N1-CDP component during EA stimulation.
CLINICAL EFFICACY OF EA STIMULATION ON NEUROPATHIC PAIN

Several clinical trials had been performed to evaluate EA stimulation as an alternative
treatment for neuropathic pain. Fortunately, in many of those tests, EA was effective in
reducing neuropathic pains of different etiologies. Among the etiology of pain, EA
stimulation reduces the symptoms of diabetic neuropathy (Abuaisha et al., 1998; Goodnick et
al., 2000), malignancy (Filshie, 1998) and phantom limb pain (Carabelli and Kellerman,
1985). Acupuncture produces a significant improvement in primary and/or secondary
symptoms in 77% of patients with diabetic neuropathy, and 67% of the patients were able to
discontinue or significantly reduce their medications (Abuaisha et al., 1998). In HIV-infected
individuals, acupuncture significantly reduced the scores for pain/aching/burning, pins and
needles, and numbness sensations in hands and feet (Phillips et al., 2004). In patients with
spinal cord injury-induced pain, 24 of 36 showed a significant improvement after EA
treatment (Rapson et al., 2003). In contrast, Penza and collaborators (2011) reports null
efficacy of EA treatment in a group of patients with chronic painful neuropathy, but the lack
of effectiveness in that study probably is related to an inadequate protocol of treatment.
Recently, Li and coworkers (2015) showed that studies with negative results found a series of
deficiencies with respect to several intervention details: (1) incompletely rational acupoint
selection; (2) inconsistent ability of acupuncturists; (3) negligible needling response to
needling; (4) use of inadequate frequencies in the acupuncture treatment (low in most
studies); and (5) irrational setting of placebo control. Indeed, the primary basis for the
negative results or placebo effects in international clinical trials of acupuncture is not related
to the quality of the methodology, which is not in compliance with the essential requirements
proposed by acupuncture theory in terms of clinical manipulation conditions.
To avoid such contradictory results, Langevin and collaborators (2011) noted the
importance of establishing a strong interrelation between different fields of medicine and
acupuncture, which must include basic research, clinical research and clinical practice. In this
way, basic research must elucidate the mechanisms of action of acupuncture and determine
the most effective EA stimulation parameters, such as frequency and intensity of stimulation,
for each clinical condition, in particular those for neuropathic pain.
In addition, basic research must characterize with precision the localization and
properties of acupoints, which are parameters of importance for the design of the EA
treatment. Meanwhile, clinical practice should indicate the development of clinically
appropriate outcome measures and biomarkers as well as the evaluation of treatments in
successful trials (Langevin et al., 2011). In addition, it is important to mention that EA
stimulation could exert a relevant synergic effect in combination with conventional clinical
treatments. For example, EA stimulation combined with low doses of conventional analgesics
results in a highly effective treatment for pain (Mi et al., 2008). According to this finding, it is
possible that low-frequency EA could enhance the analgesic effects of drugs related to
adrenergic or serotonergic mechanisms on neuropathic pain in the clinical setting, as
previously stated in this chapter. In concordance with this issue, Goodnick and collaborators
(2000) showed the possible synergism of 5-HT effects, induced by nefazodone, with
acupuncture for pain relief in patients with diabetic neuropathy.
By considering that EA also produces important influences on the neuronal activity in the
spinal cord, probably through pre- and post-synaptic GABAergic mechanisms, it is
reasonable that the analgesic effect of EA will be enhanced by the use of GABAergic drugs,
such as GABA agonists, for the clinical treatment of neuropathic pain. In summary, EA, when
combined with low dosages of conventional analgesics, provides effective pain management
that can forestall the side effects of chemical drugs.
CONCLUSION
Several lines of evidence support the notion that EA stimulation activates a complex
series of cellular and molecular mechanisms that are involved in the modulation of
neuropathic pain. Among them, those related to adrenergic, serotonergic, GABAergic and
opioidergic pathways and glial and immune cell interactions in the spinal cord had a relevant
participation in the analgesic effect of EA treatment on neuropathic pain. The effect of EA
stimulation on non-painful sensory pathways also has a substantial role in switching the
sensory input of Aβ afferents from non-painful to painful signals in the pathogenesis of
neuropathic pain. The described evidence also showed that low frequency EA stimulation
could be considered a good procedure for the treatment of allodynia, in particular when it is
applied in combination with adrenergic, serotonergic and GABAergic-related analgesic drugs.
More clinical studies are needed to elucidate the optimal parameters of EA stimulation for the
management of pain in neuropathic patients.
ACKNOWLEDGMENTS
We thank American Journal Experts for editing this manuscript. This work was partially
supported by fellowships granted to I. Jiménez-Estrada and S. Quiroz-Gonzalez from the
Sistema Nacional de Investigadores (SNI-CONACYT) and by Programa Integral de
Fortalecimiento Institucional (PIFI).
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Chapter 2
WHAT ARE THE EVIDENCE ABOUT ELECTRICAL

STIMULATION FOR PRESSURE ULCER PREVENTION
AND TREATMENT IN SPINAL CORD INJURY? –
A SYSTEMATIC REVIEW AND META-ANALYSIS
Liang Q. Liu1,∗, Dr, MBBS, PhD, Julie Moody2, MSc,

Rachel Deegan3, RN, IP, BSc and Angela Gall4, Dr, FRCP
1
Centre for Critical Research in Nursing and Midwifery,
Department of Adult, Child and Midwifery,
School of Health and Education, Middlesex University, London, UK
2
Department of Adult, Child and Midwifery,
School of Health and Education, Middlesex University, London, UK
3
London Spinal Cord Injury Centre,
Royal National Orthopaedic Hospital, London, UK
4
London Spinal Cord Injury Centre, Royal National Orthopaedic Hospital, London, UK
ABSTRACT
Context: Electrical stimulation (ES) can confer benefit to pressure ulcer (PU)
prevention and treatment in spinal cord injuries (SCIs). However, the clinical guidelines
on ES for PU prevention in SCI are limited. New NPUAP/EPUAP clinical guideline
2014 recommends the use of ES to facilitate healing in recalcitrant stage II, any stage III
and IV PU. Yet the effectiveness of different type and mode of ES for PU healing in SCI
is unclear.
Objectives: To critically appraise and synthesize the research evidence on ES for PU
prevention and treatment in SCI.
Method: Any types of interventional studies published from 1985 to June 2015 were
included. Target population included adults with SCI. Interventions of any type of ES
∗
Corresponding author: Dr Liang Q Liu, Centre for Critical Research in Nursing and Midwifery, Department of
Adult, Child & Midwifery, School of Health and Education, Middlesex University, Hendon Campus, Williams
Building Room WG36. NW8 4BT. London. UK. Tel: 0044-(0)2084112893. Email: L.Q.Liu@mdx.ac.uk.
22 Liang Q. Liu, Julie Moody, Rachel Deegan et al.
were accepted. Any outcome measuring effectiveness of PU prevention and treatment

was included. Methodological quality was evaluated using established instruments.
Pooled analyses were performed to calculate the mean difference for continuous data,
odds ratio for dichotomous data.
Results: Thirty studies were included, 17/30 studies were preventive studies, 13/30
trials were therapeutic trials. Two types of ES modalities were identified in therapeutic
studies (surface electrodes, anal probe). Four types of modalities in preventive studies
(surface electrodes, ES shorts, sacral anterior nerve root implant, neuromuscular ES
implant). ES enhanced PU healing in all 13 therapeutic studies. Pooled analyses of eight
unique controlled therapeutic trials showed ES significantly improved weekly healing
rate (WHR) (mean difference (MD by 22.5 (95% CI 5.27-15.73, p < 0.0001). Pooled
subgroup analysis of therapeutic trials showed that pulsed current ES significantly
improved WHR compared with no ES 13.1, 95% CI 5.70-20.4, p = 0.0005, I2 = 94%).
Subgroup analyses of two therapeutic trials showed significantly higher numbers of
ulcers healed (odds ratio (OR) 2.95, 95% CI 1.69-5.17, p = 0.0002, I2 = 0%) with ES
treatment. Subgroup analysis of three therapeutic trials showed that patients with ES
treatment reported significant less number of PU worsened than control group. (OR 0.30,
95% CI 0.10–0.89, p = 0.03, I2 = 9%).
Conclusion: The methodological quality of the studies was poor, in particular for
prevention studies. The findings of ES for PU prevention in SCI are inconsistent across
studies. The great variability in ES parameters, stimulating locations, and outcome
measure prevent a formal meta-analysis on ES for PU prevention. ES appears to increase
WHR in SCI. Pulsed current ES seems to confer better benefit on PU healing than direct
current. Future research is suggested to improve the design of ES devices, standardize ES
parameters alongside outcomes measures and address device-related adverse events for
PU prevention studies. To confirm the beneficial effect on the enhancement of PU
healing in SCI, more rigorous preclinical studies and clinical trials on determining the
optimal stimulation parameters and electrodes placement are warranted.
Keywords: electrical stimulation, pressure ulcer, spinal cord injury, systematic review
BACKGROUND
Incidence of PU in SCI
In the United Kingdom, approximately 1,200 people are paralysed from a spinal cord
injury (SCI) every year with a total of approximately 40,000 people living with paralysis [1].
While in the USA, there are approximately 12,000 new SCI cases each year excluding those
who die at the scene of an accident [2]. Following SCI, individuals cope with numerous
challenges that require daily attention to alleviate complications that may arise throughout
their lives. Pressure ulcers (PU) are one of the most common devastating complications in
people living with SCI [3, 4]. It can occur in SCI patients very early, often within a few days
following the injury. According to the Model SCI System Statistical Centre, the annual
incidence rate of PUs is seen at 14.7% in the first post-injury year and noted to be steadily
increasing thereafter [3]. It is estimated that up to 85% people living with SCI develop a PU
during their life time [5, 6, 7, 8]. After SCI, loss of mobility results prolonged lying or sitting
for their daily activities, consequently, the sacrum and ischial tuberosity (IT) are the most
What Are the Evidence about Electrical Stimulation …? 23
common sites for PU development [9]. An estimated 36 to 50 percent of PU incidence results

from sitting in a wheelchair [10].
Definition and Etiology of PU
A PU is otherwise and perhaps more commonly known as a pressure sore. According to

the National/European Pressure Ulcer Advisory Panel (NPUAP/EPUAP) [11], a PU is
described as an area of localised damage to the skin as a result of prolonged pressure alone, or
pressure in combination with shearing forces. It is typically categorised into four key stages
depending on severity. In stage one, the skin is not broken but is red or discoloured; the
redness or change in colour does not fade within thirty minutes after pressure is removed. In
stage two, the epidermis or topmost layer of the skin is broken, creating a shallow open sore
and drainage may, or may not, be present. At stage three, the break in the skin extends
through the dermis (second skin layer) into the subcutaneous and fat tissue and the wound is
deeper than in stage two. In stage four, the breakdown extends into the muscle and can extend
to the bone. At this stage, there is often a large amount of dead tissue and drainage.
While PU development is undoubtedly multifactorial in individuals with different
pathological conditions, the prolonged pressure loading sufficient to produce ischemia, cell
deformation, lymphatic impairment and reperfusion injury has been identified as important
process of PU formation [12, 13]. Following SCI, the interruption of spinal vasomotor
pathways results in the loss of vasomotor control over skeletal muscle and skin, which lowers
the tone of the vascular bed below the level of the lesion. The impaired vascular patency
causes the vessels to be less able to withstand normal loading conditions. Concurrent with the
loss of capillary networks due to lost muscle bulk, the volume of blood in the tissues is
reduced [14-16]. Considering microcirculation being crucial for tissue viability in terms of
supply of oxygen and nutrients and removal of waste products, interrupted microcirculation
leads to ischemia, local tissue starvation and ultimately cell necrosis.
Impact of PU on People Living with SCI
Once a PU has developed, it can be extremely difficult to achieve full repair. Those who
suffer a PU may be subjected to longer hospital stays, delayed rehabilitation and a significant
loss of independence, which adds another burden to the psychological trauma of injury and
reduced quality of life. If a PU is severe, it can lead to further disabilities, need for surgical
interventions and even fatal infections [17].
Apart from the significant personal consequences, PU also represents a significant cost
burden for health and social care systems. According to the National Institute for Health and
Care Excellence (NICE) guideline 2014 [18], in addition to the costs of standard care, the
daily costs of treating a pressure ulcer are estimated to range from £43 to £374
(£1 = approximately $1.55 USA) in the United Kingdom. Resources required for treating a
pressure ulcer include nurse time, dressings, antibiotics, diagnostic tests and high
specification pressure redistributing devices. Although exact cost of PU management in SCI
is unknown, the total cost of treating PUs has been estimated to be between £1.4bn and
£2.1bn per year, with the average cost to treat one Category IV PU being £14,108 per episode
in general population [19]. It is estimated that PUs account for approximately 25% of overall
treatment costs for people with SCI [20, 21].
Current PU Management in SCI
PU Treatment Options in SCI

Once a PU has developed, it can be extremely difficult to achieve full repair, particularly
in people living with SCI. Following SCI, immobility along with incontinence can affect the
ulcer healing. To date, treatment for PUs can vary, depending on the grade of the ulcer. While
the standard wound care of PU recommended by clinical guidelines includes offloading,
improving nutrition, cleansing, debridement and dressing [22, 23], the general principles of
Stage I ulcer treatment incorporate pressure relief, careful clinical monitoring, and dressings
to promote hydration. Stage II ulcers may require pressure relief, and sometimes antibacterial
to control infection alongside a moist dressing for re-epithelialization. Stage III and IV ulcers
usually require advanced non-surgical or surgical treatment in addition to pressure relief; and
sometimes require antibacterial to control superinfection, debridement and control of exudate
[23]. A number of non-surgical advanced treatments are documented in the literature, such as
negative pressure wound devices, oxygen, ultrasound and electrical stimulation. It often
depends on the availability of modalities and the cost and time invested when determining
which of the advanced therapies to use.
PU Prevention Strategies in SCI

Thus far, the tremendous efforts to prevent PUs tend to focus on methods to reduce
external pressure. These range from using pressure-relieving devices, to patients performing
‘pressure relief’ maneuvers themselves, such as frequent repositioning or ‘push-ups’ or
‘leaning forward [24-27]. However, these efforts are only partially effective at best in SCI.
Although it is well documented that simple pressure relief measures confer benefits on
reducing local pressures at bony prominences, they do not prevent the muscle atrophy that has
emerged as a specific risk factor for PU development in SCI [28, 29]. In fact, the incidence of
PUs remains unacceptably high [5-8].
Electrical Stimulation for PU Management
Regan et al. (2009) conducted a systematic review of preventive and therapeutic

interventions for PUs after spinal cord injury [30], and identified electrical stimulation (ES) as
being interventions for both PU prevention and treatment in the SCI population. Indeed, as
early as 50 years ago, electrical stimulation has been documented to enhance healing of
various chronic wounds including pressure ulcers in spinal cord injured individuals [31, 32],
whilst the preventive effects of ES for PU in SCI has been reported since 1980s [20, 33, 34,
35].
ES for PU Prevention
Electrical stimulation has been postulated to simultaneously impact several intrinsic and
extrinsic factors that contribute to the risk of PU formation in SCI. For instance, blood flow is
the major intrinsic factor in PU development affected by SCI, while the externally applied
pressure is extrinsic factor.
After SCI, there is a decrease in sympathetic nervous activity, which leads to reduced
systemic vascular resistance and generalized vasodilation, particularly after cervical spinal
injury. Thus, both systolic and diastolic blood pressures are decreased. ES has been shown to
increase blood flow and vascularity of muscle tissue, thus improving tissue viability and
resistance to PU development [35, 36]. For instance, as early as the 1980s, Levine and
associates investigated the effects of electrical stimulation of gluteal muscles using surface
electrodes on blood flow of gluteal muscle in eight nondisabled subjects and six subjects with
SCI [35]. They found that all subjects showed increased muscle blood flow during
stimulation. These increases in blood flow above baseline levels can be maintained for more
than 15 minutes after removal of ES.
The prolonged applied pressure, in particular non-uniform pressures, such as those
occurring in the region of bony prominences with reduced soft-tissue coverage, has been
reported cause tissue distortion that tends to collapse the regional vasculature. The interface
pressure required to occlude blood flow over hard sites, such as bony prominences, is roughly
half that required in soft sites [37, 38]. Furthermore, a large body build improves pressure
distribution at the support interface [39, 40]. These findings provide indirect evidence to
support the hypothesis that increased muscle bulk in the gluteal region after exercise with ES
will improve the pressure distribution at the buttock–cushion interface.
ES for PU Treatment
ES has been proposed as a therapeutic modality for wound healing over a century ago
and has been well documented since the 1960’s, especially for wounds not responding to
standard forms of treatment [31, 32, 41-44]. For example, early studies by Wolcott et al. [41]
showed that ischaemic ulcers healed significantly faster following electrostimulation. Their
observations were later supported by other studies [42-44]. In terms of ES for PU healing,
back to 1990s, Baker et al. [32] assessed three different forms of ES current for pressure
healing among 185 pressure ulcers in 80 patients with SCI who were treated for 45
minutes/day for 4 weeks. They reported that ES enhanced PU healing in SCI. In addition,
they identified asymmetric biphasic waveform of electrical current as the optimal wound
healing protocol in comparison with microcurrent and control. Despite the fact ES has been
demonstrated to accelerate wound healing, the understanding of the exact physiological
mechanism remains incomplete. Previous animal models and pre-clinical studies have given
some indication of the mechanism of ulcer healing being enhanced by ES [45-50]. It is known
that endogenous electrical fields which measure electrical potentials naturally exist in the
human body and are known to be vital for tissue development and repair [51-53]. The
electrical potential at the epidermis is known as ‘transepithelial potential’ and is generated by
intact skin through directional active ion transportation, leading to the concentration of
negative chlorine ions at the surface and positive sodium and potassium ions in the tissues.
The epithelial layer of intact skin acts as an electrical barrier. When a wound occurs, the
epithelial barrier is broken, allowing the current to flow out of the wound. The transepithelial
potential collapses and ions immediately begin to leak out, establishing a weak but
measurable current between the skin and inner tissues, called the ‘current of injury.’ The
current is thought to continue until the skin defect is repaired [51-53]. Application of an
external electrical current to a wound is believed to mimic the body’s natural bioelectricity
and to restart and stimulate endogenous electrical fields and as such, promotes wound healing
[54-56]. For instance, ES has been demonstrated to enhance cellular activities such as
collagen and DNA synthesis, ATP concentration, and generation of chemotaxic factors [45-
47, 57]. ES has also been shown to increase tissue perfusion, decrease oedema, and promote
angiogenesis and galvanotaxis, directing and accelerating the process of endothelial migration
in the wound tissue to promote wound healing [46-49].
Rationale of Conducting the Systematic Review
Taken together, ES has been demonstrated the potential benefit for PU prevention and
treatment in SCI. Interestingly, Mittmann et al. constructed a decision analytic model over
one-year duration, to determine the incremental cost-effectiveness of ES plus standard wound
care (SWC) in comparison with SWC alone in SCI with grade III/IV PU [58]. The authors
concluded that the ES as a conjunction treatment improved grade III/IV PU and reduced
costing in SCI population. In 2014, the new NPUAP/EPUAP clinical guideline recommends
the use of ES to facilitate wound healing in recalcitrant Category/Stage II PU and any
Category/Stage III and IV pressure ulcers in SCI [22]. Yet the lack of consistency in the use
of stimulation mode, parameters, together with the small sample size in the individual
published trial, makes it difficult for health professionals and health providers to make
clinical decisions on the implementation of ES treatment for PU in SCI. Furthermore, clinical
practice guidelines regarding the use of ES for PU prevention in SCI remain limited.
OBJECTIVES
The overall aim of this review is to critically appraise and synthesize the research
evidence available on electrical stimulation for prevention and treatment of PUs in people
living with SCI. This review therefore sought to address three specific questions:
1) What devices are used to deliver electrical stimulations for PU prevention and
treatment in SCI population?
2) What are the parameters of electrical stimulations used for PU prevention and
treatment?
3) How effective are electrical stimulations for PU prevention and treatment in SCI
population?
METHODS
An original systematic review protocol was registered in the PROSPERO database in
July 2013 (http://www.crd.york.ac.uk/PROSPERO/) and the registration number is
CRD42013005088. The author (LL) updated the search up to June 2015 by using the same
search strategy.
Search Methods for Identification of Studies
Electronic Searches
All relevant literature published from 1985 to 2015 was searched up to 5th June 2015 in
five databases without any language restrictions. Free-text and keyword/MESH terms for
each of the following databases were used: Medline, Embase, CINAL, PsycINFO and the
Cochrane Central Register of Controlled Trials. Subject sub-headings and word truncations
were entered according to database requirements in order to map all possible keywords.
Search terms for SCI included quadriplegi*, tetraplegi*, paraplegi*, spinal cord trauma* and
spinal cord injur*. Search terms for electrical stimulation included electric* stimulation,
nerve/neuro-muscular/ neuromuscular/muscular/muscle and electric* and stimulat*. Those for
PUs covered: pressure sore*, PU*, decubitus ulcer*, ischaemic ulcer*, bed sore* and skin
sore*.
Other Resources
The National PU Advisory Panel (NPUAP), European PU Advisory Panel (EPUAP),
National Institute for Health and Clinical Excellence (NICE) and Scottish Intercollegiate
Guidelines Network (SIGN) were searched for relevant published guidelines. In addition, the
reference list of included studies and other relevant papers (e.g., available reviews) were
screened for eligible studies and authors and experts in the field were contacted to identify
any additional studies.
All searched hits were exported to Endnote (Endnote version X7 for Windows Thomson
Reuters). All titles and abstracts were screened for eligibility, and then full texts of those
potentially relevant articles were retrieved and considered for inclusion by the first author
(LL). Each stage of the selection process was cross-checked by the second author (JM). Any
disagreement was discussed in order to achieve the consensus.
Inclusion Criteria
1) Types of studies: In order to capture all relevant evidence, eligible studies included
any type of primary interventional study, such as randomized controlled trials
(RCTs), non-randomized controlled trials, prospective cohort studies, case series,
case control studies and case report studies.
2) Target population: Adults with SCI irrespective of their age, gender, and degree of
severity of traumatic or non-traumatic SCI;
3) Interventions: Any type of intervention using electrical stimulations was accepted
and intervention terminology included functional electric stimulation
(surface/implant), neuromuscular electric stimulation and nerve root stimulation;
4) Outcome measurement: Any outcome measuring the effectiveness of PU prevention
and treatment was taken into account. Outcomes of prevention criteria were PU
incidence (direct), seating pressure, muscle bulk, skin blood flow and PTco2
(indirect). Outcomes of treatment were healing time, healing rate, ulcer size and the
stage of the ulcer.
Data Extraction
The following data was extracted from eligible articles by one reviewer (LL) and cross-
checked by the second reviewer (JM): year of publication, country of affiliated author and
type of study design. All other data including sample size, participants’ age, gender, type and
level of SCI, the type of electric stimulation, period of the stimulation, pattern of stimulation,
duration of study, adverse events, outcome measures and findings along with methodological
quality was assessed independently by two reviewers (LL & JM). Any disparity in assessed
findings between the two independent reviewers was resolved by discussion or through
consultation with a third reviewer.
A quality assessment was conducted for each article (except case reports). For RCTs a
Jadad score was employed together with the item allocation concealment and whether the
analysis was based on the randomized groups [58, 59, 60], and a modified Downs and Black
tool for non-RCTs [61]. Both scales are well-established tools for assessing and reporting on
the quality of clinical and health-related studies in the literature.
Jadad scale is a procedure to independently assess the methodological quality of a clinical
trial and has known reliability and external validity [60]. It contains a most important aspect
of assessment namely risk of bias (selection bias, performance bias, detection bias, attrition
bias) and has relative ease of use. The Jadad scale addresses the items relating to
randomization, blinding and description of withdrawals and dropouts, with scores ranging
from zero to five with trials scoring three or greater, considered to be of reasonably good
quality. Allocation concealment was considered adequate if patients and investigators who
enrolled patients could not foresee treatment assignment. ‘Intention to treat’ (ITT) is defined
as an analysis which demonstrates inclusivity of all randomized participants based on the
following criteria: the groups to which they were originally randomly assigned regardless of
whether they satisfied the entry criteria, and the treatment actually received and subsequent
withdrawal or deviation from the protocol [58].
The Downs and Black tool [61] consists of 27 questions, which evaluates the level of 4
domains: 1) reporting; 2) external validity; 3) internal validity (both bias and confounding); 4)
power. This was modified slightly because of what was felt to be an ambiguity in the final
question, thus, the highest score that any reviewed article could receive was 28. It should be
noted that scores increased in line with the methodological quality of the study, higher scores
indicating higher methodological quality [61].
Data Analysis
Level of Evidence
All studies were categorized by the type of study design and further grouped according to
the objectives of the intervention and intervention model used. All studies were classified
using the guidelines published by the Oxford Centre for Clinical Evidence in cooperation
with the grade of evidence published by Harding and Clucas [62, 63] as follows: 1) Grade I
(strong evidence): corresponded to RCTs: IA: RCTs with Jadad score greater than 3
combined with adequate allocation concealment and using ITT for data analysis; IB: RCTs
with Jadad score greater than 3 without AC or ITT. 2) Grade II (fairly strong evidence): RCT
with Jadad score less than 3 with/without AC & ITT, prospective non-randomized controlled
studies and cohort study. 3) Grade III (weaker evidence): retrospective case-controlled, pre-
post studies and case series. 4) Grade IV (weak evidence): cross sectional studies and case
reports.
Statistical analysis: All descriptive statistics were carried out using Excel 2007.
Quantitative pooled analysis was only performed for those therapeutic trials with control
groups. The pooled ES treatment effect was estimated by weekly healing rates, the number of
ulcers healed and the incidence of ulcers worsening. Since the daily healing rate is of limited
clinical relevance, for those trials that reported percentage of ulcer decrease per day or during
the whole study period, the weekly healing rate was calculated and used for pooled analysis.
Weekly healing rate was defined as the mean percentage change in ulcer size per week.
Review Manager (RevMan version 5.3) was used to pool the healing rate per week and the
number of ulcers completely healed and those that worsened, among the studies. We pooled
all data irrespective of the length of treatment and follow-up. Subgroup analysis was
performed for good quality RCTs. We also compared the weekly healing rate between pulsed
current and constant direct current, electrode overlaid versus placed at edge of ulcer. For
those trials with more than two arms, the ES arm with the largest sample was included for
comparison with the control arm (no ES/sham ES). When more than one report referred to the
same trial, only the major trial report with more outcome measurement was included for
pooled analysis. Treatment effect was significant if P < 0.05. Heterogeneity between studies
was tested with the use of both chi square test (significant if P < 0.1) and I2 test (with
substantial heterogeneity defined as values >50%). When studies showed significant
heterogeneity (I2 > 50%), the Mantel–Haenszel random effects model was used to calculate
mean difference. Otherwise, the fixed effects model (I2 < 50%), was used to calculate the
pooled effect sizes when studies did not show heterogeneity.
A formal quantitative meta-analysis with statistical pooling of results across preventive
studies was not possible because of the absence of both a uniform mode of intervention as
well as standardisation of outcome measures. Therefore the data was descriptively
synthesized and tabulated for preventive studies.
RESULTS
Included Studies
The literature search identified a total of 597 references that were all exported to Endnote
(Endnote version X7 for Windows Thomson Reuters), and five additional articles were
identified from other sources. Of these 602 articles, 224 were identified as duplicates, thus
resulting 378 abstracts and titles that were available for sifting for eligibility. A total of thirty
studies met the inclusion criteria and were subjected to full-data extraction. Figure 1 provides
a flow chart of the process and results for screening eligibility and study selection.
Figure 1. Flow chart of the process and results for screening eligibility and study selection.
Sample Characteristics
All 30 articles [16, 32, 34, 64-90], described the study target population as SCI, with 10
studies (30%) reporting the level of injury. Of the 30studies, 12(40%) of the studies were
conducted in the United States of America, five studies (17%) Slovenia respectively four
studies (13%) in the United Kingdom and; three studies in the Netherlands (10%) and in
Canada (10%) respectively, with the remaining three studies were from Germany, Australia
and Nigeria. In terms of study objectives, 17 studies were designed for PU prevention, whilst
13 studies were designed for PU treatment. As a whole, the number of patients per study
ranged from 1 to 150. Details of sample characteristics are shown in Table 1.
Table 1. Summary data of included studies (n = 27)
Author, year, Sample, Intervention description FES parameters Study Outcome Level of Results
Country I=Intervention, C=control period measures evidence
Therapeutic Studies
Houghton et al. 34 (20 male, 14 female) SCI HVPC with 50 µs pulse 3 months WSA RCT, Jadad 3, 1. The percentage decrease in WSA
2010, Canada with mean age of 51 yrs old; I: duration, 50-150 V AC yes, ITT was greater in the EST +SWC group
HVPC applied to the wound bed intensity was applied for yes; Grade IA (mean ± SD, 70 ± 25%) than in the
plus SWC program; C: SWC 20 min with 100Hz, SWC group (36 ± 61%; P= .048). 2.
only 10Hz and off each hour The proportion of stage III, IV or X
for 8 hr/day PUs improving by at least 50% WSA
was significantly greater in the EST
+SWC group than in the SWC group
Griffin et al. 20 SCI with pelvis Pus; Pulsed current 20 days WSA RCT, Jadad 4, Percentage reduction in WSA
1991, US I: HVPC overlaid wound; C: stimulation frequency AC yes, ITT no; achieved by HVPC group was greater
Sham HVPC given 1 hour a day and intensity was Grade IB than sham treatment group at day 5
for 20 consecutive days, both 100pps, 200v (32% vs. 14%, p = 0.03), day 15
group received standard nursing respectively (66% vs. 44%, p = 0.05),and day 20
care (80% vs. 52%, p = 0.05)
Adegoke et al. 7 SCI aged 21-60 yrs with grade Submotor pulsed current 4 weeks WSA RCT, Jadad 2, WSA decreased by 22.2% in IDC
2001, Nigeria IV pelvic ulcer; I: IDC overlaid ES, 30Hz without pulse AC yes, ITT no; group vs. 2.6% in sham treatment
wound; C: Sham IDC for 45- duration reported Grade II group. 2. most of the decrease in
minutes 3 d/wk, both group WSA occurred during the first 2
received SWC weeks of the study (15.8% vs. 1.9%
change in DC group vs. sham DC
group respectively)
Baker et al. 80 (66 male, 14 female) SCI Pulsed current ES, 4 weeks Healing rate RCT, Jadad 2, 1. No statistical differences in healing
1996, US aged 17-76 yrs with one or more I:100us,50 Hz, & WSA AC no, ITT no; rates and wound areas among 4
PUs; I: asymmetric biphasic ES; submotor; Grade II groups. 2. Subgroup analysis showed
II: symmetric biphasic ES; III: II: 300us, 50 Hz, the healing rate by ES in control
Microcurrent ES; C: No submotor; group was greater after the control
stimulation. All electrodes III: 10 us, 1Hz, 4mA, period (43.3 ± 12.5% change/wk vs
placed on intact skin. subsensory 9.7 ± 3.4% change/wk)
Each treatment last 1.5 hrs,
5d/wk
Table 1. (Continued)
Karba et al. 12 male SCI aged 29-42 yrs with Biphasic asymmetrical 14 weeks Healing rate RCT, Jadad 2, Wound healing rate significantly
1995, Slovenia PUs; I: ES delivered using pulsed currents 4:4 AC no, ITT no; higher in ES group than control group
electrodes placed on intact skin seconds on/off, submotor Grade II (7.13 ± 1.46%/day in ES group vs -
at edge of dressing two hrs daily; 0.66 ± 1.16%/day in control group)
II: Sham ES. All participants
received occlusive dressing
Karba et al. 50 SCI with PU; I: ES were Constant direct electric Not Relative RCT, Jadad 2, The relative healing rates of PU
1997, Slovenia delivered using the 1 positive current of 0.6 mA reported healing rate AC no, ITT no; treated by direct current with
stimulation electrode and 4 (%/day) Grade II electrode overlaid wound was higher
negative electrodes (DC+) . II: than those with electrodes placed on
Same electrical stimulation intact skin, or treated by sham ES
programme with one positive
and one negative pad. C: Sham
group, no ES delivered
Jercinovic et al. 73 SCI with 109 PUs aged 18-68 Pulsed current ES was 1 year Wound RCT, Jadad 1, 1. Mean healing rate for ES group in
1994, Slovenia years; I: SWC plus ES with applied with 40Hz, healing rate AC no, ITT yes; first four weeks was greater
electrodes placed around edge of 250us, amplitude Grade II comparing to control group. 2. ES
PU for 2 hours; C: SWC and adjusted up to 45mA group have 1.5-2 times shorter
standard rehabilitation. individually to achieve healing period
Crossover group after 4 weeks minimal contraction
Stefanovska 150 SCI with one or more PUs; Constant direct currents 4 weeks or Healing CCT, D&B Healing rate in pulsed currents ES
et al. 1993, I: Conventional treatment plus with low density an till wound rates score 13; Grade group was significantly better than
Slovenia direct currents ES; II: amplitude of 600µA; closure II direct currents and control group
Conventional treatment plus Pulsed currents with a (p=0.003) after excluding those with
pulsed currents 2 h daily; All pulse duration of 0.25ms very deep, superficial or long term
electrodes placed on intact skin; low frequency of 40Hz, wounds
III: conventional treatment only amplitude 15-25mA
Trontelj et al. 106 SCI with pressure ulcers; I: Pulsed current ES with 8 weeks Wound CCT, D&B ES treated wounds healed at almost
1994, Slovenia ES delivered with two electrodes pulse duration of healing rate score 8, Grade II twice the rate of those in control
placed on health skin at the edge 1.25ms, frequency of 40 group. Mean relative healing rate of
of each wound for 2 hrs daily; C: Hz was delivered 4s on ES group was higher than control
Conventional treatment only 4s off. (15-25mA) group (4.89 ±3.80 vs.2.6 ±2.59)
adjusted individually to
achieve minimal muscle
contraction
Recio et al. 3 male SCI aged 29 -51 yrs old Pulsed ES was delivered 12 months Pressure Case series, WSA decreased (11.5cm2 at baseline
2012, US with recalcitrant PUs; HVES to by twin peaked, ulcer status D&B score 4, vs. 0.4cm2 at end of treatment). 2. The
the wound bed for 60-minute monophasic, 10µs pulse Grade III long-standing PUs was completely
sessions 3–5 times per week width, 100Hz, submotor healed after 7 to 22 weeks
Allen 1 female aged 54 yrs at T7/8 No details given 12 weeks Wound size Case report Wound size reduced from 9.3cm2 to
complete SCI with a sustained 6.7cm2 at week 3; 2. Wound
stage III PU on the left ischial completed closed at 12 weeks
tuberosity; Active electrodes
applied directly to wound bed,
dispersive electrode on the intact
skin
M Lippert- 1 male SCI at C4 level, aged 27 No details given 6 weeks Size of Case report, After 2 weeks of stimulation, the size
Gruner et al. yrs who had a left ischial PU pressure Grade IV of ulcers were reduced on both side,
2003, Germany poorly responded to conventional ulcers within 6 weeks, all ulcers were
treatment; ES of gluteal muscles completed healed
was delivered using anal probe
for 15-20min 3times daily
Pollack et al. ES of bilateral gluteus hamstring Pulsed current ES with a 6.5 months Pressure Case report, After 6.5 months of ES, the PU
2004, US and quadriceps muscles twice frequency of 60Hz and a ulcer status Grade IV completed closed
weekly pulse duration of 400 µs
Author, year, Sample, Intervention FES parameters Study period Outcome Level of Results
Country description measures evidence
I=Intervention, C=control
Prevention Studies
Kim et al. 2010, 6 male SCI aged 36–75 years Biphasic, charge- 12 weeks TcPO2, Jadad 4, AC yes, 1. A 78% increase in TcPO2
US old without open ulcers; I: balanced stimulation after muscle ITT no; Grade immediately following ES in
Bilateral sub-threshold ES of was applied at 10-Hz recruitment thickness IB intervention group, but this was not
the gluteus muscles was frequency with a pulse and maintained at follow-up. 2. No
applied using surface duration of 200µs interface significant changes in regional TcPO2,
electrodes. C: Sham ES pressure gluteal muscle thickness or pressure
distribution pre- and post-treatment
using sub-threshold ES
Gyawali et al. 17 (10 male, 7 female) SCI ES with pulse duration dynamic Interface Jadad 1, AC no, Both continuous and bursting ES
2011, Canada mean age of 37 years; I: of 200µs and 40 Hz pressure ITT no; Grade II paradigms decreased pressure around
Continuous stimulation; II: frequency over the IT IT. 2. Within the continuous
bursting stimulation, 3 bursts paradigm, the 7-s of stimulation
of stimuli were delivered produced greater pressure reduction
bilaterally to the gluteus than 13-s stimulation. 3. ES increased
maximus muscles signal Intensity by MRI in the
atrophied and loaded muscles
Londen et al. 13 SCI, 20–74 years old; I: Rectangular dynamic Interface Jadad 1, AC no, 1. Both alternating and simultaneous
2008, The alternating stimulation of monophasic pulses were pressure ITT no Grade II stimulation caused a significant
Netherland 0.5s ES of one gluteal muscle applied with 50-Hz (P < .01) decrease in interface
and a 15s rest, followed by stimulation frequency pressure (–17±12mmHg, –19 ±
0.5s stimulation of the other and 80-mA current 14mmHg) and pressure gradient
side and a 15s rest. II: The amplitude (–12 ±11mmHg, –14 ±12mmHg)
simultaneous stimulation of a during stimulation periods compared
0.5s stimulation of both with rest periods. 2. There was no
gluteal muscles followed by a significant difference in effects
15-second rest between the alternating and
simultaneous stimulation
Author, year, Sample, Intervention FES parameters Study period Outcome Level of Results
Country description measures evidence
Petrofsky 124 SCI, 12–57 years old; ES ES with pulse width of 1 year Incidence of Cohort study, The incidence of PU was 5.2% in SCI
et al. 1992, US of quadriceps for 10- 350us, at frequency of PU D&B score 8; who had ES, 32% in control population
15minutes per day; After 4 40 Hz and amplitude Grade II
weeks, sequence stimulation varies from 0-150mA
of the quadriceps, gluteus
maximus and hamstring
muscles for 30min, 3d/wk
Dolbow et al. 8 male aged 22-64 yrs with ES with pulse width of 8 weeks Average and Case series; 1. Mean average seat pressure decreased
2013, US C5-T6 SCI; ES of gluteal 250400us, at frequency maximum D&B score by 3.69 ± 4.46 mm Hg (35.57 ± 11.99
maximus, quadriceps and of 33 Hz and amplitude seating 12; Grade III vs. 31.88 ± 13.02, P = .052),
hamstrings varies from 70-140mA pressure 2. Mean maximum seat pressure
decreased by 14.56 ±18.45 mm Hg (112
± 34.73 to 98.36 ± 25.89, P = .061).
3. Neither measurement was statistically
significant
Smit et al. 2012, 10 SCI, 34 ± 9 years old, no Pulsed currents with 50 on time Interface Case series; 1. Pressure reduced by 34.5% after g+h
Netherland current ischial Pus; Electrical Hz, 75-115mA to pressure over D&B score muscles activation compared with rest
stimulation to gluteal and achieve tetanic the IT and 14; Grade III pressure, 2. Pressure reduced by 10.2%
hamstring muscles using a contraction pressure after activation of g muscles only. 3.
custom-made electrode gradient Pressure gradient reduced significantly
garment with build-in only after stimulation of g+h muscles
electrodes (49.3%)
Smit et al. 2013, 12 male SCI, 26–52 years old, ES with a duty cycle of on time 4 hrs Interface Case series; 1. Pressure was significantly lower
Netherland no current ischial Pus; ES to 1 s stimulation and 4 s pressure over D&B score during ES as compared with rest. 2.
gluteal and hamstring muscles rest for 3min was the 14; Grade III There were no significant changes of
was delivered through surface delivered at standard tuberosities, oxygenation during ES as compared
electrodes 150V, with 50Hz, blood flow with rest. 3. There was a significant
amplitude ranging from and difference in peak blood flow during ES
55 to 125mA to induce oxygenation as compared with rest (p=0.007), but no
a titanic contraction significant change on mean blood flow
for ES
Author, year, Sample, Intervention description FES parameters Study period Outcome Level of Results
Country I=Intervention, C=control measures evidence
Liu et al. 11 (10 males, 1 female) Sacral electrical on time Interface Case series; 1. Peak pressure and gradient at peak
2006, UK suprasacral SCI, 23–62 years old, stimulation frequency pressure D&B score 13; pressure significantly decreased during
no current ischial PUs; Electrical was 20pps with pulse under ischial Grade III FMS as compared with baseline. 2.
stimulation to sacral nerve root with of ranging from tuberosities Peak pressure and gradient at peak
was delivered using an sacral 128-600us and skin pressure significantly decreased during
nerve root implant or a magnetic blood flow sacral nerve root via SARS implant as
stimulator compared with baseline. 3. Ischial skin
blood perfusion significantly increased
during the FMS and SARS
Liu et al. 5 suprasacral SCI (4 males, 1 Sacral electrical on time Interface Case series; Peak pressure and gradient at peak
2006, UK female), 34–62 years old, no stimulation frequency pressure D&B score 13; pressure significantly decreased during
current ischial PUs; Electrical was 20pps with pulse under Ischial Grade III sacral nerve root via a SARS implant
stimulation to the second sacral with of ranging from tuberosities as compared with baseline
nerve root (S2) was delivered 64-600us
using an sacral anterior nerve
root implant
Bogie et al. 8 (7 males, 1 female) SCI, 27–47 The exercise regime 8 weeks Interface Case series; 1. There was no significant difference
2003, US years old, had gluteal muscle included 3 different pressure and D&B score 12; in overall mean interface pressure
electrodes implanted bilaterally; stimulation patterns TcPO2 Grade III between baseline and post exercise. 2.
ES of gluteal muscles, leg and with frequency 16Hz Mean region interface pressure
back muscles was delivered by or 30 Hz. Ramp up statistically decreased post
NMES implant '2s', on time ' 5s' or conditioning as compared with
10's,' ramp down '2s' baseline. 3. Baseline mean unloaded
or '4s' off time '10s' TcPO2, increased by 1% to 36% at
post exercise assessment for five
participants, but showed a decrease in
other 3 participants. 3. Differences
between baseline and post exercise
TcPO2, levels were not statistically
significant
Author, year, Sample, Intervention FES parameters Study Outcome Level of Results
Country description period measures evidence
Mawson 32 SCI, 18–57 years old, with HVPGS of 50 volts and on time Sacral Case series; Sacral TcPO2, was increased during
et al. 1993, US or without current Pus; HVPGS 10Hz, then at 75 volts transcutaneous D&B score HVPGS and the results were
was applied using electrodes and 10Hz was applied to oxygen tension 10; Grade III reproducible
taped on the spine when the back T6 during TcPO2
participants were supine or prone. HVPGS of 75
prone volts and 10Hz was
delivered during prone
Levine et al. 6 acute SCI at or above T7 level 50 HZ with a duty cycle on time Ischia region Case series; All participants showed an increase in
1990, US who had no history of PUs of 2s on 4s off muscle blood D&B score 9; muscle blood flow during ES
under ischial tuberosities; ES of flow Grade III
gluteus maximus began with a
20 minutes rest, followed by 12
minutes stimulation
Wu et al. 2013, 7 (5 males, 2 females) SCI, 26– 20 Hz, 20 mA pulse on time Interface pressure Case series; 1. Maximum interface pressure
US 58 years old, had implanted amplitude under tuberosities D&B score 9; gradient showed a variable response
lower extremity NMES; ES to and region TcPO2 Grade III overall. 2. Subgroup analysis for sacral
bilateral lumber spinal muscle sitters, sacral interface pressure and
and gluteal muscle was maximum interface pressure gradient
delivered by NMES implant tend to decreased on ES application;
mean TcPO2 increased during ES and
remained elevated after the
intervention
Ferguson 9 SCI, 21–56 years old, had Pulse width 300us, on time Pressure at ischia Case series; Mean pressure across all participants at
et al. 1992, UK completed injury and had no frequency 20Hz and D&B score 8; both ischia reduced during the
current PU; ES of quadriceps amplitude 100mA. The Grade III stimulation as compared with resting
was applied bilaterally and stimulation was applied (55mmHg vs. 99 mmHg on the right,
simultaneously 30minutes per for 10 seconds intervals 49 mmHg vs. 76 mmHg on the left
day for at least 5 days/week with 20 seconds rest respectively). 2. Two participants had
period, which was an increase in left pressure during
repeated after a one quadriceps stimulation. 3. In general,
minute rest the greatest reductions occurred in
participants with large knee movement
Author, year, Sample, Intervention FES parameters Study Outcome Level of Results
Country description period measures evidence
Bogie et al. 1 male SCI at C4 level, 42 years Alternating left and 5 years Seated interface Case report, 1. Seating interface pressure was
2006, US old, with regular Grade II and right gluteal stimulation pressure, tissue Grade IV reduced significantly at 6 weeks, 6
occasional IV ischial PU; ES of at 20 Hz, 15 s on and oxygen, gluteal months and 40months following up.
gluteal muscles was delivered 15s off to each muscle muscle thickness 2. Tissue oxygen level improved
using an NMES implant for a 3-min period on and sitting over the study time. 3. Gluteal
and 17-min interval for tolerance muscle thickness was increased at 1
up to 10 hr/day year and 5 year. 4. Sitting tolerance
had increased from 6 hours a day to
more than 12 hours a day
Rischbieth 1 male SCI at C6 level with Frequency was 30pps, 24 months Dimension of Case report, The circumferential dimensions
et al. 1998, history of Pus; ES of gluteal duty cycle was 10:15s buttocks and Grade IV across the buttocks were increased
Australia muscles for 15 minutes tid between 0-1 months, sitting tolerance 21%
between 0-4 months, 30 10;8 between 4-24
minutes bid between 7-24 months; intensity was
months 54% at start, 80% at 1
month and 100%
between 4-24 months
Vanoncini 1 male SCI at T5 level with A train of square pulses on time Seated interface Case report, The pressure decreased on the side
et al. 2010, UK sensory and motor complete with a frequency of 50 pressure Grade IV opposite to the stimulation. 2. Sitting
injury; ES of erector spine Hz and a fixed pulse tolerance increased from 30mins to
through surface electrodes width of 450us and more than 2 hours
manually altered pulse
amplitude
AC=allocation concealment; D&B= Modified Down & Black score range from 0-28; ITT=intention to treat; Jadad score range from 0 to 5; NMES: Neuromuscular electrical stimulation;
IDC: Interrupted direct current; HVPC: High voltage pulsed current; HVPGS: High voltage pulsed galvanic stimulation; SD: Standard deviations; SWC: Standard wound care; TcPO2:
Transcutaneous oxygen tension; WSA: Wound surface area.
Review of Therapeutic Studies
Methodological Quality
All case reports were not assessed for methodological quality, as a single case report has
been considered to be of poor quality in comparison with any other type of study design
reported in this review.
RCTs
In a total of 13 therapeutic studies [32, 64-70], seven trials were RCTs [32, 64-66-70],
Two of the seven studies were double-blinded and described the method of double-blinding,
three trials adequately described allocation concealment [64, 66, 67] and two trials [67, 69]
used ‘intention to treat’ (ITT) to analyse the data. Two RCTs were considered to be of
reasonably good methodological quality according to the Jadad score [66, 67].
Non-RCTs
Three case reports were not assessed for methodological quality [65, 71, 72]. Other three
non-RCTs include one case series and two clinical control trials were assessed for their
reporting quality using the Down and Black tool [73-75]. The scores of these trials were 13, 8
and 4 out of a total achievable score of 28.
Grade of Evidence and quality assessment of each trial were shown in Table 1.
Intervention Features
1) Type of ES Device
In total, twelve of the thirteen therapeutic studies delivered ES using surface electrodes
[32, 64-70, 72-75]. One case study reported the use of an anal probe to heal large decubital
ulcers in gluteal region, which were resistant to conventional treatment [71].
2) Stimulation Sites
Eleven out of thirteen therapeutic trials placed the electrodes over the ulcer bed or the
intact skin around the wound [32, 64-70, 73-75]. While one case report used an anal probe to
activate gluteal muscles for ischial PU healing [71], and another case report placed the
surface electrodes on the bilateral gluteus, hamstring and quadriceps muscles to treat an
ischial PU, which had previously demonstrated poor response to conventional treatments
[72].
3) Parameters of ES
In a total of thirteen therapeutic studies, two case reports did not report the ES parameters
[65, 71], nine trials applied pulsed current ES as conjunctive treatment, one trial compared
constant current with no ES, remaining one trial compared constant direct current with pulsed
current ES for PU healing [74]. Details of ES parameters and stimulation sites are shown in
Table 1.
4) Intervention Effectiveness
All thirteen therapeutic studies aimed to heal the PUs by measuring the size of the wound
or the healing rate, with eleven studies reporting the follow up period as varying from twenty
days to one year. One study did not report the study period at all [70]. Four non-controlled
studies (one case series and three case report), reported that all PUs were completely healed
with stimulation by end of the study [65, 71-73].
Nine out of thirteen trials have a control group [32, 64, 66-70, 74, 75], in which
participants were given either sham simulation or no stimulation. All nine trials reported
healing rate either daily or weekly, two of them reported the numbers of PU completed closed
at end of study, three out of nine trials reported the incidence of PU worsened during study
period. Within the nine trials, two studies were reported by same group during same time
period, and therefore only the trial reported more outcomes were included for quantitative
analysis. Pooled analysis of effectiveness was performed in eight unique controlled studies.
Mean weekly healing rate
• Overall healing rate: weekly healing rate measured as average percentage change per
week in ulcer size. Pooled analyses of all eight controlled trials [32, 64, 66-70, 74]
showed that people receiving ES treatment in adjunction to standard wound care
reported higher weekly healing rate by 22.5% (95% CI 5.27-15.73, p < 0.0001)
(Appendix I), however, heterogeneity was substantial (I2 = 95%,). A subgroup
analysis of RCTs that were considered to be of good methodological quality showed
a trend towards higher weekly healing rate in people treated with ES than people
without ES treatment, but the pooled effect was not significant (p = 0.07).
• Healing rate by pulsed current or constant direct: Pooled analysis of six controlled
trials[32, 64, 66-69] -showed a significant higher weekly healing rate in people who
were treated with pulsed current ES than those without ES treatment (MD 13.1%,
95% CI 5.70-20.4, p = 0.0005, I2 = 94%. Appendix II). As per constant direct, one
trial [70] applied constant direct current in both ES treatment groups but with
different electrodes placement, another trial [74] compared constant direct versus
pulsed current ES treatment. Pooled analysis of these 2 trials showed an insignificant
higher weekly healing rate in people who were treated with constant direct than that
those without ES (MD 4.50, 95% CI -1.19-10.18, p = 0.12, I2 = 0%. Appendix III).
• Healing rate by active electrode overlaid the wound bed or both electrodes placed on
intact skin/ the edge of wound: One study [70] compared the effect of ES delivered
by applying the electrodes either directly on the wound or on the edge/intact skin
around the ulcer versus sham ES treatment. In group one, ES with positive electrode
overlaid the ulcer and 4 negative electrodes laid around the ulcer; in group two, same
ES programme with two electrodes laid on intact skin at the ulcer edge across the
wound. The authors reported that the healing of PU was significantly enhanced by
ES with positive electrode overlaying the wound surface and the negative electrodes
placed on intact skin (p = 0.028). However the ulcers treated with both electrodes
placed on the intact skin at opposite ends of the wound, healed with same average
relative healing rate as control group. We performed a meta-analysis for those four
controlled trials [64, 66, 67, 70] that applied the active electrodes directly on the
wound and found a significantly higher rate with ES than without ES (MD 9.01, 95%
CI 2.02-16.00, p = 0.01, I2 = 90%, Appendix IV). Pooled analysis for the five
trials[32,68-70,74] that applied both electrodes on the edge of the ulcer also showed
significant difference in weekly healing rate between people who received ES and
those who received standard wound care without ES (MD 17.2, 95% CI 4.09-30.4),
p = 0.01, I2 = 95%, Appendix V).
1) Number of ulcers completely healed: there were two trials [32, 67] that reported
numbers of ulcers healed during the study period. Both trials reported higher number of ulcers
completely healed in ES treatment group than sham/no ES treatment. Pooled analysis of these
2 trials showed significantly higher numbers of ulcers healed with ES treatment (OR 2.68,
95% CI 1.17–6.14, p = 0.02, I2 = 0%, Appendix VI).
2) Incidence of ulcers worsened: three studies [67, 68] reported the incidence of ulcers
worsened during the study period. Two trials reported lower number of ulcers worsened in
individuals who had ES treatment than control group; another trial reported that the size of
PUs increased in three participants in control group without ES treatment but no PU
worsened in ES group. Pooled analysis of these three trials showed that patients with ES
treatment reported significant less number of PU worsened than control group. ((OR 0.30,
95% CI 0.10–0.89, p = 0.03, I2 = 9%, Appendix VII.
3) Adverse Event. Only two studies [67] that reported adverse events in all thirteen trials.
One study indicated that some patients experienced minor adverse reactions related to ES,
which included red, raised, itchy skin beneath the large dispersive electrode. One patient had
a persistent (> 24h) redness or burn under the active electrode, which was resolved in 48
hours, presumably from too high a stimulus intensity. Another study reported that the
participant tolerated ES well, with no complications reported [71].
Review of Preventive Studies
Methodological Quality
RCTs
In a total of seventeen preventive studies, three were RCTs [78, 79, and 82]. One of the
three RCTs [79] described an appropriate method for generating the randomization sequence.
The trial was double-blinded, described the method of double- blinding and adequately
described the allocation concealment. It was the only one RCT was considered to be of
reasonably good methodological quality according to Jadad score. None of the three trials
used ‘intention to treat’ (ITT) to analyse the data.
Non-RCTs
Three case reports were not assessed for methodological quality. Eleven non-RCTs (ten
case series, one cohort study) were assessed for their reporting quality using the Down and
Black tool. Scores were range from 8 to 14 out of a total of 28. Details of methodological
quality and grade of evidence are shown in Table 1.
Intervention Features
1) Type of ES Device
Four types of ES were identified in seventeen PU preventive articles retained within this
review. The ES delivered through conventional surface electrodes was the most commonly
used stimulation intervention and was utilized in ten of sixteen studies. Other types of ES
identified for PU prevention through this review included a custom-made garment with built-
in electrodes [85], the electrical current delivered through a Sacral Anterior Nerve Root
Stimulator (SARS) implant,[80,81] or alternatively, via implanted intramuscular electrodes
[16, 87, 88].
2) Stimulation Sites
Eight out of seventeen studies stimulated gluteal muscles alone [34, 78-82, 88, 89], one
study stimulated quadriceps alone [77], and six trials activated gluteal muscles together with
other muscle groups, e.g., quadriceps, hamstrings and lumber spinal muscles [16, 76, 84-87].
The remaining two studies stimulated spine or erector spine respectively [83, 90].
3) Parameters of ES
The electrical stimulation parameters and sites varied greatly across individual preventive
studies. The use of different stimulation frequencies, intensities, pulse width, waveform, and
duration alongside diverse stimulating sites was seen in this review. Details of ES parameters
and stimulation locations were shown in Table 1.
Intervention Effectiveness
Overall, there were eleven studies that investigated dynamic effect of ES [34, 77, 78, 80-
83, 85-87, 90], six studies evaluated long-term effects [16, 76, 79, 84, 88, 89]. Within eleven
studies that investigated dynamic effect, eight demonstrated a significant reduction of
pressure under the ischial tuberosities [77, 78, 80-82, 85-87]; five studies measured local
tissue oxygenation or blood flow with three of the five studies reporting a significant increase
in regional tissue oxygenation or blood flow during the stimulation [34, 80, 83]. There were
two studies that reported an increase of tissue oxygenation in some participants, though not
all [86, 87].
In relation to the long term effect, half of the six studies demonstrated positive changes
including reduced seating pressure or incidence of PUs, increased muscle thickness, ischial
tissue oxygenation and sitting tolerance [84, 88, 89], yet other three studies reported
inconsistent findings [16, 76, 79].
Adverse Event
In total, four out of seventeen preventive studies recorded the adverse events [80, 81, 85,
86]. Among these four studies, two studies delivered ES using surface electrodes [85, 86] and
two studies used a SARS implant [80, 81]. All these four studies reported no adverse events
experienced by the participants.
DISCUSSION
ES for PU Therapy
In this systematic review of thirty studies, thirteen studies applied ES for the treatment of
PUs. Within all nine controlled trials, two studies reported higher number of PU healed
during the study period with ES treatment in comparison to sham ES or no ES treatment.
Pooled analysis showed a significant higher number of PU closed in ES group than in control
group. There were three trials that reported the incidence of ulcers worsened during the
treatment, the quantitative pooled analysis of these two trials demonstrated much less number
of PU worsened in ES in comparison with control group. A quantitative pooled analysis of all
eight unique controlled trials showed an average higher weekly healing rate during the
treatment period when patients received ES in adjunction to standard wound care. In addition
to supporting the recommendation by new NPUAP/EPUAP clinical guideline 2014 regarding
the application of ES for recalcitrant PU healing in SCI, our findings are in agreement with
previous studies demonstrating ES enhance chronic wound healing in non-SCI population
[41-44].
With regards to the stimulation mode for PU healing, two main types of ES currents have
been commonly used in the literature and were previously defined as pulsed current or direct
current. Pulsed current includes biphasic and monophasic waveforms. While monophasic PC
involves brief pulses of unidirectional flow of current followed by a finite off period, biphasic
PC consists of brief pulses of bidirectional current that has either a symmetric or
asymmetrical biphasic waveform. In symmetric biphasic PC, the bidirectional pulsed current
is equal and balanced, whereas asymmetric biphasic PC produces a bidirectional current that
is unequal and may or may not be balanced. Balanced asymmetric biphasic PC has no net
positive or negative charge, whilst unbalanced asymmetric biphasic PC creates a net positive
or negative charge over time. It is suggested that pulsed current ES more closely mimics the
“current of injury” necessary for triggering tissue healing by sustained activation of the
voltage-gated sodium channels in the surrounding tissues [91]. As compared with constant
direct current stimulation, pulsed current ES may carry a lower risk of possible skin burns and
a greater depth of penetration [91-93]. For pulsed current ES stimulus pulse settings,
stimulation duration varies from 40Hz to 100 Hz in frequency, and 50–150 V or 4mA to
45mA in intensity. Pooled analysis of seven trials showed that pulsed current ES significantly
improved weekly healing rate compared with no ES. Constant low intensity DC involves
applying continuous, unidirectional flow of current of low intensity (<1mA) for at least one
second, which has been associated with antibacterial effect in PU healing, but it can cause
chemical and thermal burns [92] This type of ES was employed by two studies in this
review., pooled analysis of these 2 trials showed an insignificant higher weekly healing rate in
people who were treated with constant direct than that those without ES. Pulsed current
appears to confer better benefit of PU healing than constant current. However, to confirm the
beneficial effect, future better both preclinical and clinical designed trials to compare these
two types of ES for PU healing is needed.
As per electrode configuration, three studies employed the active electrode directly on the
wound bed, and the negative electrode was placed on the intact skin around the edge of the
ulcer. Four studies reported that the negative and positive electrodes were placed on opposite
sides of the pressure ulcer on the intact skin. There was one study that compared electrode
placement, in which one group of participants received ES by applying the ES with positive
electrode overlaid on the ulcer and 4 negative electrodes laid around ulcer. Another group of
participants received ES by applying same ES programme with two electrodes laid on the
intact skin at the ulcer edge across wound, and the participants in the control group received
sham ES treatment with two electrodes laid on the intact skin at the ulcer edge across wound.
The authors found that electrodes overlaid on the wound bed were better than the electrode
placed surrounding the wound. In fact, the electrode polarity has long been thought a complex
issue in the literature. For instance, anodal stimulation was shown to increase fibroblast
migration [49, 94], while cathode stimulation enhanced keratinocyte migration and increased
fibroblast proliferation [91]. Both anodal and cathode stimulation have an antibacterial effect,
the cathode stimulation seems to have greater antibacterial effects [94, 95]. In our review,
only one study [70] compared electrode configuration by either placing the active electrode in
the wound and the dispersive electrode at a distance from the ulcer, versus placing both
electrodes on the edge of ulcer. Although the average weekly healing rate was significantly
higher by active electrodes overlaying the wound surface than electrodes placed surrounding
the wound, this study was not a RCT and was classified as low level of evidence with high
risk of bias. Such result should be interpreted with cautions. The different effect between two
types of electrodes placement for PU healing is inconclusive. More future pre-clinical in-vivo
models and clinical trials examining the impact of electrodes configuration for PU healing are
warranted.
ES for PU Preventions
Within the thirty studies included in this review, seventeen studies applied ES for PUs
prevention. Eleven of seventeen studies investigated dynamic effect of stimulation and six
studies assessed long-term effect of ES for PU prevention. Within the eleven studies
investigated dynamic effect, all of them demonstrated beneficial effects on decreased
interface pressure under ischial tuberosities. The underlying mechanism of reducing pressure
under ischial tuberosities during the dynamic stimulation has been suggested to be due to
pressure redistribution, which was caused by either pelvic and /or leg tilt or changes of gluteal
muscle force. In terms of tissue blood perfusion, five of the eleven studies measured tissue
oxygenation or blood flow, three of these showed a significant increase during the ES, with
the remaining two studies reporting inconsistent findings. The exact mechanism of improving
local tissue oxygenation and blood flow during the dynamic ES remains unclear, but a
dynamic ‘pressure relief’ caused by gluteus muscle contractions and/or pelvic tilt, which
dilates the micro-vessels underlying the ischial skin, may be partly attributable. Alternatively,
increased blood perfusion may result from muscle contraction allowing higher oxygen
delivery rates and metabolite removal, or neuronal excitation and cardiovascular response.
However, of the three studies which investigated the interface pressure and tissue
oxygenation or blood flow simultaneously, none of them approved the hypothesis that ES-
induced muscle activation would directly increase blood flow and oxygenation.
There were six studies which explored the long term effect of ES in PU prevention in
SCI. While three of them reported beneficial changes, such as. Increased muscle thickness,
reduced seating pressure, increased ischial tissue oxygenation and sitting tolerance or a
reduction in the incidence of PUs after a period of ES. Conversely, the other three studies
reported no statistically significant difference noted in muscle thickness, pressure distribution
under ischial tuberosities or tissue oxygenation after a period of ES applications. It is worth
noting, the follow-up period of the three studies with non-significant findings seems to be
much shorter than that in the other three studies with positive findings (eight weeks and
twelve weeks with non-significant findings versus one, two and five years in those with
positive findings). However, among the three studies with positive findings, two were single
case reports that provide the weakest evidence. The long-term beneficial effect of ES for PU
prevention is therefore inconclusive, based on the low level of evidence with diverse findings
in the six long-term studies in this review. Well-designed and large sample sized studies to
investigate the long-term of effect of ES for PU prevention is unquestionably needed.
In terms of the type of modalities to deliver ES in seventeen preventive studies, four
different types of neuromuscular ES have been identified in this review, with the traditional
surface ES system being the most commonly used intervention, with the electrodes being
placed on the skin over the nerves or over the ‘motor points’ of muscles to be activated. The
advantage of the surface ES system is that it is non-invasive and relatively technologically
simple. However, the repeated placement of the electrodes in the appropriate locations to get
the desired response requires skill and patience. Also, it can be difficult to achieve isolated
contractions or to activate the deep muscles. In addition, local skin reaction caused by
electrodes together with managing the electrodes, wires, stimulators and applying the
electrodes to the skin each session is inconvenient for long-term use.
Interestingly, one PU preventive article reported ES as capable of stimulating gluteal
muscles and hamstrings using a custom-made electrode garment with built-in electrodes (ES
shorts) [85]. The ES shorts (Axiobionics, Ann Arbor, MI, USA) were custom-developed lycra
shorts in which wires and surface electrodes were integrated. These operated by placing two
built-in surface electrodes over gluteal muscles and hamstring muscles on both sides. Whilst
this innovative device is easy to put on, and also avoids the need for skin preparation (thus
overcoming some of the common disadvantages of traditional surface ES), the authors
indicated the key limitation of such a device is that the electrodes were fixed to one place in a
one size of shorts. They suggested that the electrodes should be individually positioned in
flexible shorts to make it more practical and efficient. Future research on an improved design
of the ES shorts in order to improve the flexibility and efficiency, as well as more clinical
studies, is needed.
Within this review, two types of ES implants were identified for PU prevention in SCI.
One of them named SARS implant [80, 81], which was implanted first in SCI patients in
1976 to aid bladder management. A typical SARS implant (Fintech-Brindley SARS implant)
usually utilises electrodes that are implanted into the S2, S3 and S4 roots. Because the S2
carries only a subordinate role for the urinary bladder, patients had S3 and S4 nerve roots
implanted, as many studies confirmed the highest detrusor response was registered at these
two nerve roots. It is known that the S2 nerve roots always innervate the gluteus maximus,
triceps surae, and also innervate other glutei, the biceps femoris and the pelvic floor. In this
review, two studies conducted by Liu and colleagues explored benefit of stimulation of the S2
nerve root for activating gluteal maximus and consequently decreasing the interface pressure
under the ischial tuberosities and increasing localised blood flow to the skin. The authors
concluded that in addition to bladder and bowel management, using a SARS implant may
confer long-term benefit for tissue health in SCI. Another type of implant is the
neuromuscular electric stimulation (NMES) system [16, 87, 88]. The stimulators of NMES
system are either placed externally (percutaneous) or fully implanted within the body. The
former utilises intramuscular electrodes that pass through skin and are implanted into the
muscles to be activated. An electrode is inserted through the skin and implanted in the muscle
using a hypodermic needle and the electrode leads exit the skin and are connected to the
external stimulator. A surface electrode is used as the return electrode. The percutaneous
interface on the skin is protected by placing a junction connector over the skin surface where
the electrodes exit and the percutaneous electrodes can then activate deep muscles, provide
isolated and repeatable muscle contractions, and are less likely to produce pain during
stimulation because they bypass the sensory afferents in the skin. In this review, one study
used a 4-channel percutaneous gluteal NMES system to improve gluteus tissue health in one
patient with a SCI at level C4 in which the intramuscular electrodes were implanted
bilaterally into the gluteus maximus. Another version of an NMES implant in which the
stimulation electrodes alongside the stimulator were fully implanted was developed for
standing and transfer in SCI. The appropriate muscle groups with this implant include the
gluteus maximus, hamstrings, vastus lateralis and erector spine. The implanted electrodes are
connected by leads under the skin to the implanted stimulator, which eliminates the need for
wiring outside of the body to an external stimulator. The electrodes can be made with larger
and more durable leads because they do not pass through the skin and these may be powered
by implanted batteries, in which case, revision surgery is only required every few years to
replace the batteries.
LIMITATIONS
Systematic reviews always present a number of limitations, which include publication
bias (particularly against negative findings), language restrictions and coding of key words.
However, to minimize the potential bias, we adopted a well-structured search strategy that
was approved by a clinical librarian, and supplemented all ‘explode’ functions and utilised
hand searches as well as contacting tissue viability specialists in SCI.
Another limitation of our review was the use of the Jadad scale for assessing mythology
quality of RCTs. Although Jadad scale is a well-established tool and is widely used for
assessing and reporting on the quality of clinical and health-related studies in the literature, it
has its own limitations. The Jadad scale is criticized for being over-simplistic and placing too
much emphasis on blinding, and can show low consistency between different raters.
Furthermore, it does not take into account allocation concealment. However, in our review,
we added 2 additional assessment items, allocation concealment and intention to treat
analysis. We also assessed the methodology for each article independently by 2 authors (LL&
JM) and consensus was achieved and disagreement was discussed between 2 reviewers.
A further limitation is the inclusion of a case report in the review, which was classified as
providing a low level of evidence. Nevertheless, the aim and objectives of this current
systematic review was to identify the updated evidence, and to make recommendations for
future research, implementing neuromuscular stimulation for PU management in SCI.
Including such a case report study in our review has undoubtedly enabled provision of more
thorough and broader evidence of ES, in the application of treating or preventing PUs in SCI.
In addition, for pooled analysis of therapeutic controlled trials, higher heterogeneities

occurred across the trials in the meta-analysis can be explained by the variation of study
design and stimulation parameters (stimulation frequency, intensity, waveform) and
stimulation device used.
Finally, our review focused on assessing effectiveness of ES for PU prevention and
healing. There are many more other outcomes associated with PU healing worth of
examination such as quality of life, hospital stay, pain and cost to patients and carers.
CONCLUSION
In appraising the ES as an intervention for PU prevention and treatment in SCI, there is a
recognition of the challenges in selecting appropriate stimulation parameters e.g., stimulation
currents, stimulation frequency, length of time of stimulation and outcome measures, which
are not usually possible to validate and standardise, especially for preventive studies.
The methodological quality of the studies included in this review was generally weak, in
particular for those prevention studies, as most of them were case series without control
groups. There were only a small number of studies which assessed the long-term effect of ES
on PU prevention. It has long been established that preventing a PU occurrence is crucial for
the SCI population and the lack of Grade I evidence has undoubtedly limited the
implementation of ES for PU prevention. Future research is recommended to conduct more
rigorous long-term clinical studies, as well as improve the design of ES devices and
determine standardised outcome measures in prevention of PUs.
ES appears to increase weekly ulcer healing rate as an adjunctive therapy in SCI
population. Pulsed direct current ES confers better benefits for PU healing than constant
direct current. In order to facilitate health professionals and the health service to utilise an
optimal ES for PU healing in SCI, well-designed clinical studies are needed by using large
sample populations, on determining the optimal stimulation parameters and stimulation
location to confirm the beneficial effect on the enhancement of PU healing in SCI. However,
to better understand the exact physiological basis of ES for enhancing PU healing, future
work is urgently needed in the form of developing more preclinical in vivo models to identify
optimal mode of ES current and electrode placement (polarity).
ACKNOWLEDGMENT
We would like to thank Mr Paul Howell, a clinical librarian for helping development of
online literature search strategy. Many Thanks to Professor Sue Dyson and Professor Michael
Traynor for their academic advice and general supports on the work reported in this chapter.
Authors also would like to thank Dr Sinead Mehigan, Head of Department of Adult, Child
and Midwifery, School of Health and Education, Middlesex University, London, UK, for
providing Endnote bibliographic software.
APPENDIX I. WEEKLY HEALING RATE WITH ES TREATMENT VERSUS WITHOUT
ES TREATMENT IN SEVEN STUDIES
APPENDIX II. WEEKLY HEALING RATE USING PULSED CURRENT VERSUS

WITHOUT ES TREATMENT
APPENDIX III. WEEKLY HEALING RATE BY CONSTANT DIRECT ES VERSUS WITHOUT
ES TREATMENT
APPENDIX IV. WEEKLY HEALING RATE BY ES WITH ELECTRODE OVERLYING WOUND VERSUS WITHOUT
ES TREATMENT
APPENDIX V. WEEKLY HEALING RATE BY ES WITH BOTH ELECTRODES ON INTACT SKIN VERSUS
APPENDIX VI. NUMBERS OF PRU HEALED DURING STUDY PERIOD BY ES TREATMENT VERSUS WITHOUT
ES TREATMENT
APPENDIX VII. INCIDENCE OF PRU WORSENED WITH ES TREATMENT VERSUS
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Chapter 3
STRUCTURE AND FUNCTION

OF THE ENTORHINAL CORTEX
WITH SPECIAL REFERENCE TO
NEURODEGENERATIVE DISEASE
Richard A. Armstrong∗
Vision Sciences, Aston University, Birmingham, UK
ABSTRACT
The entorhinal cortex (EC) comprises the anterior portion of the parahippocampal
gyrus (PHG) and constitutes an important part of the medial temporal lobe (MTL) of the
brain. The EC is involved in a variety of brain functions including the analysis of
olfactory information, various types of memory, and the final integration of sensory data
before being transmitted to the hippocampus (HC) via the perforant path. Significant
pathology occurs in the EC and HC in neurodegenerative disease and therefore, these
regions may be involved in the development of the cognitive deficits characteristics of
these disorders.
Based on the severity of pathological change in the EC, neurodegenerative disorders
can be divided into three groups: (1) those in which high densities of neuronal
cytoplasmic inclusions (NCI) and/or extracellular protein deposits occur in the EC, e.g.,
Alzheimer’s disease (AD), Down’s syndrome (DS), and sporadic Creutzfeldt-Jakob
disease (sCJD), (2) those with moderate densities of NCI and/or extracellular protein
deposits in the EC, e.g., argyrophilic grain disease (AGD), dementia with Lewy bodies
(DLB), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s
disease (PiD), neuronal intermediate filament inclusion disease (NIFID), and variant CJD
(vCJD), and (3) those in which there is relatively little pathology in the EC, e.g.,
Parkinson’s disease dementia (PD-Dem), frontotemporal lobar degeneration with TDP-
43-immunoreactive inclusions (FTLD-TDP), and multiple system atrophy (MSA).
Hence, EC pathology varies significantly among disorders which could contribute to
differences in the development of memory deficits among dementias.
∗
Corresponding author: Dr. R. A. Armstrong. Vision Sciences, Aston University, Birmingham B4 7ET, UK. Tel:
0121-204-4102, fax: 0121-204-3892, e-mail: R.A.Armstrong@aston.ac.uk.
60 Richard A. Armstrong
Pathological differences among disorders could reflect either differential

vulnerability of the EC to specific molecular pathologies or variation in the degree of
spread of pathological proteins among regions of the MTL.
Keywords: entorhinal cortex (EC), hippocampal formation, perforant path, spatial memory,
neurodegenerative disease
INTRODUCTION
The entorhinal cortex (EC) comprises the anterior portion of the parahippocampal gyrus
(PHG) and constitutes an important part of the medial temporal lobe (MTL) of the brain. The
EC is involved in a variety of brain functions including the analysis of olfactory information,
which occurs at the extreme anterior portion of the EC, various types of memory, and the
final integration of sensory data before being transmitted to the hippocampus (HC) via the
perforant path. The perforant path is the major interface between the cerebral cortex, EC, and
the HC and these regions play an important role in memory including autobiographical,
episodic, and sematic memory (declarative memory), in forming memories regarding location
(spatial memory), and in consolidation of memory during sleep.
The function of the EC is intimately associated with that of the hippocampal formation
and adjacent regions. The hippocampal formation itself comprises the DG, the HC, and
subiculum; a region intermediate in morphology between the six-layered cortex and the HC
and is the source of virtually all output from the HC via the fornix. The hippocampal
formation itself is believed to be involved in many brain functions in humans including
memory (Scoville and Milner 1957), spatial location (O’Keefe and Nadel 1978), and
behavioral inhibition (Gray 1987, Gray and McNaughton 2000, Vinogradova 2001). Within
the hippocampal formation, the dentate gyrus (DG) receives information directly from the
EC, the DG being one of the few brain areas in which new neurons develop during life, a
process which may be related to the formation of new memories (Eriksson et al. 1998).
Age-related decline in episodic and working memory occurs in humans and could be
caused by neuronal losses in the EC and hippocampal formation attributable to aging. More
serious memory deficits and clinical dementia, however, are characteristic of various
neurodegenerative disorders in which pathology affecting these specific regions is evident
(Armstrong 2012, 2014). Hence, variation in the extent and distribution of pathology
affecting the EC and adjacent regions could be a factor influencing the development of
memory deficits and the course of clinical dementia. This chapter describes the basic anatomy
of the EC, its connections with the hippocampal formation, and compares the degree and
distribution of pathology affecting these regions in a range of neurodegenerative disorders.
ANATOMY OF THE ENTORHINAL

CORTEX AND RELATED STRUCTURES
In humans, the EC is located at the anterior end of the parahippocampal gyrus (PHG)
(Figure 1) and is divided into medial and lateral regions. At its anterior end it is connected to
Structure and Function of the Entorhinal Cortex with Special Reference … 61
the amygdala via the ambient gyrus (Figure 2). In addition, the EC is closely associated
anatomically and functionally with the hippocampal formation. The hippocampal formation
includes the subcallosal and supracallosal gyri, the corpus callosum, the DG, and the HC
itself (Moyer 1980). The subcallosal gyrus is a thin sheet of gray matter which curves around
the genu of the corpus callosum and is continuous with the supracallosal gyrus, while the
latter separates the corpus callosum from the cingulate gyrus. The DG (‘dentate fascia’) is a
narrow notched band of cortex that lies on the dorsal surface of the PHG and is covered by
the fimbria. Its anterior portion is located across the medial surface of the uncus and at this
point is called the ‘band of Giacomini.’
Posteriorly, the DG is continuous with the supracallosal gyrus. The HC extends along the
floor of the inferior horn of the lateral ventricle and is covered by a sheet of myelinated fibers
known as the alveus; a structure continuous with the fimbria.
The fornix is the principal output pathway of the HC, DG, and prosubiculum and
terminates in the medial nucleus of the mammillary body.
Most regions of the cerebral cortex comprise six layers or laminae. In the visual system,
for example, information arrives in the primary visual cortex (BA 17) from the lateral
geniculate nucleus (LGN) in lamina IV (Singer 1979) and is then conveyed to laminae II and
III and to a lesser extent lamina V. The output from area V1 to ‘association areas’ BA 18, BA
19, etc., where more complex visual processing takes place, is mainly via the large pyramidal
neurons in laminae II/III which constitute the feedforward cortico-cortical projections (Zeki
1971, De Lacoste and White 1993).
In addition, neurons in the lower cortex provide the output to subcortical visual areas,
e.g., from lamina VI to the dorsal and ventral LGN and from lamina V to the superior
colliculus and the pretectal area (Parnavelos and McDonald 1983).
CC = Corpus callosum, CG = Cingulate gyrus, EC = Entorhinal cortex, FP = Frontal pole, PHG =

Parahippocampal gyrus, OC = Occipital cortex, TP = Temporal pole.
Figure 1. Anatomy of the entorhinal cortex and related gyri.

Figure 2. Coronal section of the anterior entorhinal cortex (EC) showing its connection to the amygdala
(AM) via the ambient gyrus (AmGy); (Luxol fast blue/haematoxylin and eosin stain, magnification bar
= 2 mm).
By contrast, a distinctive feature of the EC is the marked lack of cell bodies in lamina IV
(‘lamina dissecans’) giving the appearance of a laminar structure in three bands, each band
having distinct properties and patterns of connectivity (Figure 3).
ANATOMICAL CONNECTIONS OF THE ENTORHINAL

CORTEX AND HIPPOCAMPUS
Neurons in laminae II/III of the EC (Figure 3) send their axons to the DG and HC, those
from lamina II primarily providing an excitatory input to the DG and sector CA3 and those
from lamina III to sector CA1 and the subiculum.
In addition, lamina V of the EC receives one of the main outputs from the HC. The DG
has a characteristic ‘horse-shoe’ appearance in coronal section (Figure 4) consisting of a
dense band of granule cells which have ramifying dendrites extending into the molecular
layer. The axons of the granule cells, viz., the mossy fibres, then pursue a course along the
hippocampal pyramidal cells making synaptic contacts with the basal parts of their apical
dendrites via the ‘mossy tufts’ which are enlarged, saclike synaptic terminals (Brodal 1981).
The hippocampal formation makes relatively few anatomical contacts with the rest of the
brain (Figure 4). Hence, projections from the EC terminate in the molecular layer of the DG.
From the DG, mossy fibers travel to sectors CA3/4, while sector CA3 fibers pass to the lateral
septal nuclei with collaterals travelling to sector CA1. Sector CA1 axons are destined for the
subiculum which represents the major output pathway of the HC via the fornix. In addition,
there may be inputs to the HC from the septum, the orbital region of the prefrontal cortex to
CA1 and CA3, and from the amygdala, locus caeruleus, and raphe nuclei.
Figure 3. Laminar anatomy of the entorhinal cortex (EC) showing the three banded structure (laminae
II/III, IV, and V/VI); (Luxol fast blue/haematoxylin and eosin stain, magnification bar = 200 μm).
The pattern of interconnections is regular and in strips, flow of information around the
hippocampal circuit being topographical with activity commencing within one strip remaining
largely confined to that strip (Rawlins and Green 1977). There are, however, inhibitory
influences of one strip on another and excitatory connections between different strips which
largely involve sector CA3 (Rawlins and Green 1977). In addition, there are direct projections
from the EC to sectors CA3 and CA1 and a return pathway from sector CA3 to the DG.
Sector CA3 is also the source of commissural fibers which connect the hippocampi in
both hemispheres and which terminate in all of the major sectors. Hence, within hippocampal
formation, sector CA3 appears to be an important ‘nodal’ point of a circuit at which many
major projections converge.
FUNCTIONS OF THE ENTORHINAL

CORTEX AND HIPPOCAMPAL SYSTEM
The EC provides the main interface between the cerebral cortex and the HC. There is
general agreement that the EC and hippocampal formation play an important role in memory,
the EC functioning as a hub in a widespread neural network concerned with memory and
spatial navigation. However, there have been three major theories concerning the functioning
of the EC-HC system in humans: (1) the memory theory, (b) the spatial theory, and (3) the
behavioural inhibition theory. The memory theory has its origin in the surgical destruction of
the HC which results in severe anterograde and partial retrograde amnesia (Scoville and
Milner 1957). Hence, the HC is believed to be important in the formation of new memories
concerning events that have been experienced (‘episodic’ or ‘autobiographical’ memory).
CA1-4 = Sectors of cornu ammonis, DG = Dentate gyrus, EC = Entorhinal cortex, Sub = Subiculum;
(Luxol fast blue/haematoxylin and eosin stain, magnification bar = 3 mm.
Figure 4. Coronal section of the anterior hippocampus and adjacent structures.
It is also involved in the detection of novel events, places, and stimuli and is part of a
larger MTL system responsible for ‘declarative’ memory, i.e., memories that can be explicitly
verbalized. Nevertheless, damage to the HC does not affect the ability to learn new motor or
cognitive skills (‘procedural’ memory).
The spatial theory has its origin in the observation that neurons in the rat EC and HC
show activity related to location within the environment. Hence, many neurons in these
regions have ‘place fields,’ i.e., there are bursts of action potentials when an animal passes a
specific location (O’Keefe and Nadel 1978). Subsequently, it was shown that the EC contains
a neural map of the spatial environment (Hafting et al. 2005).
The lateral EC appears to have little spatial selectivity but the medial EC exhibits
multiple place fields arranged in a hexagonal pattern (‘grid cells’) which increase in a dorsal-
lateral direction. In addition, the EC possesses ‘direction path’ cells which indicate directional
activity regardless of actual location whereas place cells in the HC are activated only by a
specific location (Jacobs et al. 2010). These results suggest that the EC encodes general
spatial properties of the environment which then can be used by the HC to create unique
representations of specific places.
The behavioural inhibition theory has been particularly controversial. This theory states
that the EC and HC together function in a cognitive system which acts essentially as a
comparator (Gray 1987, Gray and McNaughton 2000, Vinogradova 2001), the memory and
spatial functions being a part of a larger overall system of behavioural inhibition.
First, the EC receives information from all sensory systems via the temporal lobe
regarding ‘the state of the world,’ the latter acting as a data storage region (Stefakhina et al.
1975, Vinogradova and Bragin 1975).
Second, this information is transmitted from the EC to the DG via the perforant path and
also to the subiculum. Third, the hippocampal circuit receives information from the prefrontal
cortex and cingulate gyrus as well as from the medial and lateral septum and brain stem
nuclei including the serotonergic raphe nucleus and the noradrenergic locus caeruleus.
Fourth, in the many loops of anatomical connections which comprise the hippocampal
formation, the subiculum appears particularly important as it receives direct inputs from the
temporal lobe via the EC and the same information after it has circulated around the
hippocampal circuit via the DG, sector CA3, and sector CA1. Fifth, circulation of information
regarding the state of the world around the hippocampal circuit results in specific items being
selected that are important to a particular task. Hence, there may be a ‘gate’ located between
the DG and sector CA3 which only allows novel stimuli to pass through. Whether a stimulus
passes through the gate may be controlled by noradrenergic and serotoninergic inputs from
the locus caeruleus and raphe nuclei respectively. Hence, the HC compares current sensory
events, obtained from the image of the world, with expected or predicted events generated by
the ‘comparator.’ If an expectation continues to be verified by information derived from the
image of the world then no alteration of current behavior results. If, however, a discrepancy is
detected, the HC intervenes by initiating appropriate action and directly controlling behavior
by active inhibition of current motor activity and initiation of new data gathering.
ROLE OF ENTORHINAL CORTEX IN NEURODEGENERATIVE DISEASE

In humans, certain types of memory, including episodic and working memory, decline
with age, which could be result from an age-related loss of neurons in the EC and HC.
Nevertheless, human aging does not appear to cause a substantial loss of neurons in these
regions, although there are synaptic changes which may affect the efficiency of transmission
of information among them. In many of the neurodegenerative diseases which affect humans,
however, the EC and HC are significantly damaged by the developing pathology, thus
potentially influencing a variety of cognitive functions (Armstrong 2014). In addition,
variation in the specific regions affected by the pathology in different neurodegenerative
disorders could influence the course of clinical dementia in the various diseases.
Two types of brain pathology are important in the pathogenesis of neurodegenerative
disease in humans (Table 1). First, aggregates of abnormal or misfolded extracellular proteins
may be deposited in the form of senile plaques (SP), such as the β-amyloid (Aβ) deposits
observed in Alzheimer’s disease (AD) and Down’s syndrome (DS) (Glenner and Wong
1984), and the disease form of prion protein (PrPsc) deposits found in Creutzfeldt-Jakob
disease (CJD) (Armstrong 2006). Second, abnormal protein aggregations occur within the
cytoplasm of neurons, viz., neuronal cytoplasmic inclusions (NCI) such as neurofibrillary
tangles (NFT) in AD, argyrophilic grain disease (AGD), and progressive supranuclear palsy
(PSP), Lewy bodies (LB) in dementia with Lewy bodies (DLB) and Parkinson’s disease
dementia (PD-Dem), Pick bodies (PB) in Pick’s disease (PiD), tau-immunoreactive inclusions
in neurons and glial cells in corticobasal degeneration (CBD), transactive response (TAR)
DNA-binding protein of 43kDa (TDP-43)-immunoreactive inclusions in frontotemporal lobar
degeneration (FTLD-TDP) (Armstrong et al. 2010), and ‘fused in sarcoma’ (FUS)-
immunoreactive inclusions in neuronal intermediate filament inclusion disease (NIFID)
(Armstrong et al. 2011). Accumulation of one or more of these proteins in the form of
deposits or inclusions is likely to lead to neuronal death and subsequent neurodegeneration
within the EC and associated regions in the different disorders.
Alzheimer’s Disease (AD)
Alzheimer’s disease (AD) (Tierney et al. 1988, Mirra et al. 1981) is a degenerative
disorder of the nervous system affecting approximately 10% of individuals aged 65 or over
(Knopman 2001). The development of dementia in AD involves a decline in short-term
memory, impairment of judgment, and a loss of emotional control.
These symptoms develop slowly over a period of years resulting in a complete
breakdown of mental function. A small proportion of cases of AD (<5%) have a genetic
origin and most cases occur sporadically within the population (Hoenicka 2006). The
pathology of AD includes the deposition in the brain of abnormal aggregates of Aβ as SP and
abnormally phosphorylated tau in the form of NFT.
Within the temporal lobe in AD, the EC and sector CA1 of the hippocampus appear to be
the most vulnerable to the developing pathology (Meencke et al. 1983, Pearson et al. 1985,
Saper et al. 1987, Armstrong 1992) (Table 2).
Table 1. Pathological lesions and their molecular composition
Disorder Pathology Molecular constituent

AGD NFT, GR 4R tau
AD SP, NFT Aβ, tau
CBD NCI 4R tau
sCJD SP PrPsc
vCJD SP PrPsc
DLB LB α-synuclein
DS SP Aβ
FTLD-TDP NCI TDP-43
MSA GCI α-synuclein
NIFID NCI FUS
PD-Dem LB α-synuclein
PiD PB 3R tau
PSP NFT 4R tau
SP = Senile plaques, Aβ = β-amyloid deposits, GR = Grains; NFT = Neurofibrillary tangles, PrPsc =
Disease form of prion protein, LB = Lewy bodies, PB = Pick bodies, NCI = Neuronal cytoplasmic
inclusions, GCI = Glial cytoplasmic inclusions, FUS = Fused in sarcoma, TDP-43 = Transactive
response (TAR) DNA-binding protein of 43kDa) in various disorders (AGD = Argyrophilic grain
disease, AD = Alzheimer's disease, DS = Down’s syndrome (DS), PiD = Pick's disease, CBD =
Corticobasal degeneration, PSP = Progressive supranuclear palsy, DLB = Dementia with Lewy
bodies, MSA = multiple system atrophy, NIFID = Neuronal intermediate filament inclusion
disease, FTLD-TDP = Frontotemporal lobar degeneration with TDP-43 proteinopathy; PD-Dem =
Parkinson’s disease dementia, sCJD = Sporadic Creutzfeldt-Jakob disease, vCJD = Variant
Creutzfeldt-Jakob disease (vCJD).
Table 2. Pathways involving the entorhinal cortex (EC) and hippocampus (HC) likely to
be affected by the pathology in various neurodegenerative disorders: Not significantly
affected (-), Affected (+), Moderately affected (++), Severely affected (+++)
Hippocampal pathway affected

Initial processing HC Processing
Disorder Input (EC) Output CA1/Sub
(DG) (CA2/3/4)
AGD + - + ++
AD ++ + + ++
CBD + - + +
sCJD ++ - - -
vCJD + - - ++
DLB + - + -
DS ++ + + ++
FTLD-TDP - + - -
MSA - - - +
NIFID + ++ + +
PD-Dem - - + -
PiD + +++ + +
PSP + - +
Abbreviations: Argyrophilc grain disease (AGD), Alzheimer's disease (AD), Down’s syndrome (DS),
Pick's disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP),
dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuronal intermediate
filament inclusion disease (NIFID), frontotemporal lobar degeneration with TDP-43 proteinopathy
(FTLD-TDP); Parkinson’s disease dementia (PD-Dem), sporadic Creutzfeldt-Jakob disease
(sCJD), Variant Creutzfeldt-Jakob disease (vCJD); Regions: Entorhinal cortex (EC), dentate gyrus
(DG, hippocampus (HC), Subiculum (Sub), Cornu ammonis (CA1-4).
Damage to these regions in AD can lead to major memory problems and disorientation.
In addition, if there is extensive bilateral HC damage, it can lead to anterograde amnesia and
an inability to form or retain new memories. The pathology associated with AD is likely to
affect several processes within the hippocampal formation.
First, the perforant path from EC to DG could be significantly affected, and therefore, the
input of sensory information regarding the current state of the world. Second, NFT have been
recorded in sectors CA2 and CA3 and Aβ deposits are present in the molecular layer of the
DG potentially affecting the ‘gate’ between the DG and sector CA3 and therefore, the transfer
of information around the HC circuit. Third, the pathway from sector CA1 to the subiculum,
which constitutes the major output from the HC, is also likely to be compromised in AD.
Hence, all aspects of information processing within the hippocampal formation could be
potentially affected in AD, and may be a factor contributing to the profound dementia typical
of the disease.
Argyrophilic Grain Disease (AGD)
AGD is a controversial disease entity (Martinez-Lage and Munoz 1997, Ikeda et al. 2000)
characterised by the presence of ‘dot-like’ structures in tissue sections known as argyrophilic
grains (AG) and which are found largely in the HC and adjacent gyri including the EC and
ambient gyrus (Braak and Braak 1987, 1989; Tolnay et al. 2003). Patients with AGD exhibit
a progressive cognitive decline resulting in dementia (Tolnay et al. 2001, Saito et al. 2002),
which is associated with behavioural abnormalities, personality change, and emotional and
mood disturbances (Braak and Braak 1998, Ikeda et al. 2000).
Episodic memory loss, however, appears to be the most characteristic clinical symptom
(Ikeda et al. 2000). In addition to AG, NCI in the form of NFT (including ‘pre-tangles’) occur
in affected areas together with pathology associated with astrocytes and oligodendrocytes
(Tolnay et al. 2003, Tolnay and Clavaguera 2004). The NFT of AGD are composed largely of
four-repeat (4R) tau (Ishizawa et al. 2002).
There are moderate densities of NFT in the EC and DG in cases of AGD (Table 2)
suggesting that the input of data to the hippocampal formation could be affected in these
patients. In addition, NFT are present consistently in both CA1 and CA2 suggesting that
processing within the HC and the output of data could also be affected.
Corticobasal Degeneration (CBD)
CBD is a rare, progressive movement disorder, the most characteristic clinical features of
which include limb dysfunction (Rinne et al. 1994, Schneider et al. 1997, Wenning et al.
1998), parkinsonism (Ueno 1996), apraxia (Schneider et al. 1997), and dementia (Ueno
1996). An individual patient, however, may exhibit a wide variety of clinical symptoms
(Tsuchiya et al. 1997a) including myoclonus (Carelli et al. 1997), memory loss, behavioral
change, speech, and gait problems (Schneider et al. 1997). Neuropathologically, CBD is
characterized by a progressive cortical atrophy affecting the anterior cerebral cortex
(Tsuchiya et al. 1997a), the fronto-parietal region (Ikeda 1997), and the superior temporal
cortex (Ikeda 1997). As a consequence, the disease is regarded as a subtype of FTLD (Cairns
et al. 2007). There is atrophy of the basal ganglia, including the caudate nucleus (Markus et
al. 1995, Tsuchiya et al. 1997b) and substantia nigra (Kawasaki et al. 1996).
In addition, widespread neuronal and glial pathology is present including ballooned
neurons (BN) (Mori and Oda 1997), neuropil threads (Komori et al. 1997), tau-
immunoreactive NCI (Ikeda 1997), GI (Matsumoto et al. 1996), and astrocytic plaques (AP)
(Dickson et al. 1996). The tau-immunoreactive lesions are composed of predominantly 4R
tau, as in PSP (Trojanowski and Dickson 2001).
The density of tau-immunoreactive inclusions in the EC and HC in CBD is relatively
low, but where lesions occur, are likely to affect primarily the input of sensory data to the HC
via the perforant path, processing of information in the DG being relatively unaffected (Table
2). In addition, the output pathway from sector CA1 to the subiculum could be compromised
to some extent, a similar pattern of damage to that seen in AD, which may explain why some
patients with CBD appear to develop an AD-type dementia.
Creutzfeldt-Jakob Disease (CJD)
CJD was first described by Creutzfeldt and Jakob in the 1920s (Creutzfeldt 1921, Jakob
1921) and is the most important human form of human prion disease (Budka et al. 199,
Armstrong 2006). Patients develop a rapidly progressive dementia, which is sometimes

preceded by ataxia and loss of coordination, death resulting in about 3-24 months after the
onset of symptoms. Several subtypes of CJD have been described including those associated
with genetic factors (familial or fCJD), those resulting from the transmission of PrPsc
(iatrogenic or iCJD), and most commonly, those that occur sporadically within the population
(sCJD). In addition a new variant of CJD (vCJD) has been described, mainly in the UK (Will
et al. 1996).
The distribution of PrPsc deposits in sCJD is similar to that of Aβ deposits in AD in that
high densities occur in the EC, but differs in that the CA sectors of the HC and the DG appear
to be less affected (Table 2). Hence, it is the input of sensory information to the HC that is
primarily affected in sCJD, processing within the DG and HC, and the output pathways being
less affected than in AD.
Hence, significant differences in dementia in may be anticipated in sCJD and AD. In
vCJD, diffuse PrPsc deposits are usually present at greatest density in the EC and subiculum
compared with sectors CA1, CA3, CA4, and the DG and florid PrPsc deposits are usually
present in greater density in the subiculum compared with other regions. Hence, processing
within the DG may be affected to a greater extent in vCJD than in sCJD.
Dementia with Lewy Bodies (DLB)
DLB (also known as 'Lewy body dementia' or ‘diffuse Lewy body disease') is the second
most common form of dementia after AD (McKeith et al. 1996) and may account for up to a
quarter of all cases of late-onset dementia. This disorder is characterized by a progressive
disabling mental impairment and includes fluctuating cognition, visual hallucinations, and
parkinsonism as typical features (Armstrong 2008).
The essential feature necessary for a neuropathological diagnosis of DLB is the presence
of LB, inclusions which are also observed in patients with PD-Dem (Armstrong 2008). LB
are spherical structures found in the cytoplasm of affected cells and can be immunolabelled
by a variety of histological methods including alpha-B-crystallin, ubiquitin, and most
significantly, α-synuclein. The synucleins are small proteins (123-143 amino acids), localized
in presynaptic terminals, and may have a role in neurotransmission and/or synaptic
organization (Iwai et al. 1995).
The distribution of LB in DLB resembles that of NFT in AD in that significant densities
occur in the EC (Table 2). The distribution of LB, however, differs from NFT in AD in that in
AD, sector CA1 is the most significantly affected region while in DLB, sectors CA2, CA3,
and to a lesser extent sector CA4, are more seriously affected.
Hence, the input of sensory information from EC to the DG and the processing of
information within the HC circuit could be affected in DLB. However, few or no LB have
been observed in DG granule cells suggesting that initial processing of information within the
DG is less compromised.
In addition, the output of information from the HC is less affected than in AD. Hence,
there could be subtle differences in clinical dementia in AD and DLB resulting from these
anatomical variations.
Down’s Syndrome (DS)
Virtually all patients with DS develop Aβ deposits within the brain if they survive into
their thirties (Mann and Esiri 1989, Motte and Williams 1989, Wisniewski et al. 1985), but
particular accumulations of deposits occur between the ages of 30 and 50 years (Hyman et al.
1995). In addition, approximately 25% of DS patients develop the clinical symptoms of
dementia (Dalton and McLachlan 1984). Within the temporal lobe, there are considerable
similarities in the distribution of Aβ deposits in the temporal lobe in DS and AD (Table 2),
Aβ deposits being present in the EC and in all areas of the hippocampal formation including
the DG. In both AD and DS, Aβ deposits are present at greatest densities in the EC, in sector
CA1, and the molecular layer of the DG. Hence, similar pathways are likely to be
compromised in DS and AD, which may explain the similarities in clinical dementia observed
in the two disorders.
Frontotemporal Lobar Degeneration (FTLD)
FTLD is the second commonest form of cortical dementia of early-onset after AD

(Tolnay and Probst 2002, Josephs 2008). The disorder is associated with a heterogeneous
group of clinical syndromes including frontotemporal dementia (FTD), FTD with motor
neuron disease (FTD/MND), progressive non-fluent aphasia (PNFA), semantic dementia
(SD), and progressive apraxia (PAX) (Snowden et al. 2007). FTLD with TDP-43
proteinopathy (FTLD-TDP), previously called FTLD with ubiquitin positive inclusions
(FTLD-U), is characterized by variable neocortical and allocortical atrophy principally
affecting the frontal and temporal lobes. In addition, there is neuronal loss, microvacuolation
in the superficial cortical laminae, and a reactive astrocytosis (Cairns et al. 2007). A variety of
TDP-43-immunoreactive lesions are present in FTLD-TDP including NCI, neuronal
intranuclear inclusions (NII), dystrophic neurites (DN) and, oligodendroglial inclusions (GI).
In FTLD-TDP, the greatest densities of NCI are often observed in the DG, densities being
significantly lower in the EC and in sectors CA1/2 (Woulfe et al. 2001, Kovari et al. 2004,
Mackenzie et al. 2006) (Table 2). Hence, input of data via the EC and initial processing of
this information within the DG are likely to be most significantly compromised in FTLD-
TDP.
Multiple System Atrophy (MSA)
Multiple system atrophy (MSA) is a rare, largely sporadic movement disorder associated
with varying degrees of parkinsonism, ataxia, and autonomic dysfunction. MSA encompasses
three major subtypes, viz., olivopontocerebellar atrophy or cerebellar subtype (MSA-C),
striatonigral degeneration or parkinsonian subtype (MSA-P), and Shy-Drager syndrome (Papp
and Lantos 1994). The average annual incidence of the disorder is 3.0/100,000 of the
population and the median survival time is 8.5 years (Bower et al. 1997). The disorder is
slightly more common in males than females (1.3:1), symptoms beginning early in the fifth
decade of life (Wenning et al. 1997).
The most consistent clinical syndrome in MSA is the presence of parkinsonism, followed
by autonomic dysfunction, cerebellar ataxia, and pyramidal tract signs (Wenning et al. 1997).
The neuropathology of MSA is characterised by anatomically selective neuronal loss,
gliosis, and myelin pathology. The hallmark pathological lesion is the glial cytoplasmic
inclusion (GCI) found mainly in oligodendrocytes and first described in 1989 (Papp et al.
1989). This pathology affects the SN, striatum, inferior olivary nucleus, pontine nuclei, and
cerebellum (Dickson et al. 1999).
In addition, there may be a progressive cerebral atrophy in severe cases affecting the
frontal lobes (Konogaya et al. 1999) and the motor/premotor areas (Wakabayashi et al. 1998).
The GCI are silver positive 10-15 nm diameter coated filaments, the major molecular
constituent of which is α-synuclein. α-Synuclein is also found in the LB of PD-Dem and
DLB and unites these disorders with MSA as a major class of neurodegenerative disorders,
the synucleinopathies (Spillantini et al. 1998).
The synucleins themselves are small proteins (123-143 amino acids) and localized to
presynaptic terminals and may have a role in neurotransmission and/or synaptic organisation
(Iwai et al. 1995). Inclusions have also been observed in the nuclei, cytoplasm and cell
processes of neurons in MSA (Dickson et al. 1999). In addition, neuronal abnormalities have
been observed including enlargement and atrophy of the cell body (Armstrong et al. 2004).
In MSA, the overall density of the GCI in the EC and HC is usually low and these lesions
also have a more restricted distribution, being observed most commonly in sectors CA1,
CA2, and CA3, with little evidence for significant involvement of the DG (Table 2). Hence,
relatively little pathology is observed in the EC and HC in MSA, with the possible exception
of the involvement of sector CA1 (Armstrong et al. 2004), and these patients have been
observed to exhibit a clinical dementia more rarely.
Neuronal Intermediate Filament Inclusion Disease (NIFID)
Neuronal intermediate filament inclusion disease (NIFID) is a disease of early-onset with

a variable clinical phenotype that includes FTD, pyramidal, and extrapyramidal signs (Bigio
et al. 2003, Cairns et al. 2003, Josephs et al. 2003).
Neuropathologically, there is degeneration of the cerebral cortex, striatum, and brain stem
with neuronal loss in the frontal, parietal, and temporal cortex (Bigio et al. 2003, Cairns et al.
2003, Cairns et al. 2004a). Abnormal neuronal intermediate filament (IF) aggregates as NCI
immunoreactive for ‘fused in sarcoma’ (FUS) protein are present in NIFID (Cairns et al.
2004b). In addition, swollen achromatic neurons (SN) and gliosis are observed in affected
areas (Cairns et al. 2003).
The pathology of NIFID frequently affects the EC and HC and adjacent gyri, and
especially the DG and a lesser extent sector CA1 (Table 2).
This pathology is likely to influence varies pathologies involving the hippocampal
formation including the input of information to the HC, processing of information within the
HC, and the output pathway from the HC. Hence, pathology in the EC and HC may play a
particularly significant role in the development of dementia in NIFID.
Parkinson’s Disease Dementia (PD-Dem)
Parkinson’s disease (PD) is a chronic neurodegenerative disorder resulting in a variety of

movement problems including bradykinesia, rigidity, tremor, and postural instability. Non-
motor symptoms, however, can also play a significant role in the disease (Antal et al. 1998,
Armstrong 2008). Hence, cognitive decline in PD can range from subtle mental dysfunction
to overt dementia (Braak et al. 2005). PD-Dem is a particularly debilitating condition
substantially increasing mortality (Willis et al. 2012). The risk of PD-Dem increases with
patient age, 90% of recorded cases being over 70 years of age (Rana et al. 2012). In PD-Dem,
there is reduced gray matter volume in several brain regions including temporal cortex, most
notably the PHG and EC, parietal and frontal cortices, cingulate gyrus (CG), caudate nucleus,
HC, amygdala, and putamen (Melzer et al. 2012).
Histologically, PD is characterized by the death of pigmented neurons in the substantia
nigra (SN), neurons of the SN and cerebral cortex often containing α-synuclein-
immunoreactive LB. In the hippocampal formation of PD-Dem, LB most significantly affect
sectors CA2, CA3, and CA4, with fewer LB in the EC, sector CA1, the DG being the least
affected (Table 2). However, small numbers of α-synuclein-immunoreactive rounded
inclusions resembling small LB have been observed in DG granule cells, which could be
associated with pathological changes in the EC spreading to the DG via the perforant path.
The density of LB in PD-Dem is similar to that of DLB, i.e., LB are present in gyri adjacent
to the HC and within the HC, greater densities are present in sectors CA2/CA3 compared with
CA1 (Armstrong et al. 1998). Hence, processing of information within the HC is most likely
to be affected in PD-Dem.
Pick’s Disease (PiD)
PiD (Pick 1906) accounts for approximately 0.4-2% of all cases of dementia (Jellinger et
al. 1990). Both sporadic and familial forms of the disease exist, the latter being inherited as a
dominant gene but with polygenic modification (Slater and Cowrie 1971). Clinically, PiD
patients exhibit a primary progressive aphasia, behavioral change, extrapyramidal symptoms,
and apraxia (Kertesz and Munoz 1997). Neuropathologically, PiD is characterized by a
fronto-temporal cortical atrophy, degeneration of white matter, together with the presence of
achromatic Pick cells (PC) and cortical and hippocampal PB (Delacourte et al. 1996). PB are
eosinophilic, argyrophilic NCI immunoreactive for hyperphosphorylated tau, ubiquitin (Ub),
and Alz-50 (Love et al. 1988). They are also immunoreactive for neurofilament proteins,
clathrin, synatophysin (Nakamura et al. 1994), chromagranin-B (Bergmann et al. 1996), and
advanced glycylation end-products (Kimura et al. 1996). The tau-immunoreactive lesions are
composed of predominantly 3-repeat (4R) tau, and are therefore distinct from those of other
tauopathies such as CBD that consist mainly of either 4R tau alone, or a mixture of 3R and
4R tau as in AD (Trojanowski and Dickson 2001). PB in PiD have a characteristic
distribution in the EC and hippocampal formation (Table 2), with significantly higher
densities affecting the granule cell layer of the DG. Hence, it is the processing of sensory
information arriving from the EC via the perforant path and the functioning of the gate
between the DG and sector CA3 that are likely to be compromised in PiD.
Progressive Supranuclear Palsy (PSP)
Progressive supranuclear palsy (PSP) (Hauw et al. 1994, Litvan et al. 1996 a,b) is a
largely sporadic, multisystem disorder first described as a clinical entity in 1964 and is the
second most common parkinsonian syndrome (Papapetropoulos et al. 2005). Despite the
predominantly subcortical symptoms, some patients exhibit frontal lobe signs (‘dysexecutive
syndrome’) suggesting a degree of cortical pathology (Gomez-Haro et al. 1999).
Neuropathologically, PSP may be distinguished from normal adult brains and from PD-Dem
by symmetrical tissue loss in the frontal cortex (maximal in the orbitofrontal and medial
frontal cortices), subcortical nuclei (midbrain, caudate, and thalamic) (Cordato et al. 2005). In
addition, there is neuronal loss, gliosis, NFT, granulovacuolar change (GVC), and
demyelination, mainly affecting the globus pallidus, subthalamic nucleus, SN, dentate nucleus
of the cerebellum, and brain stem tegmentum (Lantos 1994, Daniel et al. 1995). The NFT are
structurally and biochemically different to those observed in AD. There are both globose and
flame-shaped NFT composed of 15 nm straight filaments comprising six or more
protofilaments 2-5 nm in diameter (Tellez-Nagel and Wisniewski 1973, Montpetit et al.
1985). The NFT are tau-immunoreactive and composed largely of 4R tau (Dickson 1999).
The overall density of NFT in the EC and NFT is low in most cases of PSP suggesting
fewer disturbances of hippocampal function than in other disorders (Table 2). NFT may
develop in the HC later in the disease, however, which may explain the delayed development
of dementia in some patients. Pathological changes, have also been recorded at low density in
the EC, (Braak et al. 1992), suggesting that the perforant path could be affected to some
extent. In addition, NFT occur in sector CA1 and to a lesser extent in CA2 suggesting that
processing within the HC could also be compromised. In addition, degeneration of the
cholinergic pathway from the medial septal area to sector CA3 and the subiculum has been
reported in PSP (Shinotoh et al. 1999), and it is possible that sector CA1 neurons are
secondarily affected via their connections with these regions. Pathological changes have also
been observed in the alveus (Armstrong et al. 2009) which could be a response to
degeneration of sector CA1 and the subiculum.
CONCLUSION
Based on the severity of pathological change in the EC, neurodegenerative disorders can
be divided into three groups: (1) those in which high densities of NCI and/or extracellular
protein deposits occur in the EC, e.g., AD, DS, and sCJD, (2) those with moderate densities
of NCI and/or extracellular protein deposits in the EC, e.g., AGD, DLB, PSP, CBD, PiD,
NIFID, and vCJD, and (3) those in which in there is relatively little pathology in the EC, e.g.,
PD-Dem, FTLD-TDP, and MSA. Hence, there are considerable differences in the degree to
which the EC and its connections to the DG are likely to be compromised in the various
disorders which could affect the course of clinical dementia. Two hypotheses could explain
differences in pathology affecting the EC among disorders: (1) differential vulnerability of
EC neurons to the various molecular pathologies and (2) variation in the extent to which
pathology may spread via cell to cell transfer to involve the MTL and specifically the EC as
the disease develops (Goedert et al. 2010, Steiner et al. 2011).
In AD, there is a preferential loss of neurons expressing 75KD neurotrophic receptor

p75NIr (Yaar et al. 1997) and Aβ binds specifically to this receptor.
In addition, neurons in the MTL secrete large quantities of amyloid precursor protein
(APP) in response to head trauma and that there are more APP-immunoreactive neurons in
these areas in head injury patients (McKenzie et al. 1994). These observations suggest that
EC neurons could be differentially vulnerable to different molecular pathologies and
especially to the formation of protein deposits such as Aβ or PrPsc.
Variation among disorders could also reflect differences in the spread of various
molecular pathologies within the temporal lobe. Hence, in elderly control cases, Aβ deposits
are present in temporal lobe in a significant number of cases, but only in cortical gyri, e.g.,
EC and lateral occipitotemporal gyrus (LOT), no deposits being observed in the subiculum,
CA sectors of HC, or DG (Armstrong 1994). In addition, there is considerable variation in the
density of deposits in control cases and a significant overlap with AD. Hence in AD, spread
of Aβ pathology into the HC could have occurred from the EC as the disease develops.
Recent research suggests that pathogenic proteins such as tau, α-synuclein, Aβ, and PrPsc
may be secreted from cells, enter other cells, and seed small intracellular aggregates to form
larger aggregates and inclusions (Goedert et al. 2010, Steiner et al. 2011) and is consistent
with this hypothesis. Hence, variation in the rate of spread of various pathological proteins
into the EC and subsequently to the HC, could be an important factor determining the
distribution of pathology in various disorders and therefore have a significant role in the
development of clinical dementia.
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Chapter 4
GINSENOSIDES AS BRAIN SIGNALING MOLECULES

AND POTENTIAL CURES FOR NEUROLOGICAL AND
NEURODEGENERATIVE DISEASES
Ze-Jun Wang* and Thomas Heinbockel†

Department of Anatomy, Howard University College of Medicine,
Washington, DC, US
ABSTRACT
Ginseng has been used as a traditional medicine in Asia for thousands of years and is
a popular natural medicine throughout the world. Ingredients of ginseng that have
biological activity include more than 30 different compounds known as ginsenosides.
Recent studies have reported that ginsenosides display beneficial effects on central
nervous system (CNS) processes and disorders such as aging, deficit of memory and
learning capabilities, and neurodegenerative diseases. Ginsenosides can be classified into
four types of aglycones: protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid
types.
This review will discuss recent advances in our understanding of the effects of
different ginsenosides on CNS targets and how ginsenosides can contribute to cures for
some of the most devastating neurological disorders and neurodegenerative diseases. We
focus in more detail on ocotillol as a derivate of pseudoginsenoside-F11, which is an
ocotillol-type ginsenoside found in American ginseng.
We describe the effect of ocotillol on neuronal activity in individual cells and circuits
and on locomotor behavior.
Keywords: ginsenosides, American ginseng, central nervous system, pseudoginsenoside-F11,

ocotillol, electrophysiology, olfactory bulb
*
Tel.: (202) 806-9495, Fax: (202) 265-7055, E-mail: zejunwang@hotmail.com.
†
Tel.: (202) 865-0058, Fax: (202) 265-7055, E-mail: theinbockel@howard.edu.
84 Ze-Jun Wang and Thomas Heinbockel
1. INTRODUCTION
Ginseng has been used as a traditional medicine in Asia for thousands of years and is a
popular natural medicine throughout the world. An ancient book named “Shen Nong Ben Cao
Jin” (神农本草经), an herbal book edited around 220 B.C., recorded the medical use of
ginseng for many reasons. Another old book, the earliest encyclopedia of Chinese herbs,
named “Ban Cao Gang Mu” (本草纲目), was compiled by LI SheZheng in the Ming dynasty
and published in 1590. It lists Ginseng as an ingredient for the treatment of 23 different
diseases (González-Burgos et al., 2015; Yun, 2001). In traditional Chinese medicine, the
description as a herb of Panax ginseng is “gan” (甘, sweet) and “wei wen” (微温, slightly
warm), whereas the description as a herb of Panax quinquefolius is “gan” (甘, sweet) and
“wei han” (微寒, slightly cool). The difference of the descriptions of these two herbs, Panax
ginseng and Panax quinquefolius, indicates that the use of these two ginsengs could be
slightly different in traditional Chinese medicine. It also suggests that they may display
differences in chemical composition or quantity of chemical components they contain (Chen
et al., 2015; Wang H et al., 2014).
Many pharmacological studies, conducted since the 1950s, have identified numerous
therapeutic effects of Ginseng species including cardioprotective action, inmmunomodulatory
effects, anticancer activity, hypoglycemic activity, treatment of neurodegenerative and other
neurological diseases (González-Burgos et al., 2015). Ingredients of ginseng that have these
biological activities include more than 30 different compounds known as ginsenosides. There
is increasing evidence for the beneficial effects of ginsenosides on the central nervous system
(CNS). Studies have reported that ginsenosides show beneficial effects on CNS processes and
disorders, such as aging, deficits of memory and learning capabilities, and neurodegenerative
diseases (Zhao et al., 2009; Yamaguchi et al., 1996; Mook-Jung et al., 2001; Li et al., 1999;
Van Kampen et al., 2003; Yuan et al., 2001)). Among the ginsenosides, ginsenosides Rb1,
Rb2, Rc, Rd, Rg1, Re, Rf, Ro and 24(R)-pseudoginsenoside F11 are the most important and
widely investigated compounds with respect to chemical analysis and bioactivity of ginsengs.
This review will discuss recent advances in our understanding of the effects of different
ginsenosides, especially ocotillol type of ginsenosides, on CNS targets and how ginsenosides
can contribute to cures for some of the most devastating neurological disorders and
neurodegenerative diseases.
2. GINSENGS AND GINSENOSIDES

Among the many Ginseng species, Panax ginseng C.A. Meyer (Chinese, Korean, Asian
and Oriental ginseng), Panax quinquefolius L (American ginseng) and Panax notoginseng
Burk (Sanqi ginseng) are most commonly used for medicinal purposes. These three herbs
represent the most extensively investigated species of the genus Panax. Both Panax ginseng
and Panax quinquefolius are generally known for their tonic properties, whereas Panax
notoginseng Burk (Sanqi ginseng) is mostly used for hemostasia (blood stopping) in Asia.
Thus, the ginsengs that people mostly refer to are Panax ginseng and Panax quinquefolius.
For medical purposes, Ginseng roots are processed to make white ginseng (air dried roots),
Ginsenosides as Brain Signaling Molecules and Potential Cures … 85
red ginseng (water-steamed and subsequently dried, which are commonly seen in Chinese
ginseng and Korean ginseng), and fresh, raw ginseng. American ginseng, which is the dried
root of Panax quinquefolium L., originates from the northern region of the United States and
Canada (Cruse-Sanders and Hamrick 2004; González-Burgos et al., 2015). Asian ginseng,
which is either air dried or steamed and contains the dried root of Panax ginseng, originates
from north-east of China and Korean.
The major active ingredients of Asian ginseng are similar to those of North American
ginseng, and commonly referred to as ginsenosides which are the secondary plant metabolites
produced by Ginseng species. The ginsenosides (triterpene saponins) are responsible for the
multiple activities of ginseng (Jia et al. 2009; Radad et al. 2011). Their chemical structure is
classified into three groups: dammarane type, oleanane types, and ocotillol types (Kim 2012).
Based on the chemical difference of aglycones, dammarane type ginsenosides are further
classified into two subgroups: 20(S)-protopanaxatriol and 20(S)-protopanaxadiol. Therefore,
the ginsenosides can be classified into four types of aglycones: protopanaxadiol,
protopanaxatriol, ocotillol, and oleanolic acid types (Zhu et al., 2004). Ginsenosides Rb1,
Rb2, Rb3, Rc, Rd, Rg3, Rh2, and Rs1 are protopanaxadiol type with 20(S)-protopanaxadiol
as the aglycone; Re, Rf, Rg1, Rg2, and Rh1are protopanaxatriol type with 20(S)-
protopanaxatriol as the aglycone; Ro is classified as belonging to the oleanolic acid group;
and majonoside R2 and 20(R)-pseudoginsenoside F11 (PF11) are ocotillol type with ocotillol
as their aglycone (Kim 2012; Liu et al. 2012; González-Burgos et al., 2015; Kim HJ et al.,
2013). Figure 1 shows chemical structures of ginsenosides with four types of aglycones.
Chemical differences in types and quantity of ginsenosides have been found among
Panax species (Zhang et al., 2010). For example, the ginsenosides R1 and Rf are present in
Panax ginseng and Panax notoginseng, whereas the 20(R)-pseudoginsenoside F11 is
characteristic for Panax quinquefolius (Zhang et al., 2010).
Therefore, ginsenoside such as Rf, 20(S)-pseudoginsenoside F11, which are present in
Panax ginseng and Panax quinquefolius, respectively, are used as chemical markers to
chemically identify different Panax species (Li et al. 2000; Xie et al., 2008; Park et al., 2014;
Chan et al., 2000; Leung Kelvin et al., 2007; Li et al., 2010).
3. GINSENOSIDE ACTIONS ON CNS

As major components of ginseng, ginsenosides produce restorative, immunomodulatory,
vasodilatory, anti-inflammatory, antioxidant, anti-aging, anticancer, anti-fatigue, anti-stress
and anti-depressive effects in rodents and humans (González-Burgos et al., 2015; Ong et al.,
2015).
Recently, several reviews have focused on the role of ginseng or ginsenosides on
neurological functions or possible target proteins in the CNS (Radad et al., 2011; Cho I-H
2013; Kim HJ et al., 2013; Nah et al., 2007). For example, Nah et al., reviewed how ion
channel activity is regulated by ginsenoside in the CNS (Nah et al., 2007). Many possible
target proteins of ginsenosides in the CNS have been discovered. Ginsenosides regulate
voltage- and ligand-gated ion channels including K+, Na+, and Ca2+ channels as well as N-
methyl-D-aspartate (NMDA)-, nicotine-, and serotonin-gated ion channels(Radad et al.,
2011).
R3O R3O
R1O R1O
OR2
20(S)-protopanaxadiol group 20(S)-protopanaxatriol group

OH
O
OH
OH
RO
HO
OR
oleanolic acid type ocotillol type
Figure 1. Chemical structures of ginsenosides: protopanaxadiol, protopanaxatriol, oleanolic acid type,
and ocotillol type.
Ginseng or ginsenoside modulate the neurotransmission of acetylcholine and gamma-

aminobutyric acid (GABA) by regulating the expression of synthetic enzymes,
neurotransmitter release as well as the signaling pathways involved in their respective
neurotransmitter systems (Radad et al., 2011; Kim HJ et al., 2013).
Ginsenosides can have effects on monoamine signaling. Total saponins extracted from
Panax notoginseng increased the levels of 5-hydroxytryptamine, dopamine and
noradrenaline, suggesting that it may have antidepressant effects by modulating brain
monoamine levels (Xiang et al., 2011; Ong et al., 2015). Using a wide range of in vitro and in
vivo models, researchers have attributed the benefical effects of ginsenosides to specific
pharmacological actions of ginsenosides on cerebral metabolism, oxidative stress and radical
formation, neurotransmitter imbalance and membrane stabilizing effects, and even anti-
apoptotic effects (review, see Radad et al., 2011).
Recent studies also suggested that some of ginseng’s active ingredients exert beneficial
effects on neurological disorders, aging and neurodegenerative diseases such as Parkinson’s
disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis and multiple
sclerosis (Li et al. 2015). The major ginsenosides Rb1, Rg1, Rd and Re were reported as
neuroprotective agents for Parkinson’s disease (González-Burgos et al., 2015; Liao et al.,
2002). A Ginseng extract had neuroprotective effects in traditional rodent models and a
progressive model of Parkinson's disease (Van Kampen et al., 2014). Treatment with ginseng
total saponins was able to improve the neurorestoration of rats after traumatic brain injury
(Hu et al., 2014). Ginsenosides such as Rb1 and Rg1 are the major pharmacologically active
ingredients of ginseng and their beneficial effects on Alzheimer’s disease and other
neurodegenerative disorders have been well demonstrated (Radad et al., 2006; Cheng and
Zhang, 2005; Wang et al., 2010; Li et al., 2015). Ginsenosides improve learning and memory
as determined by behavioral analysis by mechanism involving alteration of synaptic plasticity
and increase in neurogenesis, thereby affecting neuronal density in the hippocampus (Radad
et al., 2011; Kim HJ et al., 2013). Cell-based studies have shown that ginsenosides promoted
neural stem cell (NSC) proliferation in vitro and enhanced cell survival (Wang et al., 2010).
Ginsenoside Rg1 can improve cognitive ability, protect NSCs/NPCs (neural stem
cells/progenitor cells) and promote neurogenesis by enhancing the antioxidant and anti-
inflammatory capacity in the hippocampus (Zhu et al., 2014).
Ginsenosides Rg5 and Rh3 were reported to protect scopolamine-induced memory
deficits in mice probably by inhibiting acetylcholinesterase (AChE) activity and increasing
the expression of hippocampal brain-derived neurotrophic factor (BDNF) and the activation
of cAMP response element-binding protein (CREB) (Kim EJ et al., 2013). Ginsenoside Rh1
also could enhance learning and memory of mice after long-term administration probably by
promoting cell survival in the mouse hippocampus (Hou et al., 2014).
Ginsenosides also show effects on the peripheral immune system. It has been reported
that manipulation of the peripheral immune system with Rg1, a peripherally distributed anti-
inflammatory agent, could ameliorate neuroinflammation-induced behavioral deficits in rats
(Zheng et al., 2014).
Most recently, the protective effects of Ginseng and different ginsenosides in
neurological disorders have been reviewed by Ong, et al. (2015). It is becoming increasingly
evident that ginsenosides produce neuroprotective effects by reducing free radical production
and enhancing brain function (Ong, et al., 2015). Most studies and reviews have focussed on
the panaxadiol group which includes Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, and Rs1 and the
panaxatriol group which includes Re, Rf, Rg1, Rg2, and Rh1.
In this review, we will discuss recent advances in our understanding of the effects of
different ginsenosides on the CNS. Specifically, we will focus on the effects of the ocotillol
group, and how octillol-group ginsenosides can contribute to cures for some of the most
devastating neurological disorders and neurodegenerative diseases.
4. PF11 AND ITS AGLYCONE OCOTILLOL (F11)

Compared to dammarane type ginsenosides, ocotillol-type ginsenosides such as
notoginsenoside R2 and pseudoginsenoside-F11 (PF11) are minor ginsenosides. A
representative and more heavily studied ocotillol-type ginsenoside is 24(R)-
pseudoginsenoside F11, which is found in the roots and leaves of Panax quinquefolius
(American ginseng) rather than Asian ginseng (Zhang et al., 2010). It is usually used as a
chemical marker to characterize American ginseng (Chen et al., 2015; Wang H et al., 2014;
Li et al., 2009; Li et al., 2001). Actually, 24(R)-pseudoginsenoside F11 was quantified to be
more than 0.1% in north American ginseng, which is 1000 times higher than that in Asian
ginseng (less than 0.0001%) using liquid chromatography-tandem mass spectrometry (LC-
MS-MS) analysis (Li et al., 2000).
As a major member of the ocotillol-type ginsenosides, PF11 and its degraded compound
(3R,6R,12R,20S,24S)-20,24-Epoxydammarane-3,6,12,25-tetraol dihydrate show beneficial
effects on disorders of the CNS and cardiovascular system (Meng et al., 2010; Yu et al.,
2007). PF11 increased the excitability of rat cardiac cells (Yang et al., 1994), and had
beneficial effects for memory impairment (Li et al., 1999), neurotoxicity (Wang H et al.,
2014; Wu et al., 2003), and neuro-inflammation (Wang X et al., 2014). PF11 and its
triterpenoid derivatives also have the ability of enhancing nitric oxide release and
antibacterial actions (Bi et al., 2014, 2015a, 2015b). PF11 antagonized learning and memory
deficits induced by morphine, scopolamine, and methamphetamine (Wu et al., 2013). These
neuroprotective effects against morphine-induced impairments and morphine-induced
dependence suggest PF11 might be a candidate for the treatment of drug abuse (Hao et al.,
2007; Li et al., 2000: Li et al., 1999; Wu et al., 2003; Wu et al., 2013). With its anti-amnesic
effect, PF11 might also serve as a potential therapeutic target for Alzheimer’s disease (Wu et
al., 2013; Wang CM et al., 2013). In addition to be a promising compound for improving
memory performance, PF11 has attracted remarkable interest as a promising anti-Parkinson
agent (González-Burgos et al., 2015). It has been proven that PF11 antagonizes the decrease
of dopamine (DA) in the brain of methamphetamine-treated mice [Wu et al., 2003]. PF11
markedly improved the locomotor, motor balance, coordination, and apomorphine-induced
rotations in 6-OHDA-lesioned rats (Wang JY et al., 2013). The potent anti-Parkinson effect of
PF11 is possibly mediated through inhibiting free radical formation and stimulating
endogenous antioxidant release (Wang JY et al., 2013).
Ocotillol is an aglycone of pseudoginsenoside-F11 in which the attached sugar moiety
was removed (see Figure 1). Recently, it has been prepared using methods of chemical
degeneration (Ma et al., 2005). Research into the structure–activity relationship of the
antitumor effect of ginsenosides has shown that the activity of sapogenins is more potent than
that of ginseng saponins (Wang et al., 2007).
As an aglycone of pseudoginsenoside-F11, ocotillol may display similar bioactivities but
with differences in potency in vivo and in vitro. We hypothesized that ocotillol may be even
more effective in modulating neuronal activity in the CNS, as it is more lipophilic compared
with pseudoginsenoside-F11, and thus has a more favorable blood–brain barrier permeability.
Although a wide range of pharmacological activities of ginsenosides has been studied, the
bioactivity of ocotillol and the mechanisms underlying its activity have not been reported. In
this review, we focus in more detail on ocotillol as a derivate of pseudoginsenoside-F11. We
describe the effect of ocotillol on neuronal activity in individual nerve cells and neural
circuits in main olfactory bulb (MOB) and on locomotor behavior.
5. ORGANIZATION OF THE MAIN OLFACTORY BULB

Principal neurons such as mitral cells (MCs) and interneurons form neuronal circuits in
the mouse main olfactory bulb (MOB). Both MCs and interneurons highly express inhibitory
and excitatory proteins and ion channels that are involved in the modulation of neuronal
excitability. MCs display their neuronal excitability as spontaneous action potential firing,
which can be modulated by membrane receptors as well as synaptic inputs (Heinbockel et al.,
2004). Based on these characteristics of MCs and MOB, the effects of ocotillol on the activity
of neurons and the underlying mechanism of its actions were determined using patch-clamp
recording from MCs in MOB (Wang et al., 2011).
The MOB is the first relay station in the CNS for processing of sensory information that
comes from olfactory receptor cells in the nasal epithelium. Synaptic processing in the main
olfactory bulb is dominated by modulatory input. The relay from the nose to principal
neurons in the main olfactory bulb, mitral and tufted cells, and from these neurons to higher
order olfactory centers is strongly regulated by local intrabulbar circuitry as well as
centrifugal inputs to the main olfactory bulb from other brain areas. The intrabulbar circuitry
includes GABAergic interneurons, such as periglomerular cells and granule cells (Shipley and
Ennis 1996).
The glomerular layer houses the cell bodies of three neuronal subpopulations (Figure 2):
periglomerular (PG), external tufted (eTC), and short-axon (SA) cells. The GABAergic
periglomerular cells are neurochemically and functionally heterogeneous (Kiyokage et al.,
2010; Ennis et al., 2007; Shao et al., 2009).
Short-axon cells express both GABA and dopamine, and external tufted cells are
glutamatergic (Heinbockel and Heyward, 2009; Kiyokage et al., 2010; Hayar et al., 2004a).
Input from the olfactory nerve targets periglomerular cells which also receive dendrodendritic
glutamatergic input from external tufted or mitral cells, e.g., as spontaneous excitatory
postsynaptic currents (EPSCs) (Ennis et al., 2007; Hayar et al., 2004a and 2004b).
Periglomerular cells mediate presynaptic inhibition of olfactory receptor neurons through
GABAergic transmission (Aroniadou-Anderjaska et al., 2000; Murphy et al., 2005). External
tufted cells are targeted by periglomerular cells that evoke spontaneous bursts of inhibitory
postsynaptic currents (sIPSCs) at inhibitory GABAergic synapses with external tufted cells
but they also receive spontaneous glutamatergic EPSCs (Hayar et al., 2004b; Hayar et al.,
2007).
6. NEURONAL ACTIVITIES OF OCOTILLOL ON NEURON IN

OLFACTORY BULB
The excitability of neurons in the brain is an integral of intrinsic membrane conductances
and synaptic inputs. Neurons such as mitral cells (MCs) in the mouse main olfactory bulb
(MOB) display their neuronal excitability as spontaneous action potential firing, which can be
modulated by membrane receptors as well as synaptic inputs (Heinbockel et al., 2004).
Highly expressed proteins, such as GABA receptors (GABAA, GABAB), Na+ channels, and
ionotropic and metabotropic glutamate receptors in MCs, are involved in the modulation of
neuronal excitability (Ennis et al., 2007). Thus, we used slices from the MOB of mice to
record MC activity and to determine the effects of ginsenosides on the activity of neurons
using whole-cell patch-clamp recording.
Our results show that ocotillol enhanced neuronal excitability, which was mediated by
increased release of glutamate. We also show that ocotillol had a positive influence on
locomotor activity of mice in vivo (Wang et al., 2011).
Modified from Heinbockel and Heyward, 2009; original drawing by Cristina Shirley.
Figure 2. Olfactory bulb circuitry. Olfactory receptor neuron axons enter the main olfactory bulb
through the olfactory nerve laver (ONL), to synapse with periglomerular cells (PG), mitral cells (MC)
and tufted cells (of which external, eTC, and deep, dTC, tufted cells are shown) within the glomerular
layer (GL). Short Axon (SA) cell axons receive synaptic input from eTCs and form extensive
interconnections between glomeruli, while mitral cell apical dendrites convey sensory information to
deeper layers of the bulb. In the external plexiform layer (EPL), mitral and (deep) tufted cells extend
lateral dendrites which release glutamate onto the dendrites of granule cells (GC). Mitral cell bodies are
located in the mitral cell layer (MCL), which is also densely packed with granule cells. Mitral and
tufted cell axons project through the internal plexiform layer (IPL) to olfactory cortex (their axon
collaterals branching into the GCL are not shown). The granule cell layer (GCL) contains the major
population of inhibitory granule cells. Blane’s cells (BC) within the GCL make inhibitory contact with
granule cells. Centrifugal fibers (CFF) shown projecting to the GL and GCL include glutamate-
releasing axons of olfactory cortex pyramidal cells receiving mitral cell output. These contact the basal
dendrites of granule cells, while mitral cell dendrites contact their apical dendrites.
6.1. Ocotillol Increased Spiking Activity of MCs
Principal neurons, MCs, play a crucial role in processing sensory information in MOB.
They receive direct synaptic inputs from the axons of olfactory receptor neurons, send
excitatory projections to piriform cortex, and receive strong feedback inhibition primarily
through reciprocal dendrodendritic synapses (Shepherd et al., 2004; Ennis et al., 2007). In
slices, MCs generate spontaneous action potentials in the range of 1 to 6 Hz. the intrinsic
properties of MCs such as spontaneous firing, membrane potential, membrane currents, and
synaptic inputs such as spontaneous glutamatergic EPSCs were used to investigate the actions
of ocotillol on neuronal activity and the possible cellular mechanism underlying the
behavioral action of ocotillol.
Figure 3. Ocotillol enhanced the spike rate of MCs. (A) Original recording from a representative MC
illustrated the increased spike rate after bath application of ocotillol. The traces labeled (i), (ii), and (iii)
show the original recording from an MC in control condition, 1 μM ocotillol, and 10 μM ocotillol,
respectively. (B) A normalized and averaged bar graph showed the increase in spontaneous spiking of
MCs. The spike rate in the presence of 5 μM ocotillol was normalized with respect to control condition
(n=12, * P<0.001).
Bath application of ocotillol reversibly increased the MC firing rate. Figure 3A shows an
individual recording trace from one MC cell in the absence and presence of ocotillol. The
averaged bar graph showed the increase in spontaneous spiking of MCs by ocotillol (Figure
3B). The increased spiking was accompanied by depolarization of the membrane potential.
6.2. The Excitatory Effect of Ocotillol Was Abolished by Blocking

Glutamatergic Synaptic Transmission, but Rather GABAergic
Transmission
Excitatory ionotropic glutamate receptors and inhibitory GABAA receptors are highly
expressed in MCs, and play a critical role in the regulation of neuronal excitability (Ennis et
al., 2007). Either activation of ionotropic glutamate receptors or blockade of GABA receptors
may enhance neuronal excitability. To determine whether the excitatory effect of ocotillol
was mediated by ionotropic glutamate receptors or GABA receptors, the ocotillol-evoked
increase of MC activity in the presence of blockers of ionotropic glutamate receptors and
GABAA receptors were examined.
A cocktail of synaptic blockers (gabazine, 5 μM; CNQX, 10 μM; D-AP5, 50 μM) was
often used to efficiently block fast synaptic transmission to MCs (Heinbockel et al., 2004).
Among these receptor inhibitors, CNQX is a potent AMPA/kainate receptor antagonist, D-

AP5 is a potent N-methyl-D-aspartate (NMDA) receptor antagonist, and gabazine is a
GABAA receptor antagonist. In the presence of synaptic blockers, 5 μM ocotillol failed to
enhance the neuronal excitability of MCs. Compared with control condition, in synaptic
blockers, the ocotillol-evoked excitatory effect on MCs was completely blocked. Blockade of
GABAA receptor and ionotropic glutamate receptors reversed ocotillol-evoked excitatory
action, suggesting that either synaptic transmission via GABA receptors or ionotropic
glutamate receptors was involved in the excitatory effect of ocotillol.
The role of GABAA receptors for the observed ocotillol-induced excitatory effect was
examined by applying ocotillol in the presence of the GABAA receptor blocker gabazine.
Figure 4 is a normalized and an averaged bar graph, showing the increased spike rate of MCs
in response to ocotillol in the presence of gabazine or D-AP5+CNQX. With gabazine present,
ocotillol increased the spike rate and depolarized MCs. The persistence of the excitatory
ocotillol effect in gabazine indicated that GABAA receptors were not involved in the
excitatory action of ocotillol on MCs. The change in spike rate and membrane potential in the
presence of gabazine in response to ocotillol was not different from ocotillol-evoked effects
in control condition (in ACSF, see Figure 3).
The role of NMDA and AMPA/kainate receptors for ocotillol-induced excitatory activity
of MCs were determined. The NMDA receptor is an important type of ionotropic glutamate
receptor that controls neuronal excitability. AMPA/kainate receptors are non-NMDA type
ionotropic transmembrane receptors for glutamate that mediate fast synaptic transmission in
the CNS. Blockade of NMDA and AMPA/kainate receptors will inhibit the excitability of
neurons. In the presence of NMDA receptor antagonist D-AP5 and AMPA/kainate receptor
antagonist CNQX, the spike rate and membrane potential of MCs were not significantly
modified by applying ocotillol (Figure 4). The results suggested that ionotropic glutamate
receptors contributed to the excitatory effect of ocotillol.
Ocotillol was also bath applied in the presence of CNQX (10 µM) alone. In the presence
of the CNQX, ocotillol (5 µM) failed to increase the firing rate of MCs. Compared with the
control condition in CNQX, no change in MC activity was observed in response to ocotillol.
This result indicated that the blockade of AMPA/kainite receptors prevented the ocotillol-
evoked increase of MC activity. Thus, ocotillol either enhanced glutamate levels in the slice
or directly acted on AMPA/ kainate receptors.
6.3. Ocotillol Induced Inward Currents and Enhanced EPSCs
The postsynaptic dendrites and somata of MCs express high levels of ionotropic
glutamate receptors, NMDA, AMPA, and kainate receptors. MC apical dendrites respond to
glutamate released from the olfactory nerve (ON) via AMPA and NMDA receptors (Ennis et
al., 2007; Aroniadou-Anderjaska et al., 1999, 2000). Glutamate released from glutamatergic
neurons, such as olfactory receptor neurons at ON terminals or MCs/tufted cell activates
MCs, evokes inward currents, and increases sEPSC frequency (Ennis et al., 2007).
Further evidence for the excitatory effects of ocotillol was obtained by measuring MC
ionic currents. The holding potential was set to -60 mV. Ocotillol (1 µM) evoked an inward
current of about 20 pA in MCs. In the presence of D-AP5 and CNQX, the ocotillol-evoked
inward currents were blocked.
Figure 4. The ocotillol-induced increase of MC spike rate was mediated by ionotropic glutamate
receptors. The data were normalized to the control condition in the presence of gabazine (n=4; *
P<0.05, paired t-test), or D-AP5+CNQX (n=13; P>0.05, paired t-test), or CNQX (n=7, P>0.05, paired
t-test), respectively.
In addition to the evoked inward currents, bath application of ocotillol also increased the
frequency of spontaneous glutamatergic EPSCs. Ocotillol (5 µM) increased sEPSC
frequency, indicating that the ocotillol-evoked excitatory effects were mediated by increased
glutamate. The sEPSCs could be blocked by CNQX or blockers of ionotropic glutamate
receptors (CNQX plus D-AP5), indicating that sEPSCs were mediated by ionotropic
glutamate receptors, mostly by AMPA receptors.
The change of sEPSC frequency evoked by ocotillol was determined at two drug
concentrations (0.5 µM and 5 µM). Bath application of ocotillol resulted in a concentration-
dependent increase of sEPSC frequency in MCs. Figure 5A shows the original recording from
a representative MC. To determine a pre- or postsynaptic localization of the action of
ocotillol, TTX was included in the ACSF solution to block action-potential-dependent
glutamate release.
Under this condition, 1 µM TTX completely blocked sEPSCs in most of the cells tested
(Figure 5B). Only one of the eight MCs displayed a few sEPSCs, and, in this cell, ocotillol
increased the frequency of sEPSCs (Figure 5C), indicating that most spontaneous glutamate
release was action-potential dependent from MCs and not from olfactory receptor neurons
neurons at ON terminals.
Figure 5. Ocotillol affects glutamate release in MCs. (A) Ocotillol increased sEPSC frequency in a
concentration-dependent manner. Original recording from a representative MC showing sEPSCs in
control (ACSF), 0.5 µM and 5 µM ocotillol. (B) TTX (1 µM) blocked sEPSCs in most MCs. (C)
Ocotillol (5 µM) increased sEPSCs in one MC in the presence of TTX. (D) Cumulative inter-sEPSC
interval distributions. Ocotillol evoked a left shift of the curves, n = 4. (E) Cumulative amplitude
distributions in the absence and presence of ocotillol. N = 4.
With 0.5 µM and 5 µM ocotillol, the averaged cumulative inter-sEPSC interval

distribution exhibited a leftward shift, or shorter inter-sEPSC intervals (Figure 5D).
The averaged cumulative sEPSC amplitude distribution did not show a change with 0.5
µM and 5 µM ocotillol (Figure 5E). The result is consistent with the observation that MCs
release glutamate and produce self-excitation (Ennis et al., 2007).
7. OCOTILLOL INCREASED SPONTANEOUS LOCOMOTOR ACTIVITY

OF MICE
An increase in glutamate receptor activity can result in increased activity of principal

neurons in the brain, and it might be responsible for changes in locomotor activity of animals.
Therefore, we determined whether ocotillol regulates spontaneous locomotor activity of mice
in vivo.
The effects of ocotillol on spontaneous locomotor activity were measured. Ocotillol
significantly increased the spontaneous locomotor activity of mice when administered at
doses of 1–8 mg/kg. The behavioral effect of ocotillol displayed dose dependency and was
detectable even at the lowest dose of 1 mg/kg. The averaged locomotor activities induced by
varying doses of ocotillol were well fit by the Hill equation, and gave rise to an estimated
EC50 of 2.0 mM/kg. The shape of the concentration-response curve for the enhancement of
spiking of MCs by ocotillol in vitro is similar to that of the dose-response curve of the
ocotillol-evoked increase of spontaneous activity of mice in vivo, suggesting that the
ocotillol-evoked activation of principal neurons, which was mediated by glutamate, may be
responsible for the increased spontaneous locomotor activity evoked by ocotillol in vivo.
Strengthening the central glutamatergic system by ocotillol may be beneficial in learning and
for memory deficits in vivo.
CONCLUSION
Ginseng and its major active ingredients, named ginsenosides, display beneficial effects
on central nervous system (CNS) processes and disorders such as aging, memory deficits,
learning ability, and neurodegenerative diseases. Ginsenosides have four types of aglycones:
protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid types. Ginsenosides exhibit
different effects on neurons, and various molecular mechanisms are responsible for these
effects. We discussed recent advances in our understanding of the effects of different
ginsenosides on CNS targets and how ginsenosides can contribute to cures for some of the
most devastating neurological disorders and neurodegenerative diseases. As a derivate of
pseudoginsenoside-F11, which is an ocotillol-type ginsenoside found in American ginseng,
ocotillol increases both the neuronal activity in individual neuron in vitro and locomotor
behavior activity in vivo. The ocotillol-evoked enhancement of glutamate transmission may
be responsible for the increased spontaneous locomotor activities in vivo. Strengthening the
central glutamatergic system by ocotillol may be beneficial for learning and memory deficits
in vivo.
Reviewed by Dr. Naina Bhatia-Dey and Dr. Sulman Rahmat, Howard University.
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Chapter 5
NEUROPLASTICITY AND NEUROGENESIS:

TWO PATHS, ONE END
Rosmari Puerta Huertas∗

Nursing Service, Marqués de Valdecilla
University Hospital (HUMV), Cantabria, Spain
ABSTRACT
Neuroplasticity is the ability of our NS to respond structurally and functionally to
intrinsic or extrinsic stimuli. The mechanisms of response and functional adaptation to
stimuli express the onset and continuity of constant neuromodulation involved in the
process of neuroplasticity. The discovery of neurogenesis in the adult brain, generating
new neurons, demonstrated in the hippocampus and olfactory system, together with the
process of neuroplasticity in light of changes, guarantee homeostasis in our NS.
We only understand a small percentage of the brain's potential, which we still do not
fully comprehend. As our science advances each day with new lines of research in this
discovery, we adopt action strategies to favor this complex phenomenon in our NS,
which starts during our embryonic development and facilitates our survival in the adult
years, by reorganizing damaged circuits as a spontaneous response of natural plasticity.
Therapeutic intervention programs are based on brain plasticity. We can assert what
occurs on a molecular level in our complex brain microanatomy to be able to understand
this ability, but we have one question: how can we use these mechanisms in a targeted
way to be able to achieve our desired therapeutic objectives?
Keywords: neuroplasticity, neuromodulation, neurogenesis, plasticity brain, reactive

synaptogenesis, unmasking, homeostasis
∗
Rosmari de la Puerta Huertas, Nurse. Nursing Service, Marqués de Valdecilla University Hospital (HUMV),
39007 Santander, Cantabria, Spain. E-mail: rosmariph@yahoo.es, rpuerta@humv.es.
102 Rosmari Puerta Huertas
ABBREVIATIONS
NS nervous system
CNS central nervous system
LTP long term potentiation
LDP long term depression
NGF nerve growth factor
NT3 neurotrophin 3
BNDF brain-derived neurotrofic factor
NMDA N-methyl-D-aspartate
AMPA acid α-amino-3-hidroxi-5-methyl-4-isoxazolpropiónic
RMf functional magnetic resonance imaging
PET positron emission tomography
SPECT single-photon emission computed tomography
TMS transcranial magnetic stimulation
MEG magnetoencephalography
MR magnetic resonance neuroimaging
GMI graded motor imaging
PVA periventricular area
SVZ subventricular zone
5-HT serotonin (5-hidroxitriptamin)
NOR noradrenaline
INTRODUCTION
Neuroplasticity as a fundamental property of the nervous system (NS) is based on
common mechanisms in our species. Each stage of an individual's development is expressed
according to genetically programmed phenomena, such as growth and neuronal migration,
and is also associated to individual experiences such as learning or subsequent NS damage
[1]. The NS responds to intrinsic and extrinsic stimuli by reorganizing its structure, functions
and connections at molecular, cellular, systemic and behavioral levels [2].
It has several electrical, genetic, structural, biochemical and functional mechanisms that
represent a continuum more than individual and isolated elements [3].
These changes in the CNS have given rise to interesting studies in the last 30 years. We
have been able to differentiate the mechanisms used by the brain to adapt itself to the changes
imposed by the medium. These mechanisms can be summarized into three main concepts:
reactive synaptogenesis, unmasking, and long-term potentiation [4].
Neuroplasticity and Neurogenesis: Two Paths, One End 103
NEUROBIOCHEMISTRY AND PHYSIOLOGY

OF BRAIN PLASTICITY PROCESSES
Reactive Synaptogenesis
Reactive synaptogenesis corresponds to the sprouting and extension of new axonal

branches, culminating with the formation of new synaptic contacts. There branching by
growing axons and proteins such as laminins, integrins and cadherins, with multiple coupling
sites for neurons, trophic factors and glycoproteins. Collateral branches are new axonal
processes that have sprouted from an undamaged axon and grow toward a vacant synaptic
site. This occurs in the CNS and can be adaptive [5, 6].
Unmasking
Unmasking is when neural connections that are resting or inhibited in a normal state
become activated after brain damage [6]. The role of neurotrophins has been highlighted in
this process: nerve growth factor (NGF), neurotrophin 3 (NT3) and brain-derived
neurotrophic factor (BDNF) as promoters of neural maturity and viability, but also involved
in the formation of new dendrites and synapses, and in the unmasking of silent synapses or
the regulation of synaptic efficiency. BDNF acts as a limiting factor during the critical period,
as preferred afferent fibers promote its activity-dependent release in the cortex. Thus, BDNF
levels are thought to be low for stimuli that arrive afterward; this constitutes the basis of the
competitive nature of plasticity. Therefore, BDNF availability is low during the critical
period, intervening in the selection of preferred synapses; it increases once this has occurred,
and is no longer a limiting factor [7].
Long-Term Potentiation (LTP)
The efficiency of synaptic transmission can also be modified by stably increasing

excitatory (LTP) or inhibitory (LTD) functions. It is a learning and memory process of the
brain that involves synaptic plasticity. Changes in the excitation/inhibition balance of a group
of synapses are always associated with a loss of influence of the groups that could be affected
by local and remote consequences [1].
Studies attribute considerable importance to the activity of the N-methyl-D-aspartate
(NMDA) receptor in the regulation and limitation of neuroplasticity depending on use or
training, by the modification of glutamatergic excitatory synapses. The synaptic weakening
and consolidation that give rise to plasticity can be explained using the long-term potentiation
(LTP) and long-term depression (LTD) model. The induction of these forms of plasticity is
regulated by the entry of intracellular calcium mediated by glutamate activity on the NMDA
receptor, responsible for facilitating the expression of synaptic proteins. Thus, detecting
coherent and maintained transmissions (detection of synaptic concurrence), promote and
favor structural changes as preferred synapses are maintained [8].
Bruce Dobkin, an expert in the clinical field of neuroplasticity, divides plasticity

mechanisms into two groups: neural network plasticity and synaptic plasticity [9].
BIOLOGICAL MECHANISMS OF BRAIN PLASTICITY

Neural Network Plasticity Mechanisms
• Recovery of neuronal excitability (cell and axon ionic equilibrium, resorbing of

edema and blood residues, reverse transsynaptic diaschisis)
• Activity in partially spared neural pathways
• Representational plasticity within neuronal assemblies
• Recruitment of parallel networks not ordinarily activated
• Recruitment of subcomponents in distributed networks
Synaptic Plasticity Mechanisms
• Modulation of subnetwork excitability by neurotransmitters.

• Neuronal modulation of intracellular signaling (dependent on neurotrophic factors
and protein kinases)
• Synaptic plasticity (modulation of basal transmission, denervation hypersensitivity,
activity-dependent synaptic unmasking, dendritic sprouting)
• Axonal and dendritic sprouting from uninjured collateral neurons
• Axonal regeneration (gene expression for protein remodeling, modulation by
neurotrophic factors) [9].
There are several biochemical and physiological components behind a neuroplasticity

process and this leads to different proteomic, genomic and biomolecular chemical reactions
that require intra- and extraneuronal actions to generate a neuronal response [10].
CORTICAL REORGANIZATION. NEUROMODULATION

The entire cerebral cortex is organized into functional areas that take on behavioral
responsive, integrative and motor tasks. One form of materializing neuroplastic processes is
through the cerebral cortex. Many authors divide cortical plasticity into two subprocesses:
physiological cortical plasticity (substrate of learning and human memory, process in
neurodevelopment), and pathological cortical plasticity (divided into adaptive and poorly
adaptive) [11, 12].
Brain lateralization is expressed as three aspects: anatomical symmetry, unilateral
functional differences (such as location of language, speaking and analytical processing in the
left hemisphere; and spatial-temporal and musical skills and emotional and humoristic
features in the right), and contralateral sensory-motor control.
Understanding brain functionality as these three aspects is fundamental for understanding

the processes that take place in the reorganization of the brain after an injury. However, there
are studies on the matter that lead one to believe that the phenomena of neuronal plasticity
and functional reorganization are much more complex and show particularities according to
the area and function of interest.
Neural pathways and their projections undergo topographic organization, so that each
area is projected differently to the cortex via the thalamus. Somatosensory information is
distributed onto topographic or somatotopic maps (Penfield's homunculus) according to the
different functions in the primary motor area. The plasticity inherent to brain cells enables
cortical circuit repair, integrates other cortical areas to carry out modified functions and
responds to various disorders [7].
Several neurophysiological techniques have enabled this brain reorganization to be
described: functional magnetic resonance imaging (fMRI), positron emission tomography
(PET) and single-photon emission computed tomography (SPECT), transcranial magnetic
stimulation (TMS), magnetoencephalography (MEG) combined with magnetic resonance
neuroimaging (MR) [13] and graded motor imaging (GMI), to name a few.
NEUROGENESIS
Neurogenesis is the process by which new neural cells are generated in the NS from
neural stem cells and neural progenitor cells (NPC). Together, neurogenesis and
neuroplasticity lead a dynamic and complex substrate in the NS [14]. Cerebral maturation is a
process characterized by innumerable and progressive transformations that start at conception
and progress during gestation until they are subsequently completed [15]. Neurogenesis
continues in adults in certain regions of the brain, such as the hippocampus, the olfactory
mucosa and the periventricular area (PVA) [16]. Embryonic stem cells are pluripotent; that is,
they have the ability to originate different cell types in the developing body, while adult brain
stem cells lose part of this ability, becoming multipotent, which means that they can only give
rise to specific cell types [17, 18].
FACTORS THAT REGULATE NEUROGENESIS IN THE ADULT BRAIN

The microenvironment that regulates neurogenesis during embryonic and postnatal
development is preserved in the adult brain and is positively or negatively regulated by
diverse mechanisms. There are internal and external factors that participate in said regulation
[19].
Internal Factors
Genetic and Molecular Factors

The genetic factors that induce embryonic morphogenesis and neurogenesis include the
expression of Notch, BMP, Eph/ephrin, Noggin and Shh genes.
These genes also participate by regulating cellular differentiation and proliferation in

neurogenic zones in the adult brain [20].
Growth Factors
The expression of several growth factors (such as BDNF, IGF-I, FGF-2, EGF, HB-EGF,
VEGF) involved in regulating cell fate can determine the size of the neuronal or glial
population, both in the developing brain and the adult brain [21, 22].
These factors are overexpressed in different neurodegenerative models in which they
participate as protective factors of neuronal damage or as inductive factors during the
generation and differentiation of new cells that replace damaged cells [23].
It has been demonstrated that the intracerebroventricular administration of brain-derived
neurotrophic factor (BDNF) increases neurogenesis in the olfactory bulb [24]. Thus, we can
conclude that these growth factors stimulate neurogenesis in the adult brain.
Neurotransmitters
Several neurotransmitters participate as factors that regulate neurogenesis in the adult
brain. Those most studied include glutamate, monoamines, serotonin (5-HT), noradrenaline
and dopamine. Glutamate is considered to be the most important neurotransmitter for brain
function. It is known that glutamate regulates neurogenesis in the hippocampus of adult
animals. However, most studies have focused on the NMDA receptor.
These studies suggest that NMDA administration decreases cell proliferation in the
hippocampus and that neurogenesis increases when NMDA receptor antagonists are
administered (MK-801, CGP 43487) [25, 26]. The participation of 5-HT in neurogenesis has
been demonstrated in several studies [27]. The inhibition of 5-HT synthesis has enabled us to
see a decrease in the proliferation rate in both the hippocampus and the subventricular zone
(SVZ) of rats [28, 29]. The 5-HT1A receptor participates in the regulation of neurogenesis in
the adult brain; however, 5-HT can also regulate neurogenesis via other receptor subtypes,
such as 5-HT2A or 5-HT7 [30]. The noradrenergic system is another neurotransmitter
involved in adult brain neurogenesis. By inhibiting noradrenaline release, it decreases
proliferation but does not affect the differentiation or survival of new cells in the
hippocampus [31]. Dopamine depletion decreases the generation of new neurons, both in the
SVZ and in the dentate gyrus of the hippocampus [32].
Hormones
Some studies suggest that ovarian steroids, as well as endogenous estrogens, have a
stimulant effect on the cellular proliferation of granular precursors [33, 34, 46].
Age
Age is one of the most important factors in the regulation of brain neurogenesis. The
changes observed in the cellular proliferation of neural progenitor cells (NPC) present in the
hippocampus seem to be related to high levels of glucocorticoids [35].
Studies suggest that, as a result of adrenalectomy, adrenal steroid levels reduce drastically
and cellular proliferation in the hippocampus increases, in both young and old rats [36]. It can
be concluded that the rate of neurogenesis in the adult brain decreases as age increases [37,
38, 39].
External Factors
Environmental Factors
Neurogenesis is not a static biological process, its rate is variable and depends on the
microenvironment [40].
Physical activity, enriched and pleasant environments, energy restriction and modulation
of neuronal activity act as positive regulators of neurogenesis, among other factors. Animals
that live in an enriched environment present an increase in the neurogenesis of the dentate
gyrus subgranular zone [41, 42, 43]. Alterations in the hypothalamic-pituitary-adrenal axis
induced by persistent situations of stress during development decrease the generation of new
cells in the dentate gyrus [44, 45]. Thus, cellular proliferation in the dentate gyrus decreases
due to the effect of glucocorticoids, which are released in response to stress [46].
Dr. Elkhonon Goldberg, professor of neurology at the New York University School of
Medicine and renowned neurologist and scientist, wrote (Wisdom Paradox) how important it
is to keep the mind active as a defense mechanism against mental decline, that the mind may
be kept acute and efficient in older adults if the brain is used constantly.
NEUROREHABILITATION
Neuroplasticity is the theoretical basis that supports early intervention with rehabilitation
programs, minimizing an individual’s disability and increasing their quality of life.
Rehabilitation is the best known method for facilitating the expression of neuroplasticity. In
light of recent studies, several strategies have emerged for the possibility of intervening in
and modulating brain plasticity:
− From a physical standpoint, intervention, stimulation and rehabilitation programs are

accommodated based on the knowledge of different mechanisms used by the cortex
to adapt itself: the interhemispheric plasticity capacity of the motor cortex, cross-
modal plasticity for the visual and auditory cortices, homotopic reorganization or
contralateral transfer in the cortex related to language, etc.
− From a pharmacological standpoint, physical therapy can be supported by or
combined with the administration of drugs that prolong or open the critical period to
promote neuroplastic changes, such as GABAergic tone inhibitors or antagonists.
Noradrenergic stimulants, such as amphetamines, increase LTP through adrenergic
and dopaminergic pathways, favoring the synaptic plasticity surrounding amnesia
and learning processes.
− From a cognitive and behavioral approach, functions are quickly learned and
recovered by focusing on attention during the implementation of tasks. In terms of
recovering from cognitive impairment and regaining higher mental functions,
including language, before rehabilitation strategies can be devised a complete
neuropsychological assessment must be performed to determine which system
components have been affected. An assessment can also show us which components
have been preserved that can serve as a foundation and starting point for therapy. If
we can achieve better tone, behaviorally, studies suggest that this behavioral tone
might act by facilitating neuroplasticity, mainly through noradrenergic and

serotonergic stimulation.
− The use of physical techniques, such as TMS, reveals the potential for increasing the
cortical excitability of interest, which facilitates training and makes it possible to
increase the ability to learn what is taught. TMS prepares the cortex for the therapy
session, whether physical or cognitive therapy, increasing the speed and capacity for
recovery and learning [7].
Neurorehabilitation must be holistic but individualized; inclusive and participative; it

must generate independence; have lifelong applicability, if required; it must be timely
according to the patients' needs and be community oriented [47, 48]. This involves an
interdisciplinary approach, carried out by an experienced team composed of professionals
with different scientific approaches and training, indispensably led by a specialist in
neurorehabilitation. Therefore, theoretical and practical elements must be combined to help to
establish suitable sensory-motor control in patients and to optimize these individuals’
functional recovery and an improvement in quality of life. Ultimately, this is true objective of
all interventions in human health [49].
CONCLUSION
9 The discovery of neurogenesis in the adult brain, generating new neurons, together
with the process of neuroplasticity in light of changes, guarantee homeostasis in our
NS.
9 CNS-triggered responses are more complex when environmental stimuli are more
demanding or an injury is more substantial.
9 Brain plasticity enables cortical circuit repair, integrates other cortical areas to carry
out modified functions and responds to various disorders.
9 Neurogenesis is a continuous, variable and microenvironmental-dependent biological
process of the brain in our NS.
9 The CNS has a considerable reserve at its disposal; that is, the number of neurons we
have is much greater than the number we need for any normal function. The capacity
of our NS is greater than what we will use in our life.
9 When a neuron becomes functionally isolated with no possible synaptic connections,
it degenerates and dies.
9 Studies in neurobiology on memory and learning assert that each new learning
opportunity causes structural remodeling of the NS and said learning opportunity is
thus maintained.
9 Brain plasticity substantiates the application of therapeutic intervention programs for
functional recovery.
9 Subsequent to injury, neurorehabilitation involves an interdisciplinary approach by
experienced professionals from different scientific approaches and types of training,
and must indispensably be led by a specialist in neurorehabilitation.
9 There are several factors involved in brain plasticity that influence homeostasis in the
individual.
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Chapter 6
CROSS-MODAL PLASTICITY:
PRE AND POST COCHLEAR IMPLANT STUDY
IN DEAF-BLINDNESS
L. E. Charroó-Ruíz1, F. Rivero Martínez2, A. Calzada Reyes3,

M. C. Pérez-Abalo1, M. C. Hernández1, S. Bermejo Guerra4,
B. Bermejo Guerra5, B. Álvarez Rivero4, A. S. Paz Cordovés6,
M. E. Sevila Salas6, M Estévez Báez7, L. Galán García1
and A. Álvarez Amador1
1
Centro de Neurociencias de Cuba, La Habana, Cuba
2
Hospital Carlos Manuel de Céspedes, Bayamo, Granma, Cuba
3
Instituto Medicina Legal, La Habana, Cuba
4
Hospital Pediátrico Marfán, La Habana, Cuba
5
Centro Internacional de Salud “La Pradera,” La Habana, Cuba
6
Hospital “Hermanos Ameijeiras,” La Habana, Cuba
7
Instituto de Neurología y Neurocirugía,
La Habana, Cuba
ABSTRACT
Introduction: Significant neuroplastic changes occur in deaf and blind subjects as a
result of the sensory impairment that affect them. However, the changes that occur in
deaf-blind subjects, after the auditory (re)habilitation post Cochlear Implant (CI) are not
well studied.
Objective: To identify the neuroplastic changes in subjects with deaf-blindness using
the topographic distribution maps of the somatosensory evoked potential by stimulation
of the median nerve (SEP-N20) pre-CI versus post-CI.
Methods: SEP-N20 was studied in seven deaf-blind children, after 5 years of
auditory (re)habilitation post-CI. Topographic distribution maps of SEP-N20 post-CI
were compared with the SEP-N20 pre-CI and the SEP-N20 maps of a control group. In
addition, we obtained Cortical Auditory Evoked Potential (CAEP). Results: Our study
114 L. E. Charroó-Ruíz, F. Rivero Martínez, A. Calzada Reyes et al.
provides evidences of the cross-modal plasticity. The deaf-blind children implanted

before age 7 therefore, with less than 7 years of auditory and visual deprivation, did not
show changes in the topographic distribution maps of SEP-N20 pre-CI when compared
with the control group. Similar result was found after the auditory (re)habilitation post-
CI. On the contrary, in deaf-blind children implanted after age 7 (with 7 or more years of
auditory and visual deprivation) changes occur in the SEP-N20 topography, both pre-CI
and post-CI. With the responses of the CAEP it was possible to reveal the auditory area
activation by the sensory input through the CI.
Conclusions: In deaf-blind children, with 7 or more years of auditory and visual
deprivation before the CI, changes occur in the topographic distribution maps of SEP-
N20 pre-CI versus post-CI. It is evidence of cross-modal plasticity, which may be an
expression of how important in these subjects the somesthetic information is, probably
due to the relationship with tactile language, as well as the functional interaction of
auditory and somesthetic information during the auditory (re)habilitation post-CI.
Keywords: neuroplasticity, cross-modal plasticity, deaf-blindness, cochlear implant,

somatosensory evoked potential, cortical auditory evoked potential, auditory
(re)habilitation
Highlights
• Evidences of cross-modal plasticity through the Somatosensory Evoked Potentials in

deaf-blindness.
• Changes in the topographic distribution of SEP-N20, both pre and post cochlear
implant, occur in deaf-blind children after 7 years old (with 7 or more years of
auditory and visual deprivation).
• SEP-N20 topographic changes as expression of the relevance of the somesthetic
information in deaf-blindness.
INTRODUCTION
The severe-to-profound sensorineural hearing loss (SNHL), associated with a visual
impairment that classified as deaf-blindness, is a serious health problem. It is due to the lack
of adequate input through two very important sensory systems for the development of
children. It should be added, that families, teachers and doctors do not always offer the
support (stimulation) that these children need to acquire communication skills, mainly during
their first years of life. In many cases, the emotional crisis of the family, with a child with
dual sensory deprivation, who could also have an additional physical and cognitive
impairment, conspires against early stimulation of the child.
It is crucial that, once identified children with these disabilities, they are properly studied
and the diagnosis of their health condition be established. The auditory stimulation through
the use of hearing aids or cochlear implant (CI) is the recommended therapy to SNHL. CI is
the choice well-recognized treatment when children have a SNHL, and do not show any
benefit with modern hearing digitals aids (Wilson and Dorman 2008).
Cross-Modal Plasticity 115
The assessment protocol of the Cuban Cochlear Implant Program for deaf and deaf-blind
children includes a comprehensive evaluation of them, which is done by a team of
audiologists, ophthalmologists, pediatricians, neurologists, educators, psychologists and
neurophysiologists. This team has been acknowledged by other authors (Gilley 2010). The
neurophysiological evaluation includes the study of cortical reorganization, through the
description of the topographical distribution maps of cortical responses of the evoked
potentials (EPs), which help to obtain evidences of the cross-modal plasticity, in which
cortical areas with sensory input loss are invaded by others (Finney 2001; Nishimura 1999;
Sadato 1996), therefore the use of CI could be influenced by them. The inclusion in the
assessment of cortical reorganization with EPs in children with deaf-blindness in our country
began in the clinical neurophysiology laboratory of the Marfán Pediatric Hospital, as a
proposal of the Cuban Neuroscience Center in early 2005. Charroó-Ruiz et al. (2012) studied
a group of deaf-blind children candidates for CI and a group of control children. More
recently, we also evaluated deaf children candidates for CI (Charroó-Ruíz 2013).
Functional Magnetic Resonance Imaging (fMRI) is the most used tool in published
research on neuroplasticity in subjects with single sensory deprivation, visual or auditory
(Gordon 2005; Firszt 2002a, 2002b; Finney 2001; Bavellier and Neville 2002; Merabet and
Pascual-Leone 2010). However, the electrophysiological techniques such as EPs are
particularly useful for the study of neuroplasticity, because fMRI studies cannot be carried out
after the CI.
Continuing the research work of our group on neuroplasticity in deaf and deaf-blind
children, this study provides evidence of cortical reorganization in deaf-blindness after
receiving auditory (re)habilitation post-CI. This research contributes somehow to the efforts
that are being made in the search of outline criteria to choice the children candidates for CI, to
predict results post-CI, but the most important is to give evidence about the cross-modal
plasticity effects, specifically in subjects with dual sensory deprivation (auditory and visual)
post-CI, which has hardly been studied in deaf-blind subjects (Osaki 2006).
The study of the topographic distribution maps of the SEP-N20 could reflect possible
neuroplastic changes that occur at the cortical level as a result of the auditory (re)habilitation
post-CI. In this case, it is expected that changes take place in the pattern of activation of brain
regions that are reflected in the SEP-N20 topographic maps.
The main purpose of this study was to identify the neuroplastic changes that occur in
subjects with deaf-blindness, using the topographic distribution maps of the SEP-N20 pre-CI
versus post-CI.
METHODS
Seven deaf-blind children that had received a single CI with complete insertion of the
intra-cochlear electrodes were evaluated. All subjects had bilateral pre-lingual or peri-lingual
SNHL. None of these children had surgical complications or failures with the CI.
The deaf-blind children had been receiving auditory (re)habilitation for 5 years at the
moment of the post-CI evaluation. Results of the auditory (re)habilitation were assessed using
tests that were developed and validated internationally for such purposes by other authors
(Comité Español de Audiofonología 2005; Huarte 1996).
According to of auditory skills and language development, each child was assigned to the
phases of auditory (re)habilitation: (1) detection, (2) discrimination, (3) identification, (4)
recognition, and (5) comprehension (Amat and Pujol 2006).
Table 1 summarizes the clinical features of the seven deaf-blind children tested before
being implanted, it includes data on sensory deprivation of these subjects, describes also the
information regarding cochlear implantation, and the phases of auditory (re)habilitation at the
time of assessment the cortical response of the SEP-N20 post-CI.
A control group of 23 healthy subjects, with normal hearing and vision, was selected, to
create reference patterns for the evaluation of the topographic distribution maps of the SEP-
N20 (Charroó-Ruíz 2012).
Table 1. Clinical features of the seven deaf-blind children tested before the CI and
after 5 years of auditory (re)habilitation post-CI
Subjects Age Sex Causes of the Age of Ear CI Phases of

(years) hearing loss - implantation auditory
visual Impairment (years) (re)habilitation
1 8 M Unknown 3 R Identification /
Recognition
2 11 F Congenital- 6 R Recognition/
Toxoplasmosis Comprehension
3 12 M Congenital 7 L Identification /
Recognition
4 18 M Congenital 13 R Identification /
Recognition
5 19 F Congenital - 13 R Identification /
Usher´s Recognition
Syndrome
6 20 M Unknown 15 R Comprehension
7 14 F Congenital - 9 L Discrimination
Retinitis / Identification
Pigmentosa
Recordings post-CI SEP-N20 and topographic distribution maps of the cortical response
were obtained with the same protocol and procedure used in pre-CI study (Charroó-Ruiz
2012). The maps of each deaf-blind child were compared, through visual inspection, with the
average maps of the SEP-N20 obtained from the control group as reference. Also
comparisons between the pre-CI versus post-CI results were done. The progress in the
auditory (re)habilitation was considered in this analysis.
CAEP were recorded by stimulation using clicks, 90 dB, presented at a frequency of 2
Hz, with the headphone near 30 cm of the implanted side; the subjects were relaxed with
closed eyes. A Neuropack stimulator (Nihon Kohden Corporation, Japan) was attached to the
digital recording computer of the MEDICID electroencephalograph (Neuronic S.A, Cuba.

CAEP was recorded at the 19 sites of the 10/20 International System, however in this study
only two derivations (Fz and Cz) were reported. Original signals were filtered between 0.1
and 100 Hz and300 responses were averaged and plotted in a time window of 600 ms (50 ms
pre-stimulus). In this response the absolute latency of the P1 peak was measured according to
international criteria (Martin 2008; Walker 2008; Pozzobom 2009).
Ethical considerations: The first step was to explain the parents what the test was; they
agreed to participate before starting, signing voluntarily a written consent.
The research protocol was approved by both Ethics Committees of the Marfán Pediatric
Hospital and the Cuban Neuroscience Center. The study was carried out according and in
compliance with the Helsinki declaration.
RESULTS
Figure 1A shows the topographic distribution of SEP-N20 average maps of the deaf-blind
children and control group in the study pre-CI (Charroó-Ruiz 2012). The figure 1B shows the
individual maps of the seven deaf-blind children of the studies pre-CI and post-CI. Note that
the topographic distribution of SEP-N20 in the two deaf-blind children implanted before age
7 (with less than 7 years of the auditory and visual deprivation) showed no change in neither
studies: pre-CI and post-CI with respect to the control group. They showed topographic
representation of SEP-N20 similar to the healthy children (see Figure 1A, average maps of
the control group).
Figure 1. (A) Average maps of the topographic distribution of the SEP-N20 in deaf-blind children and
control group. (B) Individual maps from seven deaf-blind children showing the topographic distribution
pattern of the SEP-N20 by stimulation of the right median nerve, pre-CI and after 5 years of auditory
(re)habilitation post-CI.
In contrast, the five deaf-blind children who were implanted after 7 years of age (with 7
or more years of auditory and visual deprivation) showed over-represented topographic
distribution of SEP-N20 in the study pre-CI (Figure 1B). Changes were also found in the
post-CI study of the topographic distribution maps of the SEP-N20 after 5 years of the
auditory (re)habilitation. Four of the five deaf-blind children showed topographic distribution
of SEP-N20 post-CI similar to the healthy children (Figure 1B). However, one deaf-blind
child, whose maps correspond to the far right maps of figure 1B, showed a greater over-
representation of the topographic distribution of SEP-N20 post-CI when compared with the
pre-CI study.
Figure 2. Cortical Auditory Evoked Potential (CAEP). Records obtained with device switch-on (in left
panel, CI in ON) and with device switch-off (in the right panel, CI in OFF) from a deaf-blind child after
5 years of the auditory (re)habilitation post-CI.
Although this deaf-blind child spent 5 years in auditory (re)habilitation, she had reflected
poor progress in transited phases of (re)habilitation (see subject 7 in Table 2). Meanwhile, the
other four deaf-blind children implanted after age 7 reached the upper phases of the auditory
(re)habilitation, even one of them reached the recognition phase, the last phase of the auditory
(re)habilitation indicating a good use of the CI for this child (see subject 6 in Table 2).
It was possible to obtain the CAEP in the seven deaf-blind subjects after 5 years of
auditory (re)habilitation post-CI. The obtained records in all subjects showed the components
described for this kind of evoked response (P1 peak), but with prolonged latencies. In the left
panel of the figure 2 it is shown the evoked responses of one of the deaf-blind subjects when
the implanted device was activated (recording with CI switch-on). Note the presence of the
P1 peak in anterior midline, Fz and Cz leads. In the right panel of the figure 2 it is shown the
recordings when the device was not activated (recording with CI switch-off), thus we were
sure that the obtained answer (CAEP) corresponds to auditory cortical activation with the
presentation of the clicks and that was not a signal caused by artifact.
DISCUSSION
Our results demonstrated, through the changes in the topographic distribution of SEP-
N20, that there is a cortical reorganization in deaf-blind children after the auditory
(re)habilitation post-CI. The most interesting findings were related to the duration of deaf-
blindness. The changes were presented in deaf-blind children with 7 or more years of sensory
deprivation before the CI.
The topographic distribution maps of SEP-N20 in the two deaf-blind children implanted
before 7 years showed no changes. A central issue in the field of pediatric CI is the optimal
age for implant a deaf child (Geers 2006; Nicholas and Geers 2006; Sharma 2002). It is
possible to consider that because less extended duration of auditory and visual deprivation
before the CI in these children, the somesthetic inputs did not establish functional connections
with auditory cortex. It could be speculated that the neural networks that process tactile
stimuli in these children have a similar activation pattern to that in normal hearing children
(Charroó-Ruíz 2012). Even after being implanted the same cortical activation pattern for the
presentation of the somesthetic stimulus (SEP-N20 post-CI) remained in the cortical area to
the somesthetic representation of the hand.
In contrast, in the five deaf-blind children implanted after 7 years of age changes did
occur in the topographic distribution maps of the SEP-N20 post-CI. Four of the five deaf-
blind children showed evident changes, and the topographic distribution the SEP-N20 was not
over-represented. It could be thought that there was a new cortical reorganization in
somesthetic sensory system after the auditory (re)habilitation post-CI. These could be related
to the improvement of these children with the auditory (re)habilitation. As a result of the
adequate use of the CI, these children progressed and they communicate using words, in
classes and out with their parents. They need less the tactile communication reducing
significantly the use of their hands; therefore it could be an expression of normal
representation of the cortical response of the SEP-N20 post-CI.
Previous studies of our group revealed that changes in the topographic distribution of the
SEP were selective for the cortical response by stimulation of the median nerve (SEP-N20),
but not to the topographic distribution elicited by stimulation of the tibial nerve (SEP-P40)
(Charroó-Ruiz 2012). Specifically, an over-representation of the topographic distribution of
the SEP-N20 was observed for the evoked response by stimulation of the right median nerve,
which was related to the handedness of deaf-blind children who make a predominant use of
their hands to communicate and explore the environment before the CI, and with cross-modal
effects after long periods of deaf-blindness during the development of children. Lee et al.
(2001) report that the extent of cross-modal recruitment of the auditory cortex in humans
increases as the duration of deafness is longer.
Why do not think that the less intense use of the hands to communicate after the auditory
(re)habilitation post-CI may reflect changes in the topography of the SEP-N20? Someone
could speculate that the arrival of the auditory sensory input through the CI on temporal
cortex in these children competes with the other modalities of sensory input that were
established in these areas before the CI. It is possible that our results could be evidence of
new cortical reorganization after auditory (re)habilitation post-CI. However, the mechanism
that promote the changes is not possible to be defined by our experimental design. One could
speculate that inhibition of pre-existing cortical connections is the possible mechanism that
sustains this finding. The auditory sensory input "competes" with the somesthetic information
that activated the temporal region in the left hemisphere previously to the implantation. This
is precisely the cortical brain area where is conformed the neural basis of hearing and
language. However, the input and processing of sensory information at the cortical level is the
result of complex processes taking place, where different areas are involved in the
information processing that arrive to the brain cortex. Deaf-blindness is an interesting model
for future research to help get into the particularities that occur in cortical processing.
Nowadays, there have been reported neuroplastic changes involving the activation of the
temporal region in deaf children who received CI (Gilley 2008). Although hardly studied in
deaf-blind it has been also reported that the auditory stimulus presentation has been able to
activate the temporal region, in this case using Positron Emission Tomography (PET) (Osaki
2006).
There are few studies about the neuroplasticity in deaf-blindness (Osaki 2004, 2006;
Obretenova 2010). What previous works have in common is that they studied a single deaf-
blind adult. They reported cortical activation patterns to the tactile stimulation, which agree
with our findings. We also used a tactile stimulus to activate the somesthetic pathway, but
using the electrical stimulation, the most recommended in neurophysiological approach to
obtain the SEP (American Clinical Neurophysiology Society 2008, Chiappa and Hill 1997).
Obretenova et al. (2010) assessed the neural correlates associated with language processing in
a deaf-blind adult (early onset). This work was based on the analysis of cortical activation
with MRI, specifically studying the brain connectivity (tractography from diffusion tensor
calculation). They described that tactile stimuli (reading words presented in Braille, Print on
Palm and a haptic from of American Sign Language) resulted in a strong activation
(connectivity) between the temporal and occipital regions. Also, Osaki et al. (2004)
conducted a study of one deaf-blind adult before been implanted with
Magnetoencephalography (MEG) and PET. They presented tactile stimuli (words and non-
words) and found an extensive cortical activation in the deaf-blind subject by comparing the
pattern of activation with six control subjects. A year after the CI, they re-evaluated the deaf-
blind subject with PET, reporting a complete different pattern of activation. Occipital lobe,
which was activated during tactile language before the CI, was not activated after the auditory
(re)habilitation post-CI (Osaka 2006). It is consistent with the results obtained in the majority
of deaf-blind children in our study who were implanted after age 7. Four of the five deaf-
blind children showed an extensive cortical activation by tactile stimulation covering the
parieto-temporo-occipital regions before the CI (Charroó-Ruiz 2012), and 5 years after the CI
the activation was limited to the parietal region (cortical area corresponding to the
somesthetic representation of the hand).
On the other hand, several causes may be influencing on the change of the topographic
distribution of the SEP-N20 post-CI in only one deaf-blind child that showed a topographic
distribution of SEP-N20 even more extensive compared with the study pre-CI (Charroó-Ruiz
2012). The most extensive topographic distribution of the SEP-N20 post-CI could be
considered as the result, among other causes, of a progressive of the visual impairment in this
child. We have reported that, changes in the topographic distribution of SEP-N20 pre-CI by
right median nerve stimulation in deaf-blind children candidates for CI seem to be related to
the early-onset of dual sensory deprivation (deaf-blindness) and severity of the visual
impairment (Charroó-Ruíz 2014, 2012). This girl suffers of Retinitis Pigmentosa. The visual
test-retest by one of our co-authors showed a progression of visual loss after 5 years of being
implanted. Moreover, in the results of the auditory (re)habilitation of this girl it is important
to note that due to illness and irregularities with the (re)habilitation in her health area, this
deaf-blind child could not receive continuous and intensive auditory therapy, such as the other
deaf-blind studied children, therefore, she did not perform the auditory (re)habilitation
schedule proposal according to Cuban Cochlear Implant Program. These reasons could
explain the poor and slow progress observed in this child. She only reached the
(re)habilitation phases of discrimination/identification, and continues using the tactile
language as a primary mode of communication at home and at the school. Some authors
describe that deaf children with SNHL who receive a CI require at least 4 years of intensive
auditory (re)habilitation to achieve maximum benefit with CI (Archbold and O´Donoghue
2009; Bond 2009). In addition, the deaf-blind child that showed over-representation of the
SEP-N20 post-CI has an additional disability (cerebral palsy) which should be considered as
another possible factor that may influence on the outcomes after the CI.
It should be noted that deaf-blindness is a condition in which each subject behaves with
marked individuality, especially, when deaf-blindness begins at birth, where the two main
sensory systems to acquire information from the environment and achieve a normal
development are affected. In deaf-blind children occurs a very peculiar adaptive brain re-
organization, therefore the Central Nervous System matures in a different way. It is difficult
to form homogeneous groups of deaf-blind children to increase samples. In addition,
nowadays there are few deaf-blind children that could be candidates for CI, and definitive
candidacy criteria in patients with multiple disabilities and cognitive impairment do not exist
(Cosetti and Waltzman 2011).
In our study, the auditory cortex was activated by the arrival of sound through the CI,
which was demonstrated objectively by the CAEP records obtained in deaf-blind children, as
it has been reported by other authors in deafness after receiving CI (Gilley 2008; Sharma
2009, 2005b). Our results show that the morphology of the CAEP was comparable to that
described by other authors in subjects after the CI (Sharma 2002; Gilley 2008; Walker 2008;
Pozzobom 2009). Although Sharma and colleagues used other type of auditory stimuli [ba],
the most used to evaluate CAEP in implanted deaf children (Sharma 2005a, 2004, 2002;
Ponton 1996a, 1996b; Eggermon 1997), we used clicks because they evoked responses
comparable with the [ba] (Martin 2008). Sharma et al. (2002) investigated the effects of
auditory deprivation and use of CI in deaf children through assessment latency of the P1 peak.
They described this parameter of the CAEP as an electrophysiological biomarker of the
changes that occur at cortical level in deaf children with CI (for assessing the maturation of
the central auditory system) (Sharma 2005b). They reported that deaf children implanted
before age 7 had a good outcome, and it is correlated with a decrease in the value of the
latency of the P1 peak. The best results were observed when the age for implanting was under
3.5 years old in children with a profound hearing loss (Sharma 2002).
Deaf children implanted after age 7 had a long latency of the P1 peak, similar to that
found in deaf-blind children in our study.
Despite the differences in methodology between the group of Sharma and our group, the
results of both labs converge to indicate the age of 7 years as an important lapse of time in
order to observe changes in the cortical evoked responses of the EPs in children with auditory
sensory impairment or auditory and visual sensory impairments.
There are few published studies in children with dual sensory deprivation (deaf-
blindness) (Saeed 1998; El-Kashlam 2001; Filipo 2004). In general, publications are limited
to the description of the progress on deaf-blind children after the CI. It is argued that deaf-
blind children, generally, have poor progress in the production of words or sentences
(Dammeyer 2008). However, the improvement that these children can have from the point of
view of family interaction, with their environment, in their level of attention and emotional
state justifies any effort to works in the benefit of these children through CI programs.
In summary: Nontraditional measures such as the topographic distribution of the SEP-
N20 could be useful in assessing of the neuroplastic changes in deaf-blindness.
Our study included seven deaf-blind children, assessed by a well-developed and
systematic methodology. The study included results in the topographic distribution of the
SEP-N20 that can be considered as evidences of cross-modal plasticity after the CI and
auditory (re)habilitation in deaf-blind children, with 7 or more years of the sensory
deprivation before the CI. Evidences of cross-modal plasticity may be an expression of how
important is the somesthetic information in these subjects, probably due to the relationship
with tactile language, as well as the functional interaction of auditory and somesthetic
information during the auditory (re)habilitation post-CI. Although there is a cortical
reorganization before the CI, the findings point to the possibility that deaf-blind children
could be in favorable conditions for better outcomes after the CI, with new cortical
reorganization of the sensory cortices, unless for any reason the auditory (re)habilitation
could not be properly delivered (progression of the visual impairment, an associated disability
or an inadequate family contribution).
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Chapter 7
DEXAMETHASONE ACTIONS
ON BRAIN VASCULAR ENDOTHELIUM
IN HEALTH AND DISEASE
Malgorzata Burek∗
University Wurzburg,
Department of Anaesthesia and Critical Care,
Wurzburg, Germany
ABSTRACT
Due to their anti-inflammatory and immunosuppressive properties, glucocorticoids
(GCs) are the most often prescribed medicine with manifold physiological effects in the
human body. GC can function through genomic and non-genomic pathways. Activation
of glucocorticoid receptor (GR) by GCs leads to transcriptional activation of GC-target
genes. A lot of work has been done to identify the GC-target genes as well as factors
influencing the expression and the activity of the GR. This chapter will give an overview
about vascular functions of dexamethasone, a synthetic GC with a focus on
dexamethasone action on the blood-brain-barrier (BBB). BBB maintains homeostasis of
the central nervous system and is disrupted in many pathological disorders, e.g., multiple
sclerosis or ischemia. The results of in vitro and in vivo studies showed that GC-
treatment improved BBB-properties. Several direct targets of GCs have been found
among the endothelial cell junction proteins. GCs have been shown to stabilize their
expression under inflammatory and ischemic conditions. Better knowledge of the
mechanisms of action of this fascinating group of endogenous and synthetic molecules
will allow better future treatment and therapy in the clinics.
∗
Corresponding author: Malgorzata Burek, PhD. University Wurzburg. Department of Anaesthesia and Critical
Care. Oberduerrbacher Str. 6. 97080 Wurzburg. Tel.:+4993120130046. Fax: +4993120130019. E-mail:
Burek_M@ukw.de.
126 Malgorzata Burek
INTRODUCTION
Endogenous glucocorticoids (GCs) are built in the adrenal cortex. GCs have manifold
physiological effects in the human body, e.g., regulation of metabolism, stress-response,
blood pressure and immune system. GCs are lipophilic and can pass the plasma membranes.
In cytosol they bind the glucocorticoid receptor (GR) which in its inactive state is bound to
protein complex including amongst others heat shock protein 90 (Hsp90), Hsp56 and p23 [1,
2]. GR is organized into an N-terminus, a highly conserved central DNA-binding domain and
a C-terminal ligand-binding domain [3].
Binding of the ligand leads to the dissociation of the protein complex, dimerization of GR
and translocation to the nucleus [2]. GR binds to chromatin on palindromic repeats, so-called
glucocorticoid response elements (GRE) 5’-AGAACAnnnTGTTCT-3’. GRE half-sites are
sufficient in numerous genes to transduce the GC-signal [4]. GR can also interact with other
transcription factors, such as activator protein 1 (AP-1), signal transducer and activator of
transcription 3 (STAT3) or nuclear factor κB (NFκB) and in this way regulates gene
expression [5-7]. The GR knockout mice do not survive indicating essential role of GR and
GC-signaling in development [8-10].
Synthetic GC, dexamethasone is more powerful that the naturally produced GCs, has a
long biological half-life and low mineralocorticoid activity. Among GCs dexamethasone is
most widely used for treatment of tumor-associated cerebral edema. However, long-term
dexamethasone-treatment, as treatment with other GC, leads to multiple side-effects.
Blood-brain barrier (BBB) is an interactive interface between the brain parenchyma and
the blood compartment. Brain microvascular endothelial cells together with other cell types,
like astrocytes, pericytes, neurons, oligodendrocytes, microglia and blood cells build up an
interactive and dynamic structure called neurovascular unit (NVU) [11]. All the cells of the
NVU interact and influence each other. Also dexamethasone can influence each type of cells
in the NVU. In further sections of this chapter, dexamethasone-induced changes in
endothelial cell biology will be especially discussed.
BLOOD-BRAIN BARRIER: STRUCTURE AND FUNCTION

The highly specialized endothelial cells of brain capillaries build an interface between
blood and brain parenchyma. The brain endothelial cells are connected through tight and
adherens junctions.
Tight junctions are constituted by transmembrane proteins occludin, tricellulin, claudins
(-1, -3, -5 and -12) and junctional adhesion molecules (JAMs) [12]. The intracellular domains
of these transmembrane proteins interact with cytosolic proteins, such as zonula occludens-1,
-2 and -3 (ZO-1, -2 and -3), cingulin, 7H6 and AF-6 connecting them to the actin-
cytoskeleton (13). Adherens junction proteins are critical for the development and
stabilization of tight junctions. Vascular endothelial-cadherin (VE-cadherin) was shown to
play a role in the regulation of gene transcription of tight junction protein claudin-5 [14].
Dexamethasone Actions on Brain Vascular Endothelium in Health and Disease 127
DEXAMETHASONE ACTIONS AT THE BBB

GCs actions at the BBB lead to an increase in the barrier integrity. These barrier-
stabilizing GC-effects have been used for decades to treat tumor-associated brain edema in
the clinic [15]. Blood-retina barrier (BRB) can also be stabilized by GC-treatment which is
used to treat for example, macular edema. In vitro, GC-mediated barrier-stabilizing effects
have been shown on various cell types and BBB-models, e.g., mouse cerebral and cerebellar
microvascular endothelial cell lines cEND and cereEND, rat brain microvascular endothelial
cell line GPNT, porcine as well as human BBB-model, hDMEC/D3 [16-21]. Treatment of
mouse, rat, porcine and human brain endothelial cells with dexamethasone led to an increase
in the expression of claudin-5, occludin and ZO-1. This correlated with an increased
transendothelial electrical resistance (TEER) and decreased paracellular permeability of the
endothelial monolayer [16, 20, 22]. Occludin promoter has been studied in detail and a
functional GRE could be identified in occludin promoter sequence (Table 1). A direct binding
of GR could be demonstrated by chromatin immunoprecipitation (ChIP) [16, 23].
Table 1. Dexamethasone target genes at the BBB
Gene name/ Regulation of Direct binding of Selected

symbol GR to promoter References
Promoter mRNA Protein
Claudin-5 (CLDN5) ↑ ↑ ↑ no data [18, 24-28]
Occludin (OCLN) ↑ ↑ ↑ ChIP [16, 24, 25, 28]
TIMP-1 ↑ ↑ no data [29]

TIMP-3 ↑ ↑ no data [30]
VE-cadherin (CDH5) ↑ ↑ ↑ no data [28, 31]
ZO-1 (TJP1) ↑ ↑ no data [19, 29, 32]
TIMP-1: Tissue inhibitor of MMPs-1; TIMP-3: Tissue inhibitor of MMPs-3; VE-cadherin: vascular
endothelial- cadherin; ZO-1: zonula occludens-1; TJP1: tight junction protein 1.
Moreover, a highly conserved occludin enhancer element (OEE) was identified in

occludin and claudin-5 promoters. OEE was shown to be necessary for dexamethasone-
induction of occludin and claudin-5 promoters, however, no direct binding of activated GR to
this region was observed [24]. A heterodimer of transcription factors p54 and polypyrimidine
tract-binding protein associated splicing factor (PSF) were shown to bind directly to OEE
after dexamethasone treatment leading to induction of occludin and claudin-5 as well as
barrier properties in human retinal endothelial cells [25].
Claudin-5, a major cell adhesion molecule of the tight junctions in endothelial cells, plays
a role in BBB permeability. Claudin-5 knockout mice showed leaky BBB to molecules
smaller than 800 Da without morphological changes of tight junctions [33]. Claudin-5 was
identified as dexamethasone target gene especially at the human BBB, although also in mouse
cells the induction at the promoter and mRNA level was demonstrated, as summarized in
Table 1 [18, 24, 25, 27]. Claudin-5 was also shown to be regulated by other steroid hormone
estrogen [34, 35]. Mouse and human claudin-5 promoters were cloned and characterized [27,
36]. Although GR interactions with the claudin-5 promoter have not been experimentally
evaluated, promoter sequence analysis revealed multiple GREs, suggesting the possibility of
claudin-5 being a direct GR target [27] (Table 1). The induction of other claudins expressed at
the BBB was not studied at promoter level, however the induction of claudin-1 and claudin-
12 mRNA was shown in mouse brain microvascular endothelial cell line cEND after
dexamethasone treatment [29, 37]. In vivo studies in sheep showed the effects of maternal
glucocorticoid exposure on expression of claudin-1, -5, occludin and ZO-1 [26, 38]. VE-
cadherin, an adherens junction protein, plays a role in calcium-mediated adhesion and is
essential for vasculature development [14]. Dexamethasone induced VE-cadherin promoter,
mRNA and protein expression in brain microvascular endothelial cell line cEND as well as
redistribution of VE-cadherin into the actin-cytoskeleton [31]. Another protein involved in the
formation of tight junctions and influenced by dexamethasone is ZO-1. ZO-1 is a cytoplasmic
protein and its upregulation has been linked specifically to the proper functioning of tight
junctions and paracellular permeability (39). Dexamethasone induced ZO-1 protein in rat
GPNT BBB-model, but no induction could be observed in mouse cEND BBB-model on
protein level (19, 29, 31). However, the induction of ZO-1 mRNA was demonstrated in cEND
[29]. No data on direct binding of GR to the ZO-1-promter region is available to date (Table
1).
Due to immunosuppressant properties of dexamethasone, a number of genes involved in
inflammation can be activated or inhibited by dexamethasone. Extracellular matrix (ECM) is
one of the NVU components. Matrix-metalloproteinases (MMPs) are degrading ECM which
influences the integrity of NVU [40, 41]. Tissue inhibitors of MMPs (TIMPs) form
complexes with either activated MMPs or with their preforms and regulates the activity of
MMPs under physiologic conditions [42, 43]. TIMP-1 and -3 are induced by dexamethasone
and hydrocortisone, which has beneficial effects on barrier integrity (Table 1) [29, 30].
DEXAMETHASONE BENEFITS IN BRAIN DISORDERS

Multiple Sclerosis
In neuroinflammatory disorders such as multiple sclerosis (MS), pathogenic T cells

infiltrate the CNS leading to local inflammation (44). Dexamethasonem, due to its strong
anti-inflammatory and immunosuppressant properties, is being used along with other GCs to
treat MS. The majority of MS patients suffer initially form the relapsing-remitting form of
MS. GC-treatment is approved as first line of therapy for MS relapses [45]. The exacerbations
of the disease are initiated by decrease of BBB integrity, which allows the T cell to pass the
endothelial barrier [46, 47]. The barrier-closing effects of GCs were shown by magnetic
resonance imaging gadolinium enhancement in patients with acute inflammatory lesions [48].
Experimental autoimmune encephalomyelitis (EAE) serves as animal model of MS [49].
Studies on EAE models revealed that dexamethasone, along with other GCs has beneficial
effects by inducing T-cell apoptosis [50, 51]. Moreover, dexamethasone leads to increase in
barrier properties and to reduction of leukocyte infiltration to the CNS [47]. Endothelial cells
in response to pro-inflammatory cytokines express vascular cell adhesion molecule 1
(VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Both proteins are involved in
leucocyte adhesion and transmigration. Treatment with methylprednisolone reduced this

cytokine-induced ICAM-1 and VCAM-1 expression [52]. In vitro, microvascular brain
endothelial cells cEND treated with multiple sclerosis patient sera showed decrease in
expression of junctional proteins occludin, claudin-5 and VE-cadherin. Dexamethasone could
suppress these changes [28]. Moreover, MS-patient sera treatment led to an increase in the
level of chemokines Ccl7 and Ccl12. This increase could also be attenuated by
dexamethasone treatment [53].
Traumatic Brain Injury and Stroke
Due to their barrier-stabilizing effects, GCs were used to treat brain edema after stroke
and traumatic brain injury (TBI). However, numerous studies with patients who suffered from
severe TBI did not show substantial benefits of GC-treatment in clinical outcome [54].
Therefore, the use of GCs for TBI treatment is no longer recommended [55]. Since to date,
there is no TBI-treatment with proven efficacy in humans, development of new therapeutic
agents remains as a focus for research.
One promising strategy has been shown in mice. Treatment of mice subjected to TBI
with a combination of proteasome-inhibitor bortezomib and dexamethasone improved BBB-
integrity, reduced edema formation and limited neuronal damage [56, 57]. Inhibiting the
proteasome-mediated degradation of GR enforced the GR signaling at the BBB and led to an
increase in tight junction protein occludin expression. Another recent approach showed
beneficial effects of combination therapy of dexamethasone with melatonin in animal model
of TBI [58]. Melatonin is the main product of the pineal gland with anti-oxidant properties.
Combination of dexamethasone and melatonin therapy led to reduction of lesion size, lower
apoptosis level and better neurological scores in mice after TBI. Moreover, treatment of rats
after TBI with dexamethasone suppressed the infiltration of RhoA positive
macrophages/microglia into the brain parenchyma. These effects were transient and persisted
for 4 days [59]. Inhibition of RhoA and its downstream targets has been shown to promote
axon regeneration and to improve functional recovery after TBI in rats [60]. An in vitro
model of blast-induced TBI was developed recently and was used for the examination of
dexamethasone treatment effects [32].
In this model which was based on immortalized mouse brain microvascular cell line
b.End.3, a breakdown of endothelial barrier was observed after injury. The same effects were
seen in another in vitro TBI model based on cEND cells [61]. Dexamethasone treatment of
blast injured BBB led to an increase in TEER and hydraulic conductivity as well as in ZO-1
expression one day after injury [32]. Interestingly, these dexamethasone effects were blocked
by GR-antagonist RU486 indicating genomic actions of dexamethasone.
During TBI and stroke all the cells of NVU are exposed to hypoxic environment. The
signaling cascades initiated in one type of cells can inflluence other cells at the NVU. In vitro
study by Zhang et al. used hypoxic astrocytes conditioned medium to treat the brain
microvascular endothelial cells [62]. This led to the inflammatory activation of endothelial
cells and this activation was blocked by dexamethasone treatment.
Brain Tumor
Expanding brain tumors within the brain parenchyma often lead to tumor-associated
brain edema. Treatment with GCs has been proven as a reliable and powerful therapy of
tumor-associated brain edema and is being used for decades [15]. Primary brain tumors are
not that common as the brain metastases of tumors from the periphery which have a ten-fold
higher incidence than the primary brain tumors. Tumors such as melanoma, breast cancer and
lung cancer most often build brain metastases. On the other hand, the prostate and colorectal
cancer rarely spread to the brain. The BBB plays an essential role in the process of brain
metastases building [63]. Therefore the processes leading to brain metastases building should
be carefully evaluated, since the prevention of brain metastases building would bring a better
prognosis for the patients. Barrier-stabilizing properties of GCs might be beneficial in this
case. Indeed, dexamethasone has been shown to decrease the permeability of blood-tumor
barrier. This barrier is formed when the tumor cells are already present in the brain
parenchyma. After the tumor overcame the BBB, it spreads and induces generation of new
vessels which further support its growth. Treatment of rats with dexamethasone led to lower
Evans blue permeability in the tumor environment, as well as to an increase in occludin and
calcium-activated potassium channel expression [64, 65]. Primary rat brain endothelial cells
undergo endothelial-mesenchymal transition upon cultivation with tumor-cell conditioned
medium [66]. This process is connected with down-regulation of claudin-5 and decrease of
barrier properties which facilitates tumor cell transmigration and metastases.
CONCLUSION
Dexamethasone influences directly or indirectly a number of known and unknown targets
at the BBB. More research on molecular mechanisms underlying dexamethasone action could
lead to development of new therapies and limitation of side effects. Recent results from in
vivo and in vitro TBI models gave insights to understanding the mechanism of
dexamethasone action in injured BBB. This might renew the clinical interest in the
development of glucocorticoid-based therapies for TBI and other disorders of the CNS.
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[66] Krizbai IA, Gasparics A, Nagyoszi P, Fazakas C, Molnar J, Wilhelm I, et al.
Endothelial-mesenchymal transition of brain endothelial cells: possible role during
metastatic extravasation. PloS one. 2015;10(3):e0123845.
Chapter 8
DEXAMETHASONE SUPPRESSES NEUROSTEROID

BIOSYNTHESIS IN HUMAN GLIAL CELLS
VIA CROSS-TALK WITH VITAMINS A AND D
Hiroomi Tamura*
Keio University, School of Pharmaceutical Sciences,
Tokyo, Japan
ABSTRACT
Emerging evidence indicates that stress hormone glucocorticoids (GC) are an
important modulator of brain development and function. To investigate whether GCs
modulate neurosteroid biosynthesis in neural cells, we studied the effects of GCs on
steroidogenic gene expression in human glioma GI-1 cells. The GC dexamethasone (Dex)
reduced StAR, CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression in a
dose- and GC receptor-dependent manner. In addition, Dex reduced de novo synthesis of
progesterone (PROG). Furthermore, Dex inhibited all-trans retinoic acid (ATRA) and
vitamin D3 (VD)-induced steroidogenic gene expression and PROG production.
Metabolism of GCs is carried out by the enzymes 11β-hydroxysteroid dehydrogenases
(HSD11B1 and HSD11B2). These enzymes control the intracellular concentration of
active GCs which in turn are related to brain functions. We found that Dex inhibits the
ATRA/VD-induced HSD11B2 gene expression. Our results suggest that GC regulates
neurosteroid synthesis in neural cells via cross-talk with the two fat-soluble vitamins, A
and D. The relationship between the effects of GCs on neurosteroid biosynthesis and on
cognitive behaviors and hippocampal neural activity is also discussed.
INTRODUCTION
Glucocorticoids (GCs) are widely used as drugs to treat inflammatory and autoimmune
disorders, including neuroinflammatory conditions such as multiple sclerosis [1]. In response
*
E-mail address: tamura-hr@pha.keio.ac.jp.
138 Hiroomi Tamura
to an inflammatory reaction or to stress, the hypothalamic–pituitary–adrenal (HPA) axis is

stimulated to increase systemic levels of GCs with a consequent repression of inflammation
in a process involving nuclear factor kappa B (NF-κB) [2]. In addition, many studies have
described the effects of GCs on the central nervous system (CNS). GCs activate several
biochemical/molecular processes in the hippocampus through two receptors, the
glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) [3]. GCs influence
cognitive behaviors and hippocampal neural activity, and also increase the rate of aging-
dependent cell loss in the hippocampus [4]. During emotional and stressful situations,
activation of the HPA axis causes the adrenal cortex to release GCs, which travel through the
bloodstream and cross the blood-brain barrier to activate GRs throughout the brain [5].
Steroids that are synthesized within the central or peripheral nervous systems are termed
“neurosteroids” [6, 7]. The nervous system is an important site of steroid production, as both
neurons and glial cells can synthesize steroids de novo from cholesterol (Figure 1). To start to
synthesize of steroids, cholesterol is needed to transport into the mitochondria, mediated by
the action of steroidogenic acute regulatory protein (StAR). StAR is a mitochondrial protein
that is rapidly synthesized in response to stimulation of the cell to produce steroid. The
mitochondrial cytochrome P450scc (CYP11A1), which is the cholesterol side-chain cleavage
enzyme that catalyzes the de novo synthesis of pregnenolone (PREG), is expressed
throughout the rodent brain [8-10]. 3β-hydroxysteroid dehydrogenase (HSD3B1), which
converts PREG to progesterone (PROG), is also largely distributed throughout the brain and
spinal cord [11, 12]. In addition, primary cultures of mixed glial cells can metabolize
cholesterol to PREG and PROG [13].
Figure 1. Steroid biosynthetic pathway. StAR, steroidogenic acute regulatory protein; PBR, peripheral-
type benzodiazepine receptor; HSD3B1, 3β-hydroxysteroid dehydrogenase; SRD5A, steroid 5α-
reductase; AKR1C, aldo-keto reductase 1C.
Dexamethasone Suppresses Neurosteroid Biosynthesis in Human Glial Cells … 139
Neurosteroids are involved in the regulation of several CNS processes, specifically mood,
affective and cognitive functions by interacting with classical neurotransmitter receptors such
as the GABAA receptor and by modulating glutamate, GABA, and acetylcholine
neurotransmission [6, 7, 14]. Elevated circulating levels of glucocorticoids (GCs) are
associated with psychiatric symptoms such as depression and dementia, although it remains
unknown if this is a cause or an effect of the psychiatric condition [15]. In addition,
depression is a well-known side effect of glucocorticoid treatment, and a third of the patients
receiving glucocorticoids experience significant mood disturbances and sleep disruption.
However, GC effects on neurosteroid synthesis have not been well documented. To
investigate the effects of GCs on neurosteroid biosynthesis in GI-1 cells, we analyzed the
effect of dexamethasone (Dex) on steroidogenic gene expression within these cells.
Vitamin A and D Enhance Neurosteroid Synthesis in Human Glioma

GI-1 Cells
Retinoid compounds, particularly the retinoic acids (RAs), are known to regulate steroid
biosynthesis in classical steroidogenic tissues such as the adrenal gland, ovary and testis. The
steroids synthesized in the CNS are the neurosteroids and we investigated the effects of RAs
upon their synthesis [16].
Figure 2. ATRA induces neurosteroid synthesis in GI-1 cells. (A) GI-1 cells were treated with ATRA
(at 0, 1 and 10 μM) for 48 h. Quantitative PCR analysis was performed for StAR, CYP11A1 and
HSD3B1 mRNAs. The results are presented using arbitrary units, with the control values set at 1, and
are the means ± SD of three experiments. *, p < 0.05. Insert, an enlargement of the vertical scale for
StAR and CYP11A1. (B) GI-1 cells were treated with ATRA (0, 1, 10 μM) for 48 h. The 14C-labeled
steroids in the culture medium were extracted with ethyl acetate:isooctane (1:1 v/v) and separated by
TLC. The radioactivity of the spots on the TLC plate was visualized using a FLA7000 and quantified.
140 Hiroomi Tamura
We analyzed the effects of all-trans-retinoic acid (ATRA) upon the expression of

neurosteroid biosynthesis genes in the human glial cell line GI-1, in which the major
steroidogenic genes are expressed.
Treatment with ATRA (10 μM) induced a 4.9–fold increase in the expression of the
cytochrome P450scc (CYP11A1) gene, the product of which cleaves the cholesterol side
chain, a rate-limiting step during steroidogenesis. ATRA also strongly induce the expression
of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase
(HSD3B1) (an increase of 5- and 50-fold, respectively) (Figure 2A). ATRA induced the de
novo synthesis of neurosteroids such as pregnenolone and progesterone to levels that would
induce their target genes (Figure 2B).
Figure 3. Effect of VD3 on the neurosteroid synthesis in GI-1 cells. (A) Effect of VD3 on the expression
of steroidogenic genes. Cells were treated with or without 100 nM VD3 for 48 hrs and the expression of
steroidogenic genes was measured by qPCR analysis. (B) Effect of VD3 on the expression of the
CYP11A1 gene in GI-1 cells. Cells were treated with 100 nM VD3 with or without 10 μM ATRA for
48 hrs and the expression of CYP11A1 was monitored by qPCR. (C) Induction of neurosteroid de novo
synthesis in GI-1 cells by exposure to VD3 and ATRA. Quantitative analysis was performed as
described in Figure 2B. *p < 0.05 indicate a significant difference compared to the negative control
(DMSO). (n = 3).
These results confirmed that ATRA induces this de novo neurosteroid synthesis via the
induction of steroidogenic genes in human glial cells. The multiple effects of vitamin A upon
CNS functions might therefore be partly explained by the induction of neurosteroidogenesis,
since these molecules have also been reported to have pleiotropic effects in the CNS.
Emerging evidence also indicates that vitamin D (VD) is an important modulator of brain
development and function [17, 18]. We investigated the effect of VD3 on steroidogenic gene
expression in human glioma GI-1 cells and found that VD3 enhanced CYP11A1 and HSD3B1
gene expression (Figure 3A).
Calcipotriol, a VD3 receptor (VDR) agonist, also induced CYP11A1 gene expression in
GI-1 cells, indicating that VDR is involved in this induction. The induction of progesterone
(PROG) de novo synthesis was observed along with the induction of steroidogenic genes by
VD3 (Figure 3C). Furthermore, VD3 enhanced all-trans retinoic acid (ATRA)-induced
CYP11A1 gene expression and PROG production (Figure 3, B and C). This suggests
cooperative regulation of steroidogenic gene expression by the two fat-soluble vitamins, A
and D.
Effect of Dex on the Steroidogenic Gene Expression in GI-1 Cells
Since GCs influence cognitive behaviors and hippocampal neural activity [20], we
examined whether Dex affects neurosteroid biosynthesis in neural cells. We measured the
expression of steroidogenic genes in GI-1 cells treated with a physiological range of Dex
concentrations. Cells were incubated with Dex for 24 hrs, and the steroidogenic gene
expression was measured by real-time PCR.
The expression of StAR, CYP11A1 and HSD3B1 genes was reduced by Dex in a dose-
dependent manner (Figure 4). The expression of all three genes was almost 50% lower in
cells treated with 10 nM Dex than in control cells. Immunoblot analysis also showed that Dex
reduced the HDS3B1 proteins according to the levels of its gene expression (Figure 4D). We
could not measure the protein levels of StAR and CYP11A1 because of low amounts of
proteins. Addition of RU-486, a GR antagonist, abolished the effect of Dex on these gene
expression, suggesting that Dex regulates these gene expression via GR.
Effects of Dex on Vitamin-Induced Steroidogenic Gene Expression in GI-1

Cells
As mentioned above, ATRA and VD3 induced neurosteroid biosynthesis in GI-1 cells
through induction of steroidogenic gene expression [15, 16]. To elucidate whether Dex affects
vitamin-induced neurosteroid synthesis in GI-1 cells, we co-treated GI-1 cells with Dex and
the vitamins (ATRA and/or VD3). Dex reduced the vitamin-mediated induction of all three
steroidogenic genes tested, StAR, CYP11A1 and HSD3B1. Immunoblot analyses also
showed that Dex reduced the induction of HSD3B1 proteins mediated by ATRA and/or VD3
(Figure).
Dex Reduces de novo Biosynthesis of Neurosteroids in GI-1 Cells
We next measured the de novo biosynthesis of neurosteroids in Dex-treated GI-1. G-1

cells were treated with 1 μM Dex for 48 hrs and [1-14C]acetic acid was then added to the
142 Hiroomi Tamura
culture medium for 24 hrs prior to collection of the medium. Radiolabeled steroids were
extracted from culture media samples, separated by TLC, and visualized.
The level of steroid production was quantified based on the band intensities, and an index
of induction relative to the non-treated control was calculated (Figure 4).
Figure 4. Effect of Dex on the steroidogenic gene expression in GI-1 cells. GI-1 cells were cultivated
with or without Dex (0-1 μM) for 48 hrs and qPCR analyses was performed for the steroidogenic gene
expression. (A) StAR, (B) CYP1A1 and (C) HSD3B1 genes. *p < 0.05, **<0.01 (n = 3). (D)
Immunoblot analysis of HSD3B1 proteins.
Figure 5. Effects of Dex on the vitamin-mediated induction of the steroidogenic gene expression in GI-
1 cells. Cells were treated with or without 1 μM Dex in the presence of 10 μM ATRA and 100 nM VD3
for 24 hrs. Expression of StAR, CYP11A1 and HSD3B1 genes was measured by qPCR. *p < 0.05,
**p < 0.01, (n = 3).
Low but significant levels of pregnenolone (PREG) and progesterone (PROG) were
synthesized and secreted into the culture medium. Following Dex addition, the de novo
synthesis of PROG decreased to half the level of the control, whereas that of PREG did not
change. Dex also reduced vitamin-induced PROG production, although it slightly increased
PREG production.
Regulation of 11β-Hydroxysteroid Dehydrogenase (HSD11B) Gene

Expression by Cross-Talk between GC and RA Receptors in GI-1 Cells
Actions of GCs are mediated by GC receptors (GRs) that modify transcriptional activity
of target genes. The amount of steroid available to activate these receptors is not only
dependent on the circulating levels but also on intracellular pre-receptor metabolism of
glucocorticoids. This metabolism is carried out by the enzymes 11β-hydroxysteroid
dehydrogenases (HSD11Bs).
There are two isozymes that control the intracellular concentration of active GCs which
in turn are related to brain functions [21. 22]. 11β-HSD type 1 (HSD11B1) elevates
intracellular glucocorticoid levels by regenerating active GCs from circulating cortisone while
11β-HSD type 2 (HSD11B2) inactivates GCs to its inert 11-keto derivative. In the adult
forebrain, HSD11B1 is the predominant whereas HSD11B2 is found only in a few very
restricted sites [23].
Figure 6. Dex reduced PROG production in GI-1 cells. GI-1 cells were incubated with 1 μM Dex and
[14C]acetic acid for 24 hrs and the quantitative analysis of PROG production was performed as
described in Figure 2B. (A) Image of spots on the TLC. (B) Quantification of PROG production in the
culture medium of GI-1 cells treated with Dex, ATRA and VD3. *p < 0.05, **p < 0.01, (n = 3).
144 Hiroomi Tamura
As mentioned above, Dex reduced the neurosteroid biosynthesis in GI-1 cells via down-
regulation of enzyme genes which are involved in the biosynthesis [24]. This result suggests
that HSD11Bs activities, which modifies the intracellular concentration of active GCs, may
affect the neurosteroid biosynthesis. To elucidate the possibility, we investigated the effect of
Dex on HSD11Bs expression in GI-1 cells.
Expression of the HSD11B2 gene but not the HSD11B1 gene was detected in GI-1 cells.
The level of HSD11B2 mRNA was significantly reduced by 1 μM Dex (Figure 7A).
Interestingly ATRA and VD3 diminished the effect of Dex on the HSD11B2 gene expression
(Figure 7A). Analyses by using specific agonists for RA (TTNPB), RXR (MTPA) and VDR
(calcipotriol) indicated that Dex interfered with RXR and VDR actions (Figure 7B).
Figure 7. Effect of Dex on HSD11B2 gene expression in GI-1 cells. (A) Effect of Dex on HSD11B2
expression with or without ATRA/VD3. Cells were treated with or without 1 μM Dex in the presence of
10 μM ATRA and 100 nM VD3 for 24 hrs. Expression of HSD11B2 gene was measured by qPCR. (B)
Effect of agonists for RAR (TTNPB), RXR (MTPA) and VDR (calcipotriol) on the Dex-mediated
reduction of HSD11B2 gene expression *p < 0.05, **p < 0.01, (n = 3).
CONCLUSION
In this chapter I described the effect of Dex on the neurosteroid biosynthesis in the
human glioma GI-1 cells. Dex at physiological concentrations reduced the neurosteroid
synthesis by suppressing expression of the steroidogenic genes such as StAR, CYP11A1 and
HSD3B. Dex also reduced the ATRA/VD-induced neuroteroid synthesis. The action of Dex
on the neurosteroid synthesis was mediated by GR, presumably interfering with the action of
RXR. Recent studies reported that Dex reduced ATRA-mediated induction of RARβ
expression in mouse hippocampal HT-22 cells [25] and prolonged exposure to Dex reduced
the VDR gene expression in rat brain [26]. Together with our results, it is likely that GCs
modulate the neurosteroid biosynthesis by tuning the functions of receptors for ATRA and
VD in neuronal cells.
Dex exhibited suppressive effect on the HSD11B2 gene expression in GI-1 cells. The
reduction in HSD11B2 activity may cause to increase intracellular glucocorticoid, and,
thereby, furthered the reduction in neurosteroid synthesis (Figure 8).
Figure 8. Schematic illustration of cross-talk among nuclear receptors on the PROG synthesis.
Shortage of RA in the brain is implicated in a variety of nervous system disorders such as

schizophrenia and depression. This may be related to the ability of RA to suppress the effect
of Dex on the HSD11B2 gene expression.
The reduction in neurosteroid synthesis by Dex was remarkable in PROG synthesis.
PROG is a major active neurosteroid that has been reported to exert protective effects in
numerous experimental models that mimic a variety of pathogenic aspects of brain
dysfunction seen with advanced age or age-related neurodegenerative diseases such as
Alzheimer’s disease (AD). The suppressive effect of GCs on PROG synthesis may be tightly
linked to cognitive dysfunction and neural disorders induced by stress hormone GCs. Our
results indicate that sufficient intake of vitamins A and D is protective against the GC-
mediated suppression of neurosteroid biosynthesis in the brain.
ACKNOWLEDGMENTS
I thank Dr. K. Oka for his encouragement. I also thank Dr. M. Funakoshi-Tago for her
excellent support. This work was supported in part by a Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by a grant from
the MEXT-Supported Program for the Strategic Research Foundation at Private Universities.
Competing Interests
I have no conflict of interest.

146 Hiroomi Tamura
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[8] Sanne JL, Krueger, KF. Expression of cytochrome P450 side-chain cleavage enzyme
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[9] Kohchi C, Ukena K, Tsutsui K. Age- and region-specific expressions of the messenger
RNAs encoding for steroidogenic enzymes p450scc, P450c17 and 3β-HSD in the
postnatal rat brain. Brain Res. 801, 233-238 (1998).
[10] Tsutsui K, Ukena K, Takase M, Kohchi C, Lea RW. Neurosteroid biosynthesis in
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[11] Guennoun R, Fiddes RJ, Gouézou M, Lombès M, Baulieu EE. A key enzyme in the
biosynthesis of neurosteroids, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase
(3β-HSD), is expressed in rat brain. Mol. Brain Res. 30, 287-300 (1995).
[12] Tsutsui K, Sakamaoto H, Ukena K. Novel aspect of the cerebellum: biosynthesis of
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[13] Hu ZY, Bourreau E, Jung-Testas I, Robel P, Baulieu EE. Neurosteroids:
oligodendrocyte mitochondria convert cholesterol to pregnenolone. Proc. Natl. Acad.
Sci. USA. 84, 8215-8219 (1987).
[14] P. Zheng. Neuroactive steroid regulation of neurotransmitter release in the CNS: action,
mechanism and possible significance. Prog. Neurobiol., 89, 134-152 (2009).
[15] Wolkowitz OM, Burke H, Epel ES, Reus VI. Glucocorticoids. Mood, memory, and
mechanisms. Ann N Y Acad Sci. 1179, 19-40 (2009).
[16] Kushida A, Tamura H. Retinoic acids induce neurosteroid biosynthesis in human glial
GI-1 Cells via the induction of steroidogenic genes. J. Biochem. 146, 917-923 (2009).
[17] Bouillon, R., Carmeliet, G., Verlinden, L., van Ette, n E., Verstuyf, A., Luderer, H.F.,
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[18] Heaney, R.P., Vitamin D in health and disease. Clin. Am. Soc. Nephrol., 3, 1535-1541
(2008).
[19] Kino T. Stress, glucocorticoid hormones, and hippocampal neural progenitor cells:
implications to mood disorders. Front Physiol. 6, 230 (2015).
[20] Yagishita T, Kushida A, Tamura H. Vitamin D3 enhances ATRA-mediated neurosteroid
biosynthesis in human glioma GI-1 cells. J. Biochem. 152, 285-292 (2012).
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Chapter 9
VITAMIN B12 IMPORTANCE FOR

THE PROPER BODY CONDITION
José Luis Cabrerizo-García1 and Begoña Zalba-Etayo2

1
Department of Internal Medicine, Hospital General de la Defensa, Zaragoza, Spain
2
Intensive Care Unit, “Lozano Blesa”
Universitary Hospital, Zaragoza, Spain
ABSTRACT
Vitamin B12 or cobalamin is produced by bacteria in the large bowel of humans and
by external bacteria and fungi. However, cobalamin from the former source is not
absorbed, and humans need to introduce it solely from the diet. The major sources of
cobalamin are animal proteins, mainly meats and eggs. Vitamin B12 deficiency causes a
wide range of hematological, gastrointestinal, psychiatric and neurological disorders.
However, the clinical picture does not always accompany the analytical variations.
Hematological presentation of cobalamin deficiency ranges from the incidental increase
of mean corpuscular volume to pancytopenia or symptoms due to severe anemia.
Neuropsychiatric symptoms may precede hematologic signs and are represented by
myelopathy, neuropathy, dementia and, less often, optic nerve atrophy. The spinal cord
manifestation, subacute combined degeneration, is characterized by symmetric
dysesthesia, disturbance of position sense and spastic paraparesis or tetraparesis.
In this chapter, we present a review of the real case of a patient with subacute
combined spinal cord degeneration and pancytopenia secondary to severe and sustained
vitamin B12 deficiency. Such cases are rare nowadays and have potentially fatal
consequences. Correction of the deficit is important, especially in severe cases, and
neurological sequelae may be irreversible if treatment is not started in good time.
INTRODUCTION [1]
Vitamin B12 (cobalamin) plays an important role in DNA synthesis and neurologic
function. Cobalamin is synthesized solely by microorganisms. Ruminants obtain cobalamin
from the foregut, but the only source for humans is food of animal origin, e.g., meat, fish, and
150 José Luis Cabrerizo-García and Begoña Zalba-Etayo
dairy products. Vegetables, fruits, and other foods of non-animal origin are free from
cobalamin unless they are contaminated by bacteria. A normal Western diet contains 5-30 μg
of cobalamin daily. Adult daily losses (mainly in the urine and feces) are 1-3 μg (~0.1% of
body stores), and because the body does not have the ability to degrade cobalamin, daily
requirements are also about 1-3 μg. Body stores are of the order of 2-3 mg, sufficient for 3-4
years if supplies are completely cut off.
Two mechanisms exist for cobalamin absorption. One is passive, occurring equally
through buccal, duodenal, and ileal mucosa; it is rapid but extremely inefficient, with <1% of
an oral dose being absorbed by this process. The normal physiologic mechanism is active; it
occurs through the ileum and is efficient for small (a few micrograms) oral doses of
cobalamin, and it is mediated by gastric intrinsic factor (IF). Dietary cobalamin is released
from protein complexes by enzymes in the stomach, duodenum, and jejunum; it combines
rapidly with a salivary glycoprotein that belongs to the family of cobalamin binding proteins
known as haptocorrins. In the intestine, the haptocorrin is digested by pancreatic trypsin and
the cobalamin is transferred to IF. IF is produced in the gastric parietal cells of the fundus and
body of the stomach, and its secretion parallels that of hydrochloric acid.
Normally, there is a vast excess of IF. The IF-cobalamin complex passes to the ileum,
where IF attaches to a specific receptor (cubilin) on the microvillus membrane of the
enterocytes. Cubilin appears to traffic by means of amnionless, an endocytic receptor protein
that directs sublocalization and endocytosis of cubilin with its ligand IF-cobalamin complex.
The cobalamin-IF complex enters the ileal cell, where IF is destroyed.
After a delay of about 6 h, the cobalamin appears in portal blood attached to
transcobalamin (TC) II. Between 0.5 and 5 μg of cobalamin enter the bile each day. This
binds to IF, and a major portion of biliary cobalamin normally is reabsorbed together with
cobalamin derived from sloughed intestinal cells. Because of the appreciable amount of
cobalamin undergoing enterohepatic circulation, cobalamin deficiency develops more rapidly
in individuals who malabsorb cobalamin than it does in vegans, in whom reabsorption of
biliary cobalamin is intact.
Prevalence of Vitamin B12 Deficiency [2]
The true prevalence of vitamin B12 deficiency is difficult to estimate because reports are
based on values that vary because of inclusion criteria and individual laboratory
methodology. In 1994, the Framingham Heart Study reported the prevalence of vitamin B12
deficiency, as defined by a serum vitamin B12 level less than 200 pg per mL and elevated
levels of serum homocysteine, methylmalonic acid, or both, to be 12 percent among 548
community-dwelling older patients.
However, most deficient patients did not have hematologic manifestations, and
neurologic manifestations were not assessed. According to unpublished data from the
National Health and Nutrition Examination Survey, 3.2 percent of US adults older than 50
years are estimated to have a serum vitamin B12 level less than 200 pg per mL.
Vitamin B12 Importance for the Proper Body Condition 151
CAUSES OF VITAMIN B12 DEFICIENCY [3]

Causes of vitamin B12 deficiency can be divided into three classes: nutritional
deficiency, malabsorption syndromes, and other gastrointestinal causes.
Nutritional deficiency: Dietary sources of vitamin B12 are primarily meats and dairy
products. In a typical Western diet, a person obtains approximately 5 to 30 mcg of vitamin
B12 daily, much more than the recommended daily allowance of 2 mcg. Normally, humans
maintain a large vitamin B12 reserve, which can last two to five years even in the presence of
severe malabsorption. Nevertheless, nutritional deficiency can occur in specific populations.
Elderly patients with “tea and toast” diets and chronic alcoholics are at especially high risk.
The dietary limitations of strict vegans make them another, less common at-risk population.
Malabsorption syndromes: The classic disorder of malabsorption is pernicious anemia, an
autoimmune disease that affects the gastric parietal cells.
Destruction of these cells curtails the production of intrinsic factor and subsequently
limits vitamin B12 absorption. Laboratory evidence of parietal cell antibodies is
approximately 85 to 90 percent sensitive for the diagnosis of pernicious anemia.
However, the presence of parietal cell antibodies is nonspecific and occurs in other
autoimmune states. Intrinsic factor antibody is only 50 percent sensitive, but it is far more
specific for the diagnosis of pernicious anemia. A Schilling test, which distinguishes intrinsic
factor-related malabsorption, can be used to diagnose pernicious anemia. The phenomenon of
food-bound malabsorption occurs when vitamin B12 bound to protein in foods cannot be
cleaved and released. Any process that interferes with gastric acid production can lead to this
impairment. Atrophic gastritis, with resulting hypochlorhydria, is a major cause, especially in
the elderly. Subtotal gastrectomy, once common before the availability of effective medical
therapy for peptic ulcer disease, also can lead to vitamin B12 deficiency by this mechanism.
The widespread and prolonged use of histamine H2-receptor blockers and proton pump
inhibitors for ulcer disease also may cause impaired breakdown of vitamin B12 from food,
causing malabsorption and eventual depletion of B12 stores. Recent studies have confirmed
that long-term use of omeprazole can lead to lower serum vitamin B12 levels.
Other causes: Other etiologies of vitamin B12 deficiency, although less common, deserve
mention. Patients with evidence of vitamin B12 deficiency and chronic gastrointestinal
symptoms such as dyspepsia, recurrent peptic ulcer disease, or diarrhea may warrant
evaluation for such entities as Whipple’s disease (a rare bacterial infection that impairs
absorption), Zollinger-Ellison syndrome (gastrinoma causing peptic ulcer and diarrhea), or
Crohn’s disease. Patients with a history of intestinal surgery, strictures, or blind loops may
have bacterial overgrowth that can compete for dietary vitamin B12 in the small bowel, as can
infestation with tapeworms or other intestinal parasites. Congenital transport-protein
deficiencies are another rare cause of vitamin B12 deficiency.
CLINICAL MANIFESTATIONS OF VITAMIN B12 DEFICIENCY

Vitamin B12 deficiency is associated with hematologic, neurologic, and psychiatric
manifestations. It is a common cause of macrocytic (megaloblastic) anemia and, in advanced
cases, pancytopenia.
Neurologic sequelae from vitamin B12 deficiency include paresthesias, peripheral

neuropathy, and demyelination of the corticospinal tract and dorsal columns (subacute
combined systems disease). Vitamin B12 deficiency also has been linked to psychiatric
disorders, including impaired memory, irritability, depression, dementia and, rarely,
psychosis [4, 5]. In addition to hematologic and neuropsychiatric manifestations, vitamin B12
deficiency may exert indirect cardiovascular effects. Similar to folic acid deficiency, vitamin
B12 deficiency produces hyperhomocysteinemia, which is an independent risk factor for
atherosclerotic disease [6].
DIAGNOSIS OF VITAMIN B12 DEFICIENCY

The diagnosis of vitamin B12 deficiency has traditionally been based on low serum
vitamin B12 levels, usually less than 200 pg per mL (150 pmol per L), along with clinical
evidence of disease. However, studies indicate that older patients tend to present with
neuropsychiatric disease in the absence of hematologic findings. Furthermore, measurements
of metabolites such as methylmalonic acid and homocysteine have been shown to be more
sensitive in the diagnosis of vitamin B12 deficiency than measurement of serum B12 levels
alone [3]. The Schilling test, which was previously used to diagnose pernicious anemia, is no
longer available in the United States, and testing for elevated levels of anti-intrinsic factor
antibodies and elevated serum gastrin or pepsinogen is recommended [7]. Because of the
association between pernicious anemia and a higher incidence of gastric cancer and
carcinoids, it is important to pursue a diagnosis and, if confirmed, recommend endoscopy [8].
CASE REPORT
We present a real case of a 64-year-old patient. His diet was varied and started to
experience tingling in both arms one year before the time of this report, and it went on
gradually increasing. Five months later, he presented difficulty in walking, feelings of
instability and decreased strength in all four limbs, especially the lower limbs. He had been
losing the flavor of foods and, in the last month, presented severe fatigue and rapidly
progressive cognitive impairment, with episodes of agitation, confusion and incoherent
speech. Over the days before going to the hospital, he presented total inability to move the
lower extremities and a significant decrease in upper-limb strength, with numbness in both
the arms and the legs. At the hospital, we observed intense pallor, depressed level of
consciousness (Glasgow 10), slurred speech, blood pressure of 90/50 mmHg and heart rate
115 bpm. The neurological examination showed absence of deep sensitivity (positional and
vibratory), absence of the sense of touch in the lower limbs, motor deficit of 1/5 in the right
leg and 2/5 in the left leg, patellar and Achilles tendon reflexes absent, and left extensor
Babinski reflex present. The upper extremity motor deficit was 3/5, with diminished tactile
sensitivity and deep tendon reflexes. Among the analytical parameters, the following were
observed (normal values are in brackets): hemoglobin: 4.2 g/dl (12-18); hematocrit: 11.1%
(37-52); erythrocytes: 890,000 cell/ml (4,200-6,100); mean corpuscular volume: 115.9 fl (76-
96); reticulocytes: 1.74% (0.5-2); leukocytes: 3,030 cell/mm3 (4,800-10,800), platelets:
63,000 cell/mm3 (130,000-400,000) and indirect bilirubin: 2.12 mg/dl (<0.7). The patient was
admitted to the intensive care unit where four packed red blood cell units were transfused and
fluids were administered until clinical stabilization was achieved. A vitamin B12 test was
requested, and the concentration found was 25 pg/ml (157-1050), while the folate
concentration was 8.1 ng/ml (2.76-18.2). Vitamin B12 treatment was started intramuscularly
at a dose of 1,000 mcg/day. Gastroscopy with biopsy showed chronic superficial gastritis and
severe atrophy with intestinal metaplasia in the antral mucosa. The remaining malabsorption,
serological, radiological and immunological evaluations were negative or normal. After
several days of treatment, bone marrow aspiration showed red and white cell hyperplasia with
significant megaloblastosis, consistent with vitamin B12 deficiency in the patient, in response
to the treatment phase, without other dysplastic signs. Within the first week, the patient’s
cognitive status was fully restored to normal and the vitamin B12 dose was changed to
1,000 mcg/week, intramuscularly. After the first month of treatment and early rehabilitation,
the patient showed improvement in the lower limb motor deficit with recovery to 4/5 bilateral
tactile sensitivity, but without recovering deep sensitivity, he tolerated sitting but not walking.
The paresthesia in the legs had improved substantially.
We continued the intramuscular treatment with 1,000 mcg/month and the patient then
moved to another hospital for further specific rehabilitation treatment.
DISCUSSION [3, 9-11, 13]

We focus on neurological manifestation of cobalamin Deficiency: In the nervous system,
vitamin B12 acts as a coenzyme in the methyl malonyl-CoA mutase reaction, which is
necessary for myelin synthesis.
Neuropsychiatric symptoms may precede hematologic signs and are often the presenting
manifestation of cobalamin deficiency. The neurological syndromes associated with vitamin
B12 deficiency include neuropathy, myelopathy, neuropsychiatric abnormalities and, less
often, optic nerve atrophy.
The spinal cord manifestation, called subacute combined degeneration (SCD) is clinically
characterized by symmetric dysesthesia, disturbance of position sense and spastic paraparesis
or tetraparesis. The involvement of the posterior and lateral columns of the cervical and upper
dorsal parts of the spinal cord is responsible for the impairment of position sense, paraparesis
and tetraparesis. The first abnormality is usually sensory impairment, most often presenting as
distal and symmetrical paraesthesias at lower limbs frequently associated with ataxia. Almost
all patients have loss of vibratory sensation, often associated with diminished proprioception
and cutaneous sensation and Romberg sign. Corticospinal tract involvement is common in the
more advanced cases, with abnormal reflexes, motor impairment and, ultimately, spastic
paraparesis. A minority of patients exhibit mental or psychiatric disturbances or autonomic
signs.
Peripheral neuropathy can be seen in 25% of patients with vitamin B12 deficiency,
pathologic findings reveal axonal degeneration with or without demyelination; 76% are
axonal, while 24% are demyelinating neuropathy. An early diagnosis is critical, since the
response to treatment depends on the extent of involvement and the timing of replacement
therapy.
Optic neuropathy due to vitamin B12 deficiency occurs occasionally in adult patients.
Abnormal visually evoked responses have been reported in patients with pernicious anemia
without visual symptoms, suggesting that there may also be subclinical damage to the visual
pathway. Optic nerve disease is characterized by symmetric, painless and progressive visual
loss. Central and centrocecal scotomas are the main ophthalmologic findings. Clinical
response can be seen during the first three months of treatment.
The typical MRI finding in SCD is a symmetrical abnormally increased T2 signal
intensity, commonly confined to posterior or posterior and lateral columns in the cervical and
thoracic spinal cord. Differential diagnoses of abnormal signal lesions in the posterior
columns of the spinal cord include infectious or postinfectious myelitis, peripheral
neuropathy, lymphoma and other neoplasm, paraneoplastic myelopathy, cervical spondylosis,
radiation myelitis, multiple sclerosis, sarcoidosis, arterial or venous ischaemia, traumatic cord
injury, arterial or venous ischaemia, vascular malformations of the dura and spinal cord, and
syringomyelia, metabolic disease (including vitamin E deficiency) and acute transverse
myelitis. Brain involvement has also been reported in B12 deficiency patients with extensive
areas of a high-intensity signal in the periventricular white matter.
Cobalamin deficiency may present with neuropsychiatric syndromes also in the absence
of hematological signs. Among the psychiatric presentations mood disorders (both depression
and mania), chronic fatigue syndrome and psychosis are notable.
Psychosis may be the presenting symptom in vitamin B12 deficiency. Reported
symptoms include suspiciousness, persecutory or religious delusions, auditory and visual
hallucinations and disorganized thought-processes. An association of vitamin B12 deficiency
and depressive symptoms in elderly patients has been documented. The results obtained by
Permoda-Osip A. [11], show higher homocysteine concentration in considerable proportion of
patients with bipolar depression, especially in men.
They also confirm a connection between high homocysteine concentration and worse
performance in some neuropsychological tests. Such relationship was more marked in men.
Vitamin B12 deficiency has been also associated with attention deficits, acute mental status
and acute cognitive changes, with electroencephalography abnormalities.
The association of vitamin B12 deficiency and cognitive dysfunction has been
extensively documented, and some authors state that it can be linked to mental decline.
Symptoms described include slow mentation, memory impairment, attention deficits and
dementia. There is a linear correlation between serum cobalamin concentration and cognitive
function, both in healthy elderly people and in patients with Alzheimer’s disease. The
available evidence does not allow one to identify a specific profile of cognitive decline,
because of the few published cases with complete neuropsychological assessment.
On a retrospective study on the effects of B12 treatment on neuropsychological function
and disease progression in patients with dementia or cognitive impairment, Eastley R. [12]
found that vitamin B12 treatment improves cognitive impairment, but does not reverse
dementia.
The traditional treatment for vitamin B12 deficiency consists of intramuscular doses of
1,000 mcg/day for a week, then 1,000 mcg/week for a month and, subsequently, 1,000 mcg/
month for life. However, since as early as 1968, oral vitamin B12 has been shown to have an
efficacy equal to that of injections in the treatment of pernicious anemia and other B12
deficiency states. The actual transport mechanism used in this pathway remains unproved, but
vitamin B12 is thought to be absorbed “en masse” in high doses.
Although the daily requirement of vitamin B12 is approximately 2 mcg, the initial oral
replacement dosage consists of a single daily dose of 1,000 to 2,000 mcg. This high dose is
required because of the variable absorption of oral vitamin B12 in doses of 500 mcg or less.
This regimen has been shown to be safe, cost-effective, and well tolerated by patients.
CONCLUSION
Vitamin B12 is a water soluble substance critical for normal functioning of the nervous
system and blood cell formation. Vitamin B12 deficiency may result from pernicious anemia,
gastric resection, intestinal malabsorption or a strict vegan diet. It is often overlooked and
may cause several hematological, gastrointestinal, psychiatric and neurological
manifestations. Megaloblastic anemia is an early hematological sign, but neurological
symptoms may occur also in the absence of hematological abnormalities. SCD, peripheral
neuropathy, neuropsychiatric disorders and optic nerve atrophy are the most common
neurologic manifestations. Physicians should be alert to identifying signs or symptoms of
anemia or suspected vitamin deficiency in populations at risk, even in the absence of
hematological symptoms or signs. The concentration of serum vitamin B12 is not sufficient
for diagnosis, and the metabolites upstream (homocysteine and methylmalonic acid) should
always be looked for. All patients with vitamin B12 deficiency should be investigated to
determine its cause and whether it might be reversible. In all cases, replacement therapy
should be administered. Although oral and parenteral administration appear to be equally
effective in patients with severe neurological symptoms, it is preferable to begin the treatment
intramuscularly to ensure compliance.
REFERENCES
[1] Hoffbrand, A. V. Megaloblastic Anemias. In: Kasper, D. L., Hauser, S. L., Jameson, J.
L., Fauci, A. S., Longo, D. L., Loscazo, J., eds. Harrison´s Principles of Internal
Medicine. Vol. I, 19th ed. New York: McGraw‐Hill. 2015. pp. 640-649.
[2] Langan, R. C., Zawistoski, K. J. Update on vitamin B12 deficiency. Am. Fam.
Physician. 2011;83(12):1425-30.
[3] Oh, R., Brown, D. L. Am. Fam. Physician. 2003;67(5):979-86.
[4] Lee, G. R. Pernicious anemia and other causes of vitamin B12 (cobalamin) deficiency.
In: Lee, G. R. et al., eds. Wintrobe’s Clinical hematology. 10th ed. Baltimore: Williams
and Wilkins, 1999:941-64.
[5] Lindenbaum, J., Healton, E. B., Savage, D. G., Brust, J. C., Garrett, T. J., Podell, E. R.
et al. Neuropsychiatric disorders caused by cobalamin deficiency in the absence of
anemia or macrocytosis. N. Engl. J. Med. 1988;318:1720-8.
[6] Nygard, O., Nordrehaug, J. E., Refsum, H., Ueland, P. M., Farstad, M., Vollset, S. E.
Plasma homocysteine levels and mortality in patients with coronary artery disease. N.
Engl. J. Med. 1997;337:230-6.
[7] Carmel, R. How I treat cobalamin (vitamin B12) deficiency. Blood. 2008;112(6):2214-
2221.
[8] Kokkola, A., Sjöblom, S. M., Haapiainen, R., Sipponen, P., Puolakkainen, P., Järvinen,
H. The risk of gastric carcinoma and carcinoid tumors in patients with pernicious
anaemia. A prospective follow-up study. Scand. J. Gastroenterol. 1998;33(1):88-92.
[9] Briani, C., Dalla Torre, C., Citton, V., Manara, R., Pompanin, S., Binotto, G., Adami, F.
Cobalamin deficiency: clinical picture and radiological findings. Nutrients. 2013 Nov.
15;5(11):4521-39.
[10] Vorgerd, M., Tegenhoff, M., Kuhne, D., Malin, J. P. Spinal MRI. in progressive
myeloneuropathy associated with vitamin E deficiency. Neuroradiology 1996;38 Suppl.
1:111-3.
[11] Permoda-Osip, A., Kisielewski, J., Dorszewska, J., Rybakowski, J. Homocysteine and
cognitive functions in bipolar depression. Psychiatr. Pol. 2014;48:1117-26.
[12] Eastley, R., Wilcock, G. K., Bucks, R. S. Vitamin B12 deficiency in dementia and
cognitive impairment: The effects of treatment on neuropsychological function. Int. J.
Geriatr. Psychiatry 2000, 15, 226-233.
[13] Cabrerizo-García, J. L., Sebastián-Royo, M., Montes, N., Zalba-Etayo, B. Subacute
combined spinal cord degeneration and pancytopenia secondary to severe vitamin B12
deficiency. Sao Paulo Med. J. 2012;130:259-62.
Chapter 10
SEX-STEROID HORMONES AND NEUROPSYCHIATRIC

DISORDERS: PATHOPHYSIOLOGY, SYMPTOMOLOGY
AND TREATMENT
Anna Schroeder*, Michael Notaras* and Rachel A. Hill

Psychoneuroendocrinology Laboratory,
The Florey Institute of Neuroscience and Mental Health,
The University of Melbourne, Victoria, Australia
ABSTRACT
Neuropsychiatric disorders, as a category of illness, arise from complex interactions
that comprise both biological and environmental factors. While progress has been made
in understanding the pathophysiology of various disorders and the molecular genetic
architecture of risk, comparatively speaking, little progress has been made in
understanding the mechanisms underlying sex differences within these disorders. While
sex-steroid hormones are clearly associated with reproductive health and pathology,
emerging evidence reveals that these hormones also have a role in the pathophysiology,
symptomology and treatment of a range of psychiatric illnesses. This chapter will address
the role of sex-steroid hormones in anxiety, affective, eating and psychotic disorders. The
molecular biology of these hormones, with a particular focus on actions of 17ß-estradiol,
and their impact on the clinical phenotype, as well as risks, will be discussed in detail. In
this regard, selective estrogen receptor modulators, which have estrogen-like effects in
the brain, but lower risks for side effects in the periphery, will be evaluated as possible
adjunctive therapeutic treatments of these illnesses, where relevant.
INTRODUCTION
Neuropsychiatric disorders, such as schizophrenia, clinical depression or post-traumatic
stress disorder (PTSD), which often co-occur with anxiety and abnormal eating behaviour are
*
Authors contributed equally to this work / shared first authorship.
158 Anna Schroeder, Michael Notaras and Rachel A. Hill
detrimental and despite enormous research conducted in this field, the treatment options
remain very limited and sometimes without any beneficial outcomes. Even though sex
differences in all these illnesses were recognized as early as the 1880s when Kraeplin coined
the term dementia praecox, now known as schizophrenia (Goldstein, 1997), a disappointingly
low number of clinical and rodent studies have focused on the distinction between male and
female biological systems. Genetic studies do not support X-linkage as the overall pattern of
inheritance, however neuroendocrine disturbances, both in early brain development, puberty
and menopause have been associated with increased onset and severity of symptoms (DeLisi
et al., 1989). Early studies mostly investigated men and male species due to the hormone-
cycling nature of females, which leads to high variability and is time consuming and costly to
control for. Indeed, it has only been in recent years that studies have begun to specifically
outline the differences between male and female mechanisms and their distinct responses to
pharmacological agents as well as environmental factors, such as stress. This is of particular
relevance to the treatment of these disorders, since considerable evidence, as will be
described in this chapter, suggests differential roles of estrogens and androgens in the
aetiology, pathophysiology and symptomatology of psychiatric illnesses. While men
experience more severe symptoms of schizophrenia compared to women, women tend to
suffer more from depressive and anxiety symptoms, eating disorders or post-traumatic stress
disorder (PTSD). Albeit some evidence suggests an involvement of the female sex hormone
progesterone in the pathophysiology of schizophrenia and major depressive disorder (MDD),
the scope of this chapter will concentrate on the effects of the most potent female and male
sex hormones, 17-β estradiol and testosterone, respectively. In order to understand the role of
sex-steroid hormones in these disorders, it is primarily important to understand their
neurobiological mechanism and function in the central nervous system (CNS).
SEX-STEROID HORMONES
Although sex hormones are well established regulators of endocrine and reproductive
systems, they are now extensively studied for their profound actions in the CNS. Indeed,
while estrogens and androgens are primarily produced by the gonads (ovary and testis,
respectively), they are also synthesized in the brain, which is consistent with the strong
expression of estrogen and androgen receptors as well as steroid-synthesizing enzymes
centrally (Grimshaw et al., 2006; Axelsson et al., 2007; Stanic et al., 2014a). Aside from
sexual differentiation and reproduction, sex steroid hormones are inter alia involved in brain
development, mood, cognition, learning and memory. The most potent sex hormones are
testosterone in males and 17β-estradiol in females. Although 17β-estradiol is highly
expressed in females this hormone is also produced at lower levels in males by the enzyme
aromatase, which converts testosterone to estradiol. Testosterone can also be converted via
the enzyme 5-α-reductase to dihydrotestosterone (DHT), a more potent form of androgen that
cannot be converted to estradiol (Heinlein & Chang, 2002). Similarly, ovaries produce much
lower levels of the male hormone testosterone than in males, suggesting that both hormones
in males and females have a functional and sex specific role. Estrogens exert their
physiological effects by binding to their cognate receptors, estrogen receptor alpha (ERα) and
beta (ERβ), which belong to the steroid nuclear receptor superfamily (Mitra, 2003). The
Sex-Steroid Hormones and Neuropsychiatric Disorders 159
binding of ligand to these receptors initiates a series of responses within a target cell that
include the modulation of gene transcription within the nucleus, as well as rapid, non-
genomic effects at the membrane. Testosterone acts in the brain through both androgen
receptors (AR) and estrogen receptors (ERα and ERβ) after conversion to estradiol. In the
human and non-human primate brain, both ARs and ERs are present, with ERα more
commonly expressed than ERβ and found in higher concentrations in areas crucial to learning
and memory, such as the hippocampus, amygdala and prefrontal cortex (Pau et al., 1998;
Perlman et al., 2005), but are also present in the hypothalamus and the limbic system,
indicating a vital importance in neuroendocrine homeostasis as well as mood regulation
(Osterlund & Hurd, 2001). Several major neurotransmitter systems including dopaminergic,
serotonergic, cholinergic and glutamatergic pathways are modulated by estrogens (Bethea et
al., 2002; Cyr et al., 2002; Sanchez et al., 2010). A large number of studies suggest that
estrogens, particularly estradiol, have neuroprotective effects, such as promoting axonal
sprouting (Kadish & van Groen, 2002) and myelination (Crawford et al., 2010). Anti-oxidant
(Goodman et al., 1996) and anti-apoptotic effects (Hill et al., 2004) of estradiol have also
been noted. Similar to estradiol, testosterone has been found to induce neurogenesis (Allen et
al., 2014), protect cells against apoptosis (Nguyen et al., 2010) and modulate hippocampal
synaptic plasticity (Schulz & Korz, 2010). Distinct and important roles of estradiol and
testosterone have been recognized in psychiatric illnesses. While estradiol seems to have
beneficial effects in schizophrenia, testosterone appears to exert beneficial effects on
depressive symptoms. The following sections will focus on the role of estradiol and
testosterone in psychiatric illnesses with a focus on schizophrenia and major depression
disorder (MDD), but will also cover their role in PTSD, anxiety as well as eating disorders.
SEX STEROID HORMONES AND SCHIZOPHRENIA

Schizophrenia
Emil Kraepelin was the first psychiatrist who coined the terms dementia praecox and
manic-depressive insanity in the 1880s, conditions now known as schizophrenia and bipolar
disorder, respectively. In 1911, Eugen Bleuler first used the term “schizophrenia,”
emphasizing that this illness did not necessarily lead to mental decline as opposed to
Kraeplin’s view (reviewed in (Lake & Hurwitz, 2007)). The most recent characterization of
schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-
V) criteria includes a combination of positive symptoms (e.g., hallucinations or delusions),
negative symptoms (e.g., inability to experience pleasure, also known as anhedonia) and
cognitive impairment, mainly affecting working and verbal memory. Green et al. (2004)
reported that 75-80% of schizophrenia patients suffer from cognitive dysfunction, which
precede the onset of psychosis and are the best predictors of functional outcome. With a
lifetime prevalence of 1%, schizophrenia remains one of the most disabling mental illnesses,
with profound consequences for an individual’s personal, social and occupational functioning.
Interestingly, schizophrenia is primarily diagnosed after puberty, a period of sexual-organ
maturation and fertilization associated with highly elevated sex-hormone levels, suggesting
some role for these hormones in the aetiology of this illness. Schizophrenia is a complex
heterogeneous disorder arising from a combination of genetic and environmental insults,

which impedes the investigation and understanding of its molecular mechanisms. Although
considerable research has been conducted to unravel the molecular underpinnings of
schizophrenia, major gaps in our understanding and conflicting results exist. Nevertheless,
converging lines of evidence point to glutamatergic synaptic dysfunction and aberrant
interaction between dopamine and glutamate as major, albeit not unique, structural and
functional players in the pathophysiology of the illness (Schwartz et al., 2012b, 2012a;
Snyder & Gao, 2013). This view is also supported by findings that a number of
genes/proteins, including COMT, dysbindin, DISC-1 and the inhibitory amino acid
transmitter, GABA, which mainly affect the dopaminergic, glutamatergic and inhibitory
signalling have been linked to schizophrenia (Prasad et al., 2010).
The genetic component of schizophrenia is quite substantial, with heritability estimates of
60-80% (McGue et al., 1983; Kremen et al., 2006). Environmental insults, such as stress, are
believed to contribute to schizophrenia in genetically predisposed individuals by disrupting
normal wiring of neurons during development.
While positive symptoms are associated with excessive dopamine release in the midbrain,
negative symptoms are believed to arise from abnormal serotonergic or GABAergic
signalling in brain areas regulating mood, such as the cingulate cortex, ventral hippocampus
or amygdala (Silver, 2004; Howes & Kapur, 2009; Carbon & Correll, 2014; Steeds et al.,
2015). It is widely recognized that cognitive deficits in patients with schizophrenia are
associated with dysfunction of the prefrontal cortex, resulting in difficulties in high-order
information processing in conjunction with problem solving and executive function (Marumo
et al., 2014; Pasternak et al., 2015). A large number of studies suggest that an imbalance of
inhibitory and excitatory firing of neurons in frontal cortex of schizophrenia patients
contributes to cognitive decline. This imbalance in firing has been linked to abnormal
synchronization of oscillatory activity, particularly in the gamma range (30-80 Hz), which is
highly relevant for higher brain function (Uhlhaas et al., 2010; Kikuchi et al., 2011).
Indeed, the fast spiking GABAergic interneurons containing the calcium-binding protein
parvalbumin (PV) were shown to generate gamma oscillations (Sohal et al., 2009) and were
shown to be reduced in number in post-mortem schizophrenia brains (Senitz, 1999; Zhang &
Reynolds, 2002; Konradi et al., 2011). Importantly, disruptions in the synchrony of neuronal
firing in the gamma range were observed in schizophrenia and bipolar patients, but were
absent in patients with affective or personality disorders (Lenz et al., 2011), constituting a
more confined endophenotype. Even though cognitive impairment is a consistent symptom of
schizophrenia, no pharmacological treatment is available to target it. All currently approved
antipsychotic drugs target dopamine D2 receptors and are often effective at treating positive
symptoms, but have little or no impact on negative symptom domains of schizophrenia
(Leucht et al., 2009). However, only 30–45% of patients experience an adequate response to
these drugs (Andreasen et al., 2005; Bertelsen et al., 2009). Despite many years of drug
research, we still require better, more targeted and more affective pharmacological treatment
options for schizophrenia including stage-dependent, sex-dependent and personalized
approaches. Estrogen significantly interacts with dopaminergic, serotonergic and
glutamatergic systems and has neuroprotective and neurotrophic properties (Begemann et al.,
2012). More importantly, it is proposed to play a protective role in schizophrenia, which is
supported by considerable data and may constitute a new target for pharmacological treatment
(Chua et al., 2005; Kulkarni et al., 2008a; Kulkarni et al., 2015; Weickert et al., 2015). The
next section will summarize the most recent insights on the role of estrogen in positive,
negative and cognitive symptom domains of schizophrenia, detailing what we know, what we
should focus on in future studies and new promising therapeutic approaches in the treatment
of this mental illness.
Estrogens and Schizophrenia
The evidence that estrogens play a protective role in schizophrenia stems from gender
differences in the clinical manifestation of this illness. While men display an earlier onset,
poorer premorbid history and larger deficits (Abel et al., 2010), women have a second peak of
onset around post-menopausal ages which coincide with reduced estrogen levels (Haefner,
2003). It has also been noted that premenopausal women experience a more favourable course
with fewer negative symptoms and show better treatment responses than men (Morgan et al.,
2008; Cotton et al., 2009). Population studies reveal that the incidence of schizophrenia is
significantly higher in men, with a 1.4 male to female ratio (Aleman et al., 2003; McGrath et
al., 2008). Reduced plasma-estrogen levels were also reported in schizophrenia patients
(Riecherrossler et al., 1994; Spencer et al., 2008). With regard to the genetic contribution,
polymorphisms in the estrogen receptor-α (ESR1) were linked to schizophrenia and
alterations in ESR1 mRNA levels were detected in schizophrenia brains (Perlman et al., 2004;
Perlman et al., 2005; Weickert et al., 2008). In humans, estrogen modulates emotional
responses (Amin et al., 2005), working memory (Keenan et al., 2001) and frontal cortex
activity (Berman et al., 1997), all of which are altered in schizophrenia.
Given the numerous brain abnormalities found in schizophrenia patients, it is often
proposed that the pathogenesis involves a progressive neurodegenerative component (Iritani,
2007) and estrogens could therefore have a modifying role in the development of
schizophrenia. Furthermore, estrogens influence dopaminergic, serotonergic and
glutamatergic systems, displaying properties similar to those of atypical antipsychotic
medications (Hughes et al., 2009; Kulkarni et al., 2012).
Estrogens in the Positive and Negative Symptom Domains
The potential involvement of estrogens in schizophrenia was recognized a long time ago,
when an association was noticed between phases of the menstrual cycle and changes in
psychopathology (Ripley & Papanicolaou, 1942; Riecher-Rossler, 2002; Haefner, 2003).
More recent studies indicate that the high-estrogen luteal phase is associated with significant
improvements in psychopathology and functioning compared to the low-estrogen follicular
phase (Ko et al., 2006; Bergemann et al., 2007). Adjunctive treatment with transdermal 17β-
estradiol (100 μg per day for 28 days) led to significant improvements in total Positive and
Negative Syndrome Scale (PANSS) scores, as well as cognitive performance (Kulkarni et al.,
2008a). The same group recently demonstrated that estrogen therapy improved recovery from
acute psychotic symptoms by reducing both positive and general anxiety symptoms of
schizophrenia primarily in older females, but also in women of childbearing age (Kulkarni et
al., 2015). Interestingly, two independent studies observed lower circulating 17β-estradiol
levels in acutely psychotic men compared to controls as well as an inverse correlation

between estrogen levels and negative symptoms (Huber et al., 2005; Kaneda & Ohmori,
2005), suggesting its clinical relevance in both sexes. This was supported by findings of
Kulkarni et al. (2011) demonstrating that adjunctive estrogen treatment had no adverse effects
and resulted in quicker improvement in men.
The potential therapeutic utility of estrogens in schizophrenia is increasingly being
recognized, but estrogens alone cannot be given for long periods without inducing certain
risks, such as endometrial hyperplasia and cancer (Lethaby et al., 2004). Raloxifene, a
selective estrogen receptor modulator (SERM), appears to act similarly to estrogens in the
brain, and constitutes a better treatment option, since it lacks the negative effects of estrogen
on breast and uterine tissue. Raloxifene is approved for use as an osteoporosis preventative at
a daily oral dose of 60 mg. In fact, 120 mg per day of adjunctive oral raloxifene for 12 weeks
in postmenopausal women with schizophrenia resulted in significantly greater recovery and
general psychopathology PANSS scores compared to placebo (Kulkarni et al., 2010). In line
with these results, Usall et al. (2011) showed that 60mg per day of raloxifene for 12 weeks in
addition to antipsychotic treatment resulted in a significant improvement of positive and
negative symptoms in schizophrenia patients. These data strongly suggest that the female sex-
hormone 17β-estradiol is involved in regulating the positive as well as the negative symptom
domains of schizophrenia.
The molecular mechanisms by which estrogen interacts with the dopaminergic and
serotonergic systems, which are believed to play a key role in these particular symptom
domains of schizophrenia, is poorly understood. Higher D2 levels in the striatum were
associated with estrogen treatment in ovariectomized (OVX) rats (Sanchez et al., 2010).
Another study found that 17β-estradiol treatment in OVX rats increased mRNA levels of
dopamine receptors and serotonin receptor (2A) in the hypothalamus and midbrain relative to
OVX controls (Zhou et al., 2002). 17β-Estradiol was also shown to upregulate glutamatergic
NMDA receptor levels and change their subunit composition (Adams et al., 2004). Given that
NMDA receptor antagonists, such as MK-801 or PCP induce a psychosis-like phenotype,
estrogens may act as antipsychotics by restoring glutamate signalling through NMDA
receptors. Indeed, 17β-estradiol treatment was able to reverse the psychotic behaviour
induced by the NMDA antagonist, MK-801 in OVX rats (Gogos et al., 2012). 17β-Estradiol
treatment was similarly able to reverse the psychotic phenotype induced by a D2 receptor
agonist and a serotonergic 5-HT1A receptor agonist in OVX rats (Gogos et al., 2010). In
agreement with these data, Arad and Weiner further demonstrated that 17β-estradiol could
reverse an amphetamine-induced psychotic phenotype in both OVX and intact female rats
comparably to clozapine or haloperidol (Arad & Weiner, 2010a, 2010b). Interestingly, 17β-
estradiol could also reverse psychotic behaviour in male rats (Arad & Weiner, 2010b), which
supports the clinical data on its relevance in both sexes. The protective effects of estrogen on
psychotic behaviour may also arise from the maintenance and enhancement of neuronal
mitochondrial function, as mitochondria are responsible for regulating the viability and death
of neurons (Simpkins et al., 2010) and are disrupted in schizophrenia (Rezin et al., 2009).
Estrogens in the Cognitive Symptom Domain
Apart from its effect on psychosis and mood, a large number of studies conducted over
the past twenty years demonstrated that 17β-estradiol affects cognitive functioning in humans
(Janowsky et al., 2000; Lacreuse et al., 2001; Gasbarri et al., 2008) and animals (Warren &
Juraska, 1997; Lacreuse et al., 2001; Pompili et al., 2010). In agreement with these studies
cognitive performance highly correlated with 17β-estradiol levels in schizophrenia patients as
well as in healthy individuals (Hoff et al., 2001; Ko et al., 2006). As mentioned, transdermal
17β-estradiol treatment in schizophrenia patients not only resulted in improvement of positive
and negative symptoms, but also improved cognitive function (Kulkarni et al., 2008b). A
daily dose of 120 mg raloxifene has also been shown to be effective in preserving brain
activity and maintaining cognitive function in older men and women (Yaffe et al., 2005;
Goekoop et al., 2006). More recent studies showed a beneficial effect of adjunctive raloxifene
treatment on cognitive performance in postmenopausal women with schizophrenia (Kulkarni
et al., 2008b; Jacobsen et al., 2010; Huerta-Ramos et al., 2014). Weickert et al. (2015)
recently demonstrated that raloxifene improved cognition in young and middle aged women
as well as men with schizophrenia symptoms.
The influence of estrogens on cognition could be explained by an abundance of
intracellular estrogen receptors (ER) α and β in the hippocampus, a brain region critical for
learning and memory (McEwen & Alves, 1999). GPR30, a membrane-bound receptor that
binds 17β-estradiol (Thomas et al., 2005) is also expressed in the hippocampus and central
amygdala (Hazell et al., 2009) and is involved in hippocampal-dependent spatial memory
(Hammond & Gibbs, 2011). Estrogen signalling has common downstream pathways such as
tropomyosin-related kinase B (TrkB) signaling and the MAPK cascade, which can activate
CREB to drive transcription of brain-derived neurotrophic factor (BDNF) (Scharfman &
MacLusky, 2005). BDNF regulates cell maturation and migration during development as well
as synaptic plasticity, and learning and memory during adulthood (Buckley et al., 2007).
BDNF was shown to be reduced in plasma and brains, particularly in the hippocampus
(Thompson et al., 2011) and dorsolateral prefrontal cortex (DLPFC) (Weickert et al., 2003) of
schizophrenia patients. More specifically, mature BDNF, but not pro-BDNF, was shown to
modulate GABAergic activity and synaptic plasticity by binding to TrkB (Holm et al., 2009).
Several studies suggest BDNF is a key mediator of the enhancing effects of 17β-estradiol
(Aguirre & Baudry, 2009; Hill, 2012; Wu et al., 2013). This hypothesis is based on the
observation that the BDNF gene contains a sequence with close homology to the estrogen
receptor element, indicating that estrogen can directly regulate the expression of BDNF
(Sohrabji et al., 1995). Rodent studies have demonstrated that preadolescent ovariectomy
decreased hippocampal BDNF expression, which was associated with cognitive deficits,
while 17β-estradiol replacement reversed these deficits (Singh et al., 1995; Berchtold et al.,
2001; Solum & Handa, 2002; Wu et al., 2015). The link between BDNF and schizophrenia is
strengthened by studies assessing the Val66Met BDNF polymorphism observed in
schizophrenia patients, which leads to disrupted secretion of BDNF (Chen et al., 2004) and
has been associated with cognitive functioning (Rybakowski, 2008). These data suggest that
estrogen is required to maintain BDNF at normal levels, at least in females, and thus has a
protective effect on cognitive function, which is disrupted in schizophrenia. Wu et al. (2015)
not only demonstrated that estrogen is needed during development to maintain BDNF levels,
but is also required for proper development of PV-containing interneurons. This study
showed that preadolescent OVX in female mice resulted in a significant reduction in PV+
interneurons in the dorsal hippocampus, which correlated with spatial memory deficits.
Importantly, simultaneous 17β-estradiol implantation rescued these deficits. The regulation of
PV+ neurons could be directly mediated by 17β-estradiol as ERα is expressed in these
interneurons (Wu et al., 2014) or via BDNF-TrkB signalling, as PV+ neurons express TrkB
receptors (Hashimoto et al., 2005). As mentioned, this particular cell type is responsible for
generation of neuronal oscillations in the gamma frequency, which in turn is highly relevant
for cognitive functioning. Schizophrenia post-mortem studies revealed abnormal levels of PV
expression (Senitz, 1999; Zhang & Reynolds, 2002; Konradi et al., 2011; Marin, 2012),
which is in agreement with observed gamma-oscillation deficits in schizophrenia patients.
While the ongoing gamma frequency oscillations measured by EEG during resting-state were
increased in schizophrenia patients (Kikuchi et al., 2011), gamma oscillations during the
execution of cognitive tasks were reduced (Uhlhaas et al., 2010). The latter has been
demonstrated for a wide range of cognitive and perceptual paradigms, such as working
memory, executive control and perceptual processing in non-treated and medicated
schizophrenia patients (Uhlhaas & Singer, 2012). Taken together, the clinical studies suggest
that 17β-estradiol plays a major role not only in the pathophysiology of positive and negative
domains but also has a significant impact on the cognitive domain of schizophrenia. Animal
studies conducted to understand the molecular mechanisms reveal that 17β-estradiol,
particularly during development, is needed to maintain the expression of BDNF as well as
PV+ interneurons in the hippocampus, which are required for normal cognitive functioning
and were shown to be disrupted in schizophrenia. A very recent study demonstrated that
depending on their time of birth (appearance) during development, the PV-containing cells
exert a different function (Donato et al., 2015). Hence it is important to understand not only at
what time window 17β-estradiol may affect the development of these interneurons, but also
through which exact mechanism. In addition, it is important to understand the action of
SERMs in respective brain areas involved in the pathophysiology of schizophrenia. This will
not only help to understand the molecular mechanism underlying schizophrenia symptoms,
but will also result in development of more targeted treatment options for all symptom
domains of schizophrenia.
Testosterone in Schizophrenia
While female sex hormones receive enormous attention in schizophrenia research, the
literature on the role of the main male sex hormone, testosterone, in the pathophysiology of
schizophrenia is somewhat controversial. While some studies reported lower testosterone
levels in adult males with schizophrenia compared to healthy controls (Akhondzadeh et al.,
2006; Ruble et al., 2010), more recent studies did not detect this correlation (Moore et al.,
2013; Vercammen et al., 2013; Li et al., 2015). A few studies showed that men with
schizophrenia had increased negative-symptom severity with lower testosterone levels
(Akhondzadeh et al., 2006; Ko et al., 2007) and exogenous testosterone treatment improved
negative symptoms in men with schizophrenia (Ko et al., 2007). Moore et al. (2013), on the
other hand, reported no significant relationship between serum testosterone levels and PANSS
total, positive and negative symptom scores. The same study, however, demonstrated that
serum testosterone levels correlated with verbal and working memory in men with
schizophrenia, which was supported by (Li et al., 2015). Conversely, Hoff et al. (2002)
demonstrated that lower testosterone levels were associated with increased cognitive function
in almost every cognitive domain in male schizophrenia patients. Moreover, while Moore et
al. (2013) could not detect any relationship between testosterone and cognition in healthy
men, Moffat et al. (2002) reported that relatively high levels of testosterone are related to
worse performance on tests of attention and spatial ability in healthy elderly men. Although
testosterone was shown to have protective properties, such as modulating neuronal damage
caused by oxidative stress and reducing neuronal apoptosis (Hammond et al., 2001), the
literature does not provide convincing results for its protective role in the pathophysiology of
schizophrenia. However, since testosterone can be converted to estrogen via the enzyme
aromatase in the male brain, some of the described beneficial effects of testosterone may be
mediated through estrogenic actions. Indeed, as mentioned, lower levels of estrogen were
found in men with schizophrenia (Huber et al., 2005; Kaneda & Ohmori, 2005) and the
selective estrogen receptor modulator, raloxifene not only improved cognition in young and
middle aged women, but also in men with schizophrenia (Weickert et al., 2015).
Administration of raloxifene in older men also delayed age-associated cognitive decline
(Goekoop et al., 2006). Other studies in men with schizophrenia have found associations
between memory and dehydroepiandrosterone (DHEA), a steroid precursor of both
testosterone and estrogen (Halari et al., 2004; Strous et al., 2007; Ritsner & Strous, 2010).
These clinical studies suggest the optimal balance of androgen and estrogen receptor
signalling rather than testosterone signalling per se is involved in the pathophysiology of
schizophrenia in men. The beneficial effects of 17β-estradiol in the male hippocampus were
shown to be particularly dependent upon aromatization of testosterone to 17β- estradiol
during the neonatal period (McEwen et al., 1997).
All this evidence cumulatively suggests that estrogens play a significant and protective
role in this mental disorder and further studies are needed to investigate their molecular
mechanisms in the positive, negative as well as cognitive domains. SERMs appear to
constitute a novel and promising agent for targeting all symptom domains. Understanding
their mechanism of action in CNS will help to develop more targeted treatment options as
well as a better understanding of this mental illness.
SEX STEROID HORMONES AND MAJOR DEPRESSIVE DISORDER

(MDD)
Major Depressive Disorder (MDD)
Major depressive disorder (MDD) is one of the most prevalent neuropsychiatric disorders
occurring twice as often in women than men (Kendler et al., 1993; Breslau et al., 1995; Gater
et al., 1998). Although MDD is primarily considered a mood disorder, depressed patients
commonly experience cognitive impairments that may contribute to their functional disability
(reviewed in (McIntyre et al., 2013)). Indeed, women display an age-independent decline in
verbal recall during the menopause transition indicating that changes in hormone levels affect
cognitive function in addition to mood in women (Epperson et al., 2013). Similar to
schizophrenia, the onset of depressive and anxiety disorders, which are often co-morbid
(Hoyer et al., 2001), peaks during adolescence and early adulthood, further supporting an
involvement of sex hormones in the pathophysiology of these illnesses. Despite a significant
genetic component in MDD (Fava & Kendler, 2000), stress constitutes a major focus in the
basic research on depression (Sterner & Kalynchuk, 2010) and it is believed that a
combination of genetic predisposition and environmental insults lead to the onset of MDD.
At the neurobiological level, stress reactivity is regulated by the hypothalamic-pituitary-
adrenal (HPA) axis via secretion of glucocorticoids. More specifically, the paraventricular
nucleus (PVN) produces corticotrophin releasing hormone (CRH) in response to stress, which
triggers the release of adrenocorticotropic hormone (ACTH) from the adenohypophysis of the
anterior pituitary. ACTH reaches the adrenal glands via the bloodstream and activates the
synthesis and release of cortisol in humans and corticosterone (CORT) in rodents into the
systemic circulation, which enter the brain to bind to their cognate glucocorticoid (GR) and
mineralocorticoid (MR) receptors. This neuroendocrine circuit is consistently hyperactive in
clinical depression (Pariante & Lightman, 2008; Stetler & Miller, 2011). Neurobiological
evidence for this hyperactivity is characterized by increased cortisol secretion, (Deuschle et
al., 1997), enlarged adrenal glands (Rubin et al., 1996) and elevated CRH levels in
cerebrospinal fluid (Nemeroff et al., 1984). The number of CRH neurons was also found to be
increased 4-fold in the PVN of post-mortem brains of depressed patients (Raadsheer et al.,
1994). Interestingly, bidirectional activation of the HPA axis and hypothalamic-pituitary-
gonadal (HPG) axis has been noted (Handa & Weiser, 2014), suggesting sex differences in
regulating the stress response (see discussion below).
The most common antidepressants currently used for the treatment of MDD are the
selective serotonin or serotonin/noradrenaline reuptake inhibitors (SSRIs or SNRIs),
tricyclics, and monoamine oxidase inhibitors (MAOIs). SSRIs or SNRI specifically target the
serotonin or serotonin/norepinephrine transporters whereas tricyclics inhibit norepinephrine
and serotonin reuptake and antagonize many neurotransmitter receptors, which is likely
related to their multiple side effects. MAOIs prevent the degradation of monoamines
(Fernandez-Guasti et al., 2012). Despite the availability of diverse antidepressants, only 50–
70% of patients respond to their first antidepressant treatment (Fava, 2000) and less than 40%
undergo remission (Trivedi et al., 2006). Even after several antidepressant treatments, a large
number of patients remain depressive (Souery et al., 1999; Rush et al., 2006).
Estrogens and MDD
Sex differences in the manifestation of clinical depression have been widely noted. While
women diagnosed with depression report greater symptom severity, comorbid anxiety, more
increased appetite, weight and hypersomnia, men are more likely to report comorbid alcohol
and substance abuse (Marcus et al., 2008). Clinical studies of patients with MDD reveal that
the sex differences arise at adolescence and more closely coincide with androgen and
estrogen levels than physical changes associated with puberty (Angold & Worthman, 1993;
Angold et al., 1999). Several studies have identified that MDD is more likely to occur in
women during menopausal transition than in premenopausal women (Freeman et al., 2006;
Harsh et al., 2009; Freeman, 2010) further supporting the idea that hormone levels in women
are a contributing factor to the incidence of depression. A polymorphism in the ERα gene has
also been associated with MDD (Ryan et al., 2012). Other studies have noted better responses
to SSRIs in women but a better response to tricyclic antidepressants in men and older women
(Kornstein et al., 2000; Khan et al., 2005). The HPG axis, which strongly interacts with the
HPA-axis, regulates the development and function of the gonads and is abnormal in depressed
patients (Baischer et al., 1995; Meller et al., 2001). This evidence suggests that stress
reactivity is highly regulated by sex-hormones and thus may explain the differential
susceptibility to depression in men and women. With regard to hormone-based treatments,
two studies could not detect any beneficial effect of estrogen on depressive symptoms
(Coope, 1981; Pearce et al., 1997). More recent studies, however, proved that estrogen can
mitigate various perimenopause-associated depressive symptoms (de Novaes Soares et al.,
2001). In line with this study, oral, transdermal as well as intranasal administration of adjunct
17β-estradiol treatment had beneficial effects on depression in postmenopausal women
(Baksu et al., 2009; Studd & Panay, 2009) as well as in women after giving birth (Dennis et
al., 2008). In agreement with these studies two double-blind, placebo-controlled studies using
transdermal patches of 17β-estradiol (Smith et al., 1995; Gregoire et al., 1996) and case
studies in which patients were treated with sublingual 17β-estradiol (Ahokas et al., 1999)
reported that estrogen improves mood in women with postpartum depression and severe
premenstrual syndrome.
In order to understand the neurobiological mechanism underlying the effect of gonadal
steroid hormones on depressive behaviour, animal models have been studied. Most animal
models of depression are based on the exposure to various types of acute or chronic stressors.
These paradigms generate changes similar to the symptoms of depression, which can be
reversed by antidepressant treatment (Willner & Mitchell, 2002). Sex differences in terms of
HPA-axis activity are already evident at baseline as female rats have higher resting levels of
CORT and display greater diurnal changes in both ACTH and CORT than males (Handa et
al., 1994). In addition, female rats have higher CORT levels following acute and chronic
stress exposure than males and this seems to be dependent on circulating gonadal hormones
(Seale et al., 2004a; Seale et al., 2004b). Furthermore, OVX in mice resulted in increased
sensitivity to stress-induced depression-like behaviour (Bekku et al., 2006; Nakagawasai et
al., 2009) suggesting a protective role of female sex hormones on depression. Interestingly,
depression-like behaviour correlated with HPA-axis dysregulation, which was normalized by
17β-estradiol treatment in male rats (Ferrini et al., 1999) demonstrating that 17β-estradiol
may have protective effects on depression-like behaviour not only in females but also in
males. 17β-Estradiol was also shown to have anxiolytic effects in animal models. During the
proestrus cycle, when 17β-estradiol levels peak, rats spent more time in the open arms of the
elevated plus maze, more time in social interaction with a conspecific, and less time freezing
in response to shock than did females in other phases of the estrous cycle or male rats
(Fernandez-Guasti et al., 1999). With regard to the antidepressant response, chronic treatment
with fluoxetine (SSRI antidepressant) reduced immobility at the dose of 5 mg/kg in female
rats, while males did not respond to this dose (Dalla et al., 2005a). This observation is in line
with the previously mentioned clinical studies showing that women respond better to SSRI
treatment than men.
How gonadal hormones interfere with the HPA axis has been investigated by a variety of
research groups. The α- and β- ERs have been shown to have opposing actions in the
regulation of the HPA-axis. The ERβ agonist diarylpropionitrile (DPN) infused into the
central nucleus of the amygdala of rats ameliorated anxiety-like behaviour and stress-induced
CORT levels (Weiser et al., 2010). Furthermore, a single acute injection of DPN was able to
reduce depression and anxiety-like behaviours in mice (Walf et al., 2009b). Clark et al. (2012)
demonstrated that two ERβ agonists, the selective oestrogen receptor modulator (SERM)- β1
and SERM-β2, were both able to reduce depression-like behaviour in the forced swim test
(FST) in mice, as well as increase neurogenesis in rats. Conversely, ERα agonists were shown
to have an enhancing effect on CORT and ACTH (Weiser & Handa, 2009). Lund et al. (2005)
similarly demonstrated that rats treated with the ERβ-agonist (DPN) displayed anxiolytic
behaviour, while rats treated with ERα-selective agonist, propyl-pyrazole-triol (PPT)
displayed anxiogenic behaviour. In line with these observations, ERβ-KO mice exhibit
anxiety and depression-like behaviours, as well as reduced serotonin levels in various brain
regions (Krezel et al., 2001; Imwalle et al., 2005b), effects, which were not seen in ERα-KO
mice (Krezel et al., 2001). A direct link between ERβ and the serotonergic system is further
supported by the expression of ERβ in the dorsal raphe nucleus, which contains a high density
of serotoninergic neurons (Mitra et al., 2003).
ERα gene expression level was elevated in the hypothalamus of depressed patients
(Wang et al., 2008) supporting its role in HPA-axis hyperactivity. These data strongly suggest
that SERMs selective for ERβ, but not ERα, decrease depression-like behaviour as well as
anxiety in rodents. Although recent studies demonstrated beneficial effects of raloxifene on
cognitive function in schizophrenia patients, no such studies have been conducted in patients
with MDD. This may be due to the fact that raloxifene appears to act via ERα rather than ERβ
(Rzemieniec et al., 2015), and based on available evidence, it may elicit a negative effect on
depressive symptoms.
The role of brain-derived neurotrophic factor (BDNF) in the pathophysiology of
depression has received a lot of attention during recent decades. Several clinical studies have
shown that serum levels of BDNF are significantly lower in antidepressant-naive depressed
patients compared to treated patients or healthy controls (Aydemir et al., 2005). Soledad
Rojas et al. (2011) demonstrated that an early increase in serum BDNF levels in response to
antidepressant treatment predicts success of the treatment in MDD patients (Soledad Rojas et
al., 2011). BDNF was also shown to promote the release of the excitatory neurotransmitter,
glutamate, and this effect was suppressed by a GR agonist (Numakawa et al., 2009)
suggesting its involvement in HPA-axis activity. In the rat, increased levels of glucocorticoids
or chronic stress have also been shown to reduce BDNF mRNA levels (Smith & Cizza, 1996;
Bravo et al., 2009). This is of particular interest, because the BDNF gene contains a sequence
with close homology to the estrogen receptor element, indicating that estrogen may directly
regulate the expression of BDNF (Sohrabji et al., 1995), which in turn appears to play a
significant role in depression. Hence, estrogens may modulate the HPA-axis and thus stress
reactivity and susceptibility to depression indirectly via BDNF expression.
Testosterone and MDD
As mentioned, the protective role of testosterone in depression becomes evident with the
observation that fewer men suffer from this illness compared to women. Indeed, testosterone
has antidepressant effects both in humans (Howell & Shalet, 2001; Kaminetsky, 2005) and
rodents (Edinger & Frye, 2005). Clinical studies associated low testosterone levels with
symptoms of fatigue, irritability, dysphoria, sexual dysfunction and an increased risk of
depressive symptoms and depression in men as well as in women (Shores et al., 2005;
Almeida et al., 2008; Hintikka et al., 2009). However, other studies could not confirm these
results (Araujo et al., 1998; Amiaz & Seidman, 2008). The role of testosterone in women is
not well established, but there is some evidence that low levels of DHEA (precursor of
androgens and estrogens) are involved in depression in women (Schmidt et al., 2005;
Maninger et al., 2009). Rodent studies demonstrate that while 17β-estradiol treatment
enhances HPA reactivity, testosterone treatment appears to inhibit it (Handa et al., 1994; Viau
& Meaney, 1996). These findings are also supported by several mechanistic studies, showing
that gonadectomy of male rats increased neuronal activity in PVN neurons measured by c-fos
mRNA expression, suggesting increased neuroendocrine responses to stress (Viau et al.,
2003; Lund et al., 2004a). 5α-androstane-3β,17β-diol (3β-diol), which is a metabolite of
testosterone was found to suppress HPA-axis activity via binding specifically to ERβ and it
has been proposed as the mediator of testosterone's antidepressant effects (Lund et al., 2004b;
Pak et al., 2005; Lund et al., 2006a). Seale et al. (2005a) demonstrated that adult female rats
had a larger and longer lasting rise in plasma ACTH following acute stress than males, but
females treated with testosterone at birth displayed ACTH secretion similar to males.
Conversely, prenatal aromatase inhibition in males resulted in ACTH secretion which
resembled female patterns in response to stress (Seale et al., 2005b). Together, these results
indicate that the HPA axis is a target of steroid hormones during development and provide
another layer of complexity to the mechanisms underlying sex differences in the aetiology of
neuropsychiatric disorders.
Anxiety Disorders
As well as being twice as likely to suffer from MDD, females appear to be particularly
susceptible to anxiety disorders. Indeed, by age six, females are already twice as likely to
have suffered from an anxiety disorder than males (Lewinsohn et al., 1998). Anxiety
disorders comprise several distinct diagnoses, including generalized anxiety disorder,
obsessive compulsive disorder (OCD), and panic disorder. There are also various types of
trait and syndrome anxiety such as phobias which often require intervention but may not meet
the diagnostic criteria of an anxiety disorder for a range of reasons. Each of these anxiety-
related conditions, however, maintains their own unique set of symptomology. For instance,
OCD is diagnosed based on intrusive thought obsessions that increase anxiety and by the
presence of behavioural compulsions which can decrease the anxiety (Stein, 2002). Panic
disorder is a psychiatric condition characterized by uncontrollable panic attacks derived from
extreme anxiety (McNally, 1994), while generalized anxiety disorder is characterized by
excessive worry and the persistent expression of anxiety-related traits that do not fit another
anxiety disorder diagnosis (Rowa & Antony, 2008). Females are more likely to suffer from
generalized anxiety and panic disorder (Crowe et al., 1983; Wittchen, 2002), while in OCD
prominent sex differences also exist (e.g., females typically have a later age of onset) (Castle
et al., 1995). These epidemiological data have raised the interest of behavioural
neuroscientists, who have extensively explored a role for sex-steroid hormones in the
modification of anxiety-related behaviour.
Estrogens and Anxiety Disorders
Apart from the behavioural neuroscience literature, there has been only limited studies of
sex-steroid hormones in anxiety disorders (Ryan et al., 2011), much of which have focused on
the genetic variation within the estrogenic signalling system. For instance, anxiety-related
traits in a cohort of 680 healthy adolescents have been shown to be associated with variation
within the gene encoding the ER-α receptor, but only weakly through effects that predict
1.6% to 2.8% of the variability in anxiety (Prichard et al., 2002). Similarly, carrying a ESR1
variant haplotype comprising both rs2234693 and rs9340799 polymorphisms is associated
with anxiety in older women but not men (Tiemeier et al., 2005). A replication study
extended these results by finding that the ESR1 rs2234693 and rs9340799 polymorphisms are
both associated with phobic anxiety, where the G allele of ESR1 rs9340799 was found to
decrease risk of phobic anxiety by 31% in the sample of older women using hormone therapy
(Ryan et al., 2011). This same study also reported an association of variant rs1256049 of
ESR2, the gene which encodes the ER-β receptor, with risk of generalized anxiety disorder
(Ryan et al., 2011). As far as we are aware, only one study has assessed the role of estrogen
receptor variants in OCD. In this study of 229 OCD patients and 279 controls, 20 tag single
nucleotide polymorphisms (SNPs) in ESR2 and 9 tag SNPs in ESR1were assessed, and no
association between genotype or allele frequency was detected (Alonso et al., 2011).
However, the ESR1 rs34535804 polymorphism and a haplotype containing five other ESR1
SNPs was found to be associated with contamination obsessions and cleaning compulsions
(Alonso et al., 2011); a result which suggests symptom-specific modulation by estrogenic
signalling. While interesting, and despite being widely studied in the cancer, neurology and
psychiatry fields, the functional effects of these gene variants on receptor activity remain
unclear and continue to be the subject of debate (Gennari et al., 2005).
Rodent studies have provided considerable evidence for a bottom-up role of estrogens in
the modification of anxiety-related behaviour. ERα knockout female mice display evidence of
increased aggression, fear and anxiety, relative to males (Ogawa et al., 1998). In addition,
ERβ knockout mice display deficient hippocampus-dependent memory (Day et al., 2005) and
are unresponsive to the acute anxiolytic effects of 17β-estradiol or DPN, suggesting that
anxiety-related behaviour may also be mediated through this receptor (Walf et al., 2008).
Interestingly, the anxiolytic effects of high estrogen phases of the estrous cycle – specifically
those seen during pro-estrous – are not observed in female ERβ knockout mice (Walf et al.,
2009a). Of interest, given the reduced synthesis of serotonin in the brain of females relative to
males (Nishizawa et al., 1997), decreased serotonin concentrations have been observed in the
bed nucleus of the stria terminalis, preoptic area and hippocampus in ERβ knockout mice and
may be a ‘molecular mediator’ of these phenotypes (Imwalle et al., 2005a). Apart from
estrogen receptor involvement, and perhaps of relevance to OCD, male aromatase knockout
mice display unaltered anxiety-related exploratory drive (Dalla et al., 2005b), but develop
compulsive behaviour (Hill et al., 2007). Of interest, several reports indicate that estrogenic
compounds mimic the anxiolytic effects of estradiol in rodents. In particular, agonists which
show greater affinity for ERβ (coumestrol or DPN) decrease anxiety in the open field test and
elevated-plus maze when infused into the hippocampus, while agonists with a greater affinity
for ERα (17α-E2 or PPT) were no more effective than vehicle in the modification of anxiety-
related phenotypes in ovariectomized rats (Walf & Frye, 2007). Furthermore, acute injection
of the ERβ modulators DPN or coumestrol also produces anxiolytic effects when
administered subcutaneously up to 48 hours prior to testing (Walf & Frye, 2005). The
commercially available SERM, raloxifene, has also been shown to reduce anxiety-related
behaviour on the elevated-plus maze in ovariectomized rats (Walf & Frye, 2010). It would be
of interest to assess whether the anxiolytic effects of SERMs are also effective in humans
with high trait anxiety, and whether these compounds can reduce symptomology in diagnosed
anxiety-disorder cases.
Androgens and Anxiety Disorders
We could only identify one study that examined the influence of genetic variation within
the androgen receptor gene on anxiety-related traits. Specifically, this study closely examined
the relationship between a CAG microsatellite repeat polymorphism of the androgen receptor
gene, which can range from 8-31 repeats (Walsh et al., 2005) with anxiety levels. Findings
revealed that amongst a sample of older males, the presence of this CAG repeat
polymorphism correlates with higher anxiety levels (Schneider et al., 2011). It was also
determined that cases with higher anxiety, panic and phobic levels were more likely to carry
longer CAG repeats (Schneider et al., 2011). No difference in circulating androgen
concentrations were detected in the subjects in this study. Given that salivary testosterone
levels are reduced in women, but not men, suffering from generalized anxiety disorder, social
phobia and agoraphobia (Giltay et al., 2012), it would be of interest to examine whether this
androgen receptor variant, as well as others, may be associated with anxiety-related disorders
or symptomology in females. Furthermore, it would be of interest to model the functional
effects of this CAG repeat in a cell line or transgenic animal to better understand how this
variant may affect androgen signalling in the brain. Less is known about the effects of
androgens on anxiety, but human studies have implicated low testosterone levels in the
expression of anxiety and disruptive behavioural problems (Granger et al., 2003).
One animal model which has explored the role of androgens in the mediation of anxiety-
related behaviour is male mice carrying a testicular feminization mutation, where a single
nucleotide deletion within the androgen receptor gene alters the reading frame of the gene
which results in attenuated androgen receptor protein expression in males (Zuloaga et al.,
2008). This trait is X-linked, and therefore is particularly relevant for studying the effects of
androgen receptor signalling in males. In particular, these mice have been shown to have an
anxiety-related phenotype in the light-dark box test and do not display anxiolytic effects in
response to testosterone, suggesting a possible AR mediated effect on anxiety (Zuloaga et al.,
2008). Although, in the same study, no other significant anxiety-related phenotypes were
apparent in this model in the large open field or elevated-plus maze tests (Zuloaga et al.,
2008). Given the limited number of behavioural studies which have utilized this model,
further research is required to examine the involvement of androgens in the maintenance of
anxiety-related behavioural phenotypes given the non-concordant results between behavioural
paradigms.
ANXIETY DISORDERS: A FOCUS ON PTSD

Sex Bias of Susceptibility to PTSD
Post-Traumatic Stress Disorder (PTSD) is a stress-related psychiatric disorder that is

characterized by overarching feelings of worry, recurring flashbacks, social avoidance, and
hyperarousal that develop following exposure to a single or series of traumatic events. PTSD
is a disorder often associated with significant lifetime disability, and is associated with an
increased risk of comorbidity (such as suicidal behaviour) that may be exacerbated by social
isolation. The fact that severe stress or trauma exposure is relatively common, but that the
lifetime prevalence of PTSD is believed to be only between 7-12% (Broekman et al., 2007),
suggests innate vulnerability factors must be present for PTSD to develop. Not surprisingly,
PTSD is typically believed to arise via gene-environment interactions, although the often
estimated two-fold higher lifetime prevalence of females relative to males (Breslau, 2001)
also highlights a definitive female-specific vulnerability to this disorder, as well as a potential
endocrine mechanism (Saxe & Wolfe, 1999). As females are more likely to experience
trauma than males (Perkonigg et al., 2000), sex-differences in susceptibility to PTSD at both a
social and biological level are important to identify. An effect of sex-steroid hormones has
become a ‘hot topic’ within the fear and PTSD field, with renewed interest in female-specific
vulnerability leading to many recent discoveries about how sex-steroid hormones affect fear-
related behaviour in both rodents and humans. In this section, the neuroendocrinology of fear
behaviour and the clinical PTSD literature will be reviewed in the context of potential sex-
steroid mechanisms, with an emphasis on the emerging role of ovarian hormones in this
disorder.
Estrogens in Fear and PTSD
Studies assessing the biological underpinnings of PTSD rely heavily, with good reason,
on fear circuitry in modelling the effect of potential PTSD risk factors on the brain. Firstly,
imaging studies have shown that fear circuitry is altered in PTSD, leading to the
conceptualization of PTSD as a disorder of ‘fear inhibition’ (Jovanovic & Ressler, 2010).
Secondly, behavioural measures of fear are also prominently altered in patients (Milad et al.,
2009b). Thirdly, the brain regions involved in the acquisition, maintenance and expression of
fear are largely conserved (or, are at least, maintain analogous functions) in both human and
mouse (Cover et al., 2014). This effectively allows for the translational modelling of PTSD
risk factors, and potentially allows for parallel studies assessing cellular function and
physiology in mice that can be contrasted with human data.
An effect of sex-steroid hormones, particularly of ovarian hormones, is likely but remains
unclear – although research within the past decade has advanced to where the underlying
effects of hormones within the fear circuitry have become more defined. Brain regions
involved in the expression of fear, namely the ventromedial prefrontal cortex and amygdala,
contain both ER-α and ER-β receptors (Zeidan et al., 2011; Cover et al., 2014), while in vivo
biological imaging of estrogen receptor luciferase (ERE-Luc) mice confirms that estrogen-
initiated gene transcription fluctuates over the estrous cycle but peaks during the high-
estrogen proestrus phase within the amygdala (Stell et al., 2008). Interestingly, and further to
these studies, aromatase is also highly expressed within the medial amygdaloid nucleus –
particularly in males (Stanic et al., 2014b). These results support the notion that estrogen is
not only present but is active within these brain regions. Furthermore, these data also support
the observation that stage of estrous/menstrual cycle determines efficacy of extinction
learning - defined as the learned suppression of a fear response to a conditioned stimulus - in
both female mice (Milad et al., 2009a) and humans (Milad et al., 2006). In PTSD patients low
estradiol levels have been shown to predict the degree of conditioned responding during an
extinction learning test (Glover et al., 2012); a result which has since been replicated in
healthy females and extended to reveal that low estradiol is associated with strength of
intrusive memories in daily life (Wegerer et al., 2014). Similarly, the retention of extinction
learning as observed during a post-extinction retention test has been shown to be increased
when extinction learning takes place during high estradiol menstrual phases in healthy female
humans (Zeidan et al., 2011). Interestingly, intra-hippocampal infusion of estradiol following
extinction learning decreases transcriptional activity in the amygdala, but increases it in the
ventromedial prefrontal cortex of rats (Zeidan et al., 2011), which suggests a role for ovarian
hormones in the control of extinction learning consolidation in these brain regions.
The question stemming from these studies is quite simple – if estrogen contributes to fear
reactivity, what receptor mechanism mediates this effect? Does one particular pathway
mediate the effects of estrogenic signalling in the expression of fear? While conflicting
evidence has been reported, there appears to be a strong case for the involvement of ERβ in
many fear-related behaviours. For instance, fear conditioned memory – the process of
conditioning animals to become fearful of neutral stimulus (typically a tone, or a light) and
then assessing that retention of fear over a short exposure period – appears to be particularly
reliant upon ERβ receptor signalling. Specifically, both male and female ERβ knockout mice
display deficient contextual fear conditioning, a form of hippocampus-dependent memory,
which is accompanied by deficits in fast synaptic transmission as well as deficient
hippocampal LTP relative to wild-type controls (Day et al., 2005). Further to this, it has also
been reported that intra-hippocampal infusion of the ERβ agonist DPN, but not the ERα
agonist PPT, is as effective as estradiol in the suppression of fear in ovariectomized rats tested
on a contextual fear extinction paradigm (Chang et al., 2009). It would thus appear that
contextually encoded fear is reliant on ERβ receptor signalling mechanisms, which has been
found to facilitate hippocampal synaptic plasticity and the expression of several synaptic
markers such as PSD-95, GluR1 and synaptophysin (Liu et al., 2008). The physiological
functions of this receptor in other fear-specific brain regions remains poorly defined, but
should be a fruitful avenue of further research.
Potential receptor signalling pathways aside, the exact mechanism which links estrogen
with risk of PTSD remains unknown and, outside those already discussed, few studies have
examined sex-specific risk from an endocrine perspective (Glover et al., 2013). One early
study implicated chronic neuroendocrine dysfunction that is dichotomous between male and
female PTSD probands, finding that females suffering from abuse-related PTSD have
significantly elevated daily levels of norepinephrine, epinephrine, dopamine, and cortisol
relative to those with a history of abuse but no diagnosis of PTSD; a distinct difference to
previously reported high catecholamine levels but relatively normal (or slightly reduced)
adrenocortical activity reported amongst men (Lemieux & Coe, 1995). Despite the strong
evidence implicating ovarian hormones in the expression of fear, other biological evidence
suggesting sex-specific vulnerability to PTSD is largely lacking. One notable exception is the
female-specific risk associated with function and expression of a receptor for pituitary
adenylate cyclase-activating polypeptide, a regulator of the stress response. Specifically, it
was reported that blood concentrations of PACAP correlate with the expression of fear in
female PTSD patients, while a polymorphism (rs2267735) within an estrogen response
element of the PACAPR type 1 gene (ADCYAP1R1) was also found to be associated with risk
and symptom severity of female PTSD patients (Ressler et al., 2011). In a more recent report,
this gene variant was also found to increase amygdala reactivity, but decrease functional
connectivity with the hippocampus, in female PTSD patients (Stevens et al., 2014). The effect
of the ADCYAP1R1 genotype on amygdala reactivity was larger than that of PTSD in this
study. Further research aimed at identifying sex-specific vulnerability to PTSD may help the
development of biomarkers of value for early intervention treatments.
Androgens and Risk of PTSD – A Focus on Combat Veterans
The majority of studies on male PTSD cases focus on combat-related PTSD, and have
implicated a potential role of androgens in the stress-response axis as well as in PTSD. For
instance, there is evidence that circulating testosterone may be decreased when under
psychological stress (Kreuz et al., 1972), as well as when soldiers are anticipating imminent
combat (Rose et al., 1969). Several androgen metabolites, such as DHT and 3β-diol, have also
been shown to suppress the stress response in rats (Lund et al., 2006b). Integrating the role of
androgens in the stress response with risk of PTSD, a recent longitudinal study of 918 male
soldiers found that plasma testosterone concentrations prior to, but not during, deployment
predicted the development of PTSD symptoms 1 and 2 years following deployment for
combat duty (Reijnen et al., 2015). Further, combat-related PTSD inpatients have increased
serum testosterone relative to controls (Mason et al., 1990). A more recent report suggested
that alterations in serum testosterone in PTSD is likely specific to whether other comorbid
disorders are present, finding that soldiers diagnosed with PTSD without comorbid MDD
symptoms had significantly elevated testosterone concentrations (Karlović et al., 2012).
However, analysis of circulating testosterone in untreated combat-related PTSD probands has
failed to produce evidence that plasma testosterone is specific to the pathophysiology of this
form of PTSD prior to treatment (Spivak et al., 2003). It may be that circulating
concentrations are not sufficient to accurately detect a correlation of testosterone with PTSD,
and at the very least these data are not likely to be representative of testosterone and androgen
concentrations within the brain. In support of this, one study found that plasma concentrations
of testosterone were unchanged in their sample, but that there was a decrease in cerebrospinal
fluid concentrations of testosterone in combat-related PTSD cases (Mulchahey et al., 2001).
While the results of these studies remain somewhat ambiguous, they do collectively suggest a
role of androgens in response to stress and possibly an association with combat-related PTSD.
Rodent studies has also indicated that testosterone may enhance contextual fear memory
(Edinger et al., 2004; Agis-Balboa et al., 2009). However, it remains unclear whether an
effect of testosterone occurs via androgen receptor signalling or if its cleavage by aromatase
then results in the activation of an estrogen receptor signalling pathway.
The above studies assessing the role of androgens in PTSD are not without their
limitations. Given that PTSD in males is commonly studied in war veterans, these studies
cannot disambiguate a potential long-term effect of combat on neuroendocrine reactivity that

may confound the correlation of circulating androgen concentrations with PTSD diagnosis.
For instance, a positive testosterone-cortisol coupling has been observed in veterans with
‘dangerous combat’ experience (Bobadilla et al., 2014). Interestingly, this effect was recently
‘replicated’ in a cohort of incarcerated male adolescents, where it was reported that this HPA-
HPG coupling of testosterone, DHEA and cortisol was ‘tighter’ amongst those with a history
of life adversity (Dismukes et al., 2014). These results suggest long-term effects of adversity
and combat experience on the expression of circulating androgens during laboratory-
simulated stress, which may result in the conflated analysis of androgen hormones in
correlational studies of combat-exposed veterans which assess an association with PTSD and
related diagnoses. Future studies should therefore consider the use of a covariate-based
analysis to help control for these long-term effects, which may increase the capacity to detect
subtle interaction factors. Further, the molecular genetic risk of androgen receptor gene
variants in combat-related PTSD cases remains relatively understudied. It may be that gene
variants within the androgen receptor are not associated with PTSD, and that risk may be in
other gene variants further up- or down-stream of sex steroids. One notable example
illustrating this effect is the association of a gene variant (rs523349) in SRD5A2, a gene
encoding 5-α-reductase type 2 – an enzyme involved in the conversion of testosterone to
DHT, with risk and symptom severity of PTSD amongst males (Gillespie et al., 2013).
Further studies may yet provide evidence that male-specific genetic risk factors exist within
sex-steroid hormone systems.
Eating Disorders
Eating disorders (EDs) are characterized by extreme body dysmorphia, anxiety about
food intake and, in acute cases, low body mass index (BMI), and are a leading source of
morbidity amongst adolescent and young adult women (Fairburn & Harrison, 2003). Females
are more susceptible than males to this class of disorder, with only 5-10% of cases being
observed in males (Garner, 1993). This epidemiological statistic highlights that social factors,
such as body dissatisfaction and the representation of beauty in the media, plays a strong role
in susceptibility to EDs, but that biological factors are also likely to play a role in their
pathogenesis. EDs are heterogeneous in their clinical presentation, and while they can be
grouped into specific types based on the avoidance behaviours displayed it is also possible for
patients to ‘migrate’ between diagnostic categories (Fairburn & Harrison, 2003). Common
diagnoses include Anorexia Nervosa, which is often characterized by severe caloric
restriction and/or excessive exercising, and Bulimia Nervosa, which is associated with
chronic binge eating and purging. In high-risk populations, caloric intake and BMI can also
be monitored as mitigating risk factors. EDs can present heterogeneously and with varying
severity, and are believed to be amongst the most lethal of psychiatric disorders (with
Anorexia Nervosa holding the highest mortality rate of all disorders due to the cumulative
effects of starvation (Klump et al., 2009) and suicide (Arcelus et al., 2011)). In acute phases
of illness, acute hospitalization is often required due to risk of medical complication as a
result of malnourishment as well as risk of refeeding syndrome, a condition associated with
electrolyte shifts, abnormal glucose metabolism, hypophosphatemia, hypomagnesaemia and
hypokalaemia which occurs when malnourished patients undergo rapid ‘refeeding’;
a condition further associated with significant risk of mortality within ED populations (Crook
et al., 2001). In this respect, EDs are a particularly severe class of psychiatric disorders
characterized by widespread physiological dysfunction, including that of endocrine systems.
As endocrine abnormalities have been predominantly studied in Anorexia Nervosa probands,
it will be the focus of the following review.
Estrogens and Eating Disorders
The frequency of cases in females relative to males provides an enigmatic but important
clue to the cause of these disorders, and potentially implicates a role of sex-steroid hormones.
Such a theory is feasible given that females are ten times more likely than males to develop
Anorexia Nervosa (AN) (Procopio & Marriott, 2007), and that endocrine abnormalities are a
major component of EDs (Lawson & Klibanski, 2008). For instance, amenorrhea (the
irregular absence of menstruation) is a core component of restrictive EDs such as Anorexia
Nervosa (Gendall et al., 2006), and is believed to arise from decreased frequency and
amplitude of gonadotropin-releasing hormone secretion in the hypothalamus (Reame et al.,
1985). While still the topic of debate, changes in the expression of many hormones are
believed to be due to the effects of starvation and are not necessarily associated with the
pathophysiology of EDs per se, even though some alterations may fail to recover following
recovery and subsequent weight gain (Lawson & Klibanski, 2008). Lower serum
concentrations of estradiol have been observed amongst female Anorexia Nervosa patients
(Ohwada et al., 2007), although reports have been inconsistent (Newman & Halmi, 1989).
While a contribution of sex-steroid hormones in the pathophysiology of EDs and not just
starvation alone remains unclear but likely, the administration of exogenous sex-steroid
hormones does appear to hold at least some therapeutic merit. Most of these applications are
focused on using exogenous sex-steroid hormones as a potential treatment for the low bone
density and osteoporosis (DiVasta et al., 2012) which often develops in restrictive disorders
as a result of starvation, sex-steroid hormone deficiencies (Ohwada et al., 2007) and
hypercortisolemia (Newman & Halmi, 1989). Conflicting results have been published as to
the efficacy of sex-steroid derived treatments for correction of bone mineral density in EDs,
with estrogen-based treatments often not reporting statistical evidence to support their
efficacy (Klibanski et al., 1995; Golden et al., 2002; Munoz et al., 2002). It is also believed
that genetics play an important role in risk of EDs (Klump et al., 2009), but the molecular
genetic architecture of EDs remain relatively understudied compared to other psychiatric
conditions. Given the sex difference in frequency of EDs, variation within genes encoding for
sex-steroid receptors represent ideal gene candidates for screening. Early studies implicated
that the ERβ receptor gene G1082A polymorphism may be associated with AN (Rosenkranz
et al., 1998), a result which was replicated four years later (Eastwood et al., 2002). Likewise,
several ERβ gene variants have also been independently associated with Bulimia Nervosa and
the ‘Not Otherwise Specified’ diagnosis, including one rare ERβ variant (A661G) which
changes the primary structure of the ERβ protein (Nilsson et al., 2004). Based on these early
studies it was argued that there was no evidence that variation within ESR1, the gene which
encodes ERα receptors, was associated with EDs, and that variation within ESR2, the gene
which encodes ERβ, was more likely to be a candidate gene of interest in risk of EDs
(Eastwood et al., 2002). However, a more recent study of ERα receptor variants found
significant over-transmission of the rs726281 and rs2295193 variants in Anorexia Nervosa,

which were also associated with the restrictive type of Anorexia Nervosa (Versini et al.,
2010). In this same study, the ERα variant rs3798577 was also associated with EDs amongst a
cohort of 693 women (Versini et al., 2010). One further report has also provided evidence that
the ERα rs9340799 variant may also predict risk of bone mineral density in Anorexia Nervosa
patients (Stergioti et al., 2013), but it remains unclear whether this variant affects response to
ovarian hormone treatments to treat osteoporosis in AN patients. While cumulatively
suggestive of a genetic role of sex-steroid hormones in risk of EDs, particularly Anorexia
Nervosa, due to the limited number of studies which have examined variants within the ESR1
and ESR2 genes, further study is warranted within larger cohorts to assess replication, effect
size and transmission of these gene variants within EDs.
Other genes, however, have also emerged from genetic association studies that are known
to interact with sex-steroid hormones, and therefore cannot be ruled out as potential
phenotype determinants. These include variants within the serotonergic system, and BDNF
(Monteleone & Maj, 2008). Serotonin has widespread functions in the brain, but is best
known for its effects on mood. In the case of EDs, it appears that variants within the 5HT-2A
receptor gene may mediate several ED-related phenotypes (Monteleone & Maj, 2008),
although inconsistent results necessitate further study. Likewise, BDNF also has widespread
functionality in the brain and is also known to modulate many endocrine factors within the
hypothalamus, as well as control food-seeking behaviour. For instance, BDNF heterozygote
knockout mice develop metabolic abnormalities such as insulin resistance (Duan et al., 2003)
and hyperphagia (Lyons et al., 1999). While these preliminary studies of BDNF are
suggestive of a phenotype that may be of greater relevance to binge eating or obesity, genetic
association studies of the relatively common BDNF Val66Met (rs6265) polymorphism have
reported consistent evidence of association with body fat percentage and low BMI (Hong et
al., 2012), Anorexia Nervosa (Ribases et al., 2003) and Bulimia Nervosa (Ribasés et al.,
2004). Promisingly, many of these reports derive from studies comprising large samples (e.g.,
for BMI analysis, n = 20,270) and replicate the results of several smaller studies of more
homogenous patient groups as recently reviewed (Notaras et al., 2015). Given the strong
replication track record for a genomic role of BDNF in EDs, it would be of benefit for future
genetic association studies to consider sex-steroid receptor variants as potential interaction
factors.
Androgens and Eating Disorders
Decreased serum DHEAS has also been observed in Anorexia Nervosa patients who were
concurrently receiving oral contraceptives (Miller et al., 2007), while decreased serum
testosterone has also been observed amongst male and female Anorexia Nervosa patients
(Misra et al., 2003). Interestingly, deficient testosterone levels have been reported to recover
to levels consistent with healthy controls following weight gain (Andersen et al., 1982). These
reports therefore appear to suggest that these abnormalities in circulating sex-steroid hormone
concentrations in EDs such as Anorexia Nervosa are likely the result of starvation as
previously discussed. Further, it also remains unclear if these alterations reflect reduced
adrenal or sex organ output (Lawson & Klibanski, 2008). Although, nonetheless, these data
do not rule out an effect of sex-steroid hormones during critical periods of development,
which may shape or determine phenotypes or risk of disordered eating in adulthood. In

support of this hypothesis, there is evidence that sex-steroid hormone exposure in utero may
predispose to disordered eating amongst females (Culbert et al., 2008) and to disorders such
as Anorexia Nervosa (Procopio & Marriott, 2007) in adulthood. In particular, it would appear
that, in utero, testosterone may protect against discorded eating in adulthood (Culbert et al.,
2008). Fewer reports have examined a role of sex-steroid hormones as a potential treatment or
adjunctive treatment for the cognitive, mood and related symptoms in EDs, however those
which have done so have reported promising preliminary data. One early report found that
short-term, low-dose, transdermal testosterone administration improved depressive
symptoms, spatial memory and several bone formation markers in Anorexia Nervosa
(Andersen et al., 1982), while another found that low-dose testosterone therapy could recover
hypofunction of the posterior cingulate cortex (Miller et al., 2004). Further research which
assesses the effects of sex-steroid hormones as potential adjunct treatments for comorbid
symptoms of EDs in the long-term would not only be insightful, but also timely given these
preliminary reports.
General Discussion
This chapter has focused on the role of sex-steroid hormones in several classes of
psychiatric illness, with a particular emphasis on those disorders with an established or
emerging role of sex-steroid hormones in their pathophysiology or potential treatment.
Particular emphasis was placed on reviewing the role of estrogen and androgens in
schizophrenia, as an emerging role of sex-steroid hormones in the pathophysiology and
treatment of this disorder is currently a hot topic for research. Specifically, several reports
now implicate that SERMs may hold therapeutic potential for the treatment of cognitive and
negative symptoms of the disorder. However, only a limited number of studies have been
conducted, and it remains unknown whether genetic factors within target receptor genes (e.g.,
ESR1 and ESR2) may determine responses to these therapeutic agents. In this respect, further
double-blind studies comprising larger cohorts (ideally, multi-center in origin) which also
collect genetic data would be useful in determining the efficacy of SERMs in the treatment of
schizophrenia.
Unfortunately, largely due to a lack of studies on the topic, it is unclear whether a similar
treatment approach can be adapted to other disorders. As far as we are aware, the effects of
SERMs as potential adjunctive treatments have not been examined in most other disorders. In
MDD, it appears although SERMs which target ERα make symptoms worse, those which
target ERβ have had more success. It is clear that without better resolving the action of sex-
steroid hormones in this disorder, further study is required to deduce whether SERMs
represent viable therapeutic agents within mood disorders such as MDD. In anxiety disorders
such as PTSD, an effect of SERMs has not been assessed. While it is well established that
circulating ovarian hormones affect the consolidation of extinction learning, putatively via
ERβ receptors, further study is required to disambiguate the effect of sex-steroid hormones on
the onset and maintenance of symptomology in both males and females. While low estradiol
levels have been suggested as a vulnerability factor for the disorder (Lebron-Milad et al.,
2012), this evidence derives from a limited number of experimental reports and requires
further investigation. Furthermore, a challenge persists to improve our understanding of a role
of sex-steroid hormones in PTSD, as studies assessing differences in androgens and ovarian

hormones often do not sample or compare both sexes. This limitation is compounded by the
divergent sampling of forms, type and sources of trauma that may predispose to PTSD
between males and females, with much of the male literature focusing on combat-related
PTSD, while the female literature focuses on abuse and maltreatment. Further study, utilizing
larger samples and more heterogeneous patient groups, is therefore required before a
conclusion can be made about the potential therapeutic use of sex-steroid hormones in anxiety
disorders such as PTSD. Likewise, a role for sex-steroid hormones in EDs such as Anorexia
Nervosa also remains a possibility, however clinical assessments of hormone levels remain
confounded by the effects of starvation on endocrine homeostasis, while only a handful of
association studies have targeted gene candidates of relevance to sex-steroid signalling.
Furthermore, like PTSD, a major limitation in our understanding of biological risk factors for
EDs, of all forms, also derives from the relatively few studies focused on male-specific
mechanisms. In this respect, the action of both estrogenic, androgenic and related sex-
hormone signalling within the brain of those at high risk or with a current ED diagnosis
remains unclear, and should be the topic of further research.
In closing, sex-steroid hormones represent a likely component of the pathophysiology of
a range of psychiatric conditions. Despite females being more susceptible to many types of
disorder, few studies have investigated possible mechanisms and even fewer have focused
specifically on understanding a role of sex hormones. SERMs represent an emerging and
potentially viable form of treatment for several symptom domains across multiple disorders,
but unfortunately only a handful of clinical studies have investigated their efficacy. It is
therefore important that future studies continue to test novel sex-steroid compounds as
possible adjunctive treatments. Likewise, it is important that researchers continue to derive a
better understanding of the signalling mechanisms of sex hormones in psychiatric illness so
that new therapeutics can be developed and applied appropriately.
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This chapter has been reviewed by
A/Pr Andrew L. Gundlach

Neuropeptides and Behavioural Neuroscience Divisions
The Florey Institute of Neuroscience and Mental Health
30 Royal Parade, Parkville, Victoria 3052, Australia
and
Florey Department of Neuroscience and Mental Health
Department of Anatomy and Neuroscience
The University of Melbourne, Victoria 3010, Australia
<andrew.gundlach@florey.edu.au> OR <andrewlg@unimelb.edu.au
INDEX
amenorrhea, 176, 185, 192

A amino, 6, 9, 20, 69, 71, 102, 160
amino acid(s), 6, 9, 20, 69, 71, 160
acetic acid, 141, 143
amnesia, 107
acetylcholine, 11, 86, 139
amphetamines, 107
acetylcholinesterase, 80, 87
amplitude, 4, 5, 32, 34, 35, 37, 38, 94, 176
acid, ix, xi, 6, 17, 83, 85, 86, 95, 102, 137, 140, 141,
amygdala, 61, 62, 72, 159, 160, 163, 167, 172, 174,
150, 151, 152, 155, 197
194, 199, 201
action potential, 64, 89, 90, 99
amyloid beta, 186
acupoints, vii, 1, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14
amyloid deposits, 66
acupuncture, vii, 1, 3, 4, 10, 12, 13, 14, 15, 16, 17,
amyotrophic lateral sclerosis (ALS), 75, 86
18, 19, 20
analgesic, 3, 6, 10, 11, 12, 14, 15, 18, 184
acute stress, 169
anatomy, 60, 63, 133
adaptation, ix, 101
androgen(s), 158, 165, 166, 169, 171, 174, 175, 178,
adenosine, 6
179, 186, 187, 190, 191, 194, 198, 202
adhesion, 126, 127, 129, 134
anemia, xi, 149, 151, 152, 154, 155
adjunctive therapy, 47, 189
angiogenesis, 26
adolescent development, 201
anorexia, 180, 181, 184, 185, 189, 190, 192, 193,
adolescents, 170, 175, 183, 184, 190, 193, 194
194, 195, 198, 200
adrenal gland(s), 139, 166
anorexia nervosa, 181, 192, 193, 195
adrenoceptors, 7
antagonism, 131
adrenocorticotropic hormone (ACTH), 166, 167,
anterograde amnesia, 67
168, 169
antibody, 151
adults, viii, 21, 56, 105, 107, 122, 134, 150, 191
anti-cancer, 98
adverse effects, 162
anticancer activity, 84
adverse event, viii, 22, 28, 41, 42
anticonvulsant, 2
aetiology, 158, 159, 169
antidepressant(s), 86, 166, 167, 168, 181, 182, 185,
age, x, 27, 28, 31, 34, 52, 65, 72, 75, 77, 78, 106,
188, 198
110, 111, 114, 117, 118, 119, 120, 121, 124, 145,
antigen, 134
161, 165, 169, 189, 195, 197
anti-inflammatory drugs, 2
aggregation, 132
antioxidant, 85, 87, 88
aggression, 170
antipsychotic, 160, 161, 162, 190
aggressiveness, 191
antipsychotic drugs, 160, 190
aglycones, ix, 83, 85, 95
antitumor, 88
agonist, 141, 162, 167, 168, 173
anxiety, vii, xi, 55, 157, 159, 161, 165, 166, 167,
agoraphobia, 171
168, 169, 170, 171, 175, 178, 182, 183, 184, 185,
alcoholics, 151
188, 189, 190, 191, 192, 194, 196, 199, 200, 201,
allele, 170
202
alters, 111, 132, 171, 182, 197
204 Index
anxiety disorder, 165, 169, 170, 178, 185, 190, 196, biomarkers, 14, 174
199, 201 biopsy, 153
aphasia, 70, 72, 77 biosynthesis, vii, xi, 137, 139, 140, 141, 144, 145,
apoptosis, 81, 128, 129, 134, 159, 187 146, 147
appetite, 166 bipolar disorder, 159
apraxia, 68, 70, 72 bleeding, 190
argyrophilic grain disease (AGD), ix, 59, 65, 66, 67, blindness, vii, x, 113, 114, 115, 119, 120, 121, 122
68, 73, 77 blood, vii, x, 23, 25, 27, 35, 36, 37, 42, 44, 45, 53,
ARs, 159 57, 84, 88, 104, 125, 126, 130, 131, 132, 133,
Asia, ix, 83, 84, 190 134, 135, 138, 150, 152, 155, 174, 181, 195
aspartate, 6, 9, 85, 92, 102, 103, 179 blood flow, 25, 27, 35, 36, 37, 42, 44, 45, 53, 57
assessment, 28, 36, 39, 46, 55, 79, 107, 115, 116, blood pressure, 126, 152
121, 154, 192 blood-brain-barrier (BBB), vii, x, 125, 126, 127, 128,
astrocytes, 9, 68, 126, 129, 134 129, 130, 131, 132, 133, 134, 138
ataxia, 69, 70, 71, 153 bloodstream, 138, 166
atmospheric pressure, 97 body dissatisfaction, 175
ATP, 6, 9, 26 body fat, 177
atrophy, ix, xi, 59, 66, 67, 68, 70, 71, 72, 75, 76, 78, body mass index (BMI), 175, 177, 187
79, 80, 81, 149, 153, 155 bone, 18, 23, 153, 176, 178, 185, 189, 193
auditory cortex, 119, 121, 123, 124 bone cancer, 18
auditory evoked potentials, 124 bone form, 178
auditory stimuli, 121 bone marrow aspiration, 153
authentication, 96 bottom-up, 170
autoimmune disease, 151 bowel, xi, 45, 149, 151
autonomic nervous system, 19 Braille, 120, 124
autopsy, 81 brain, vii, ix, x, xi, xii, 1, 59, 60, 62, 65, 66, 70, 71,
avoidance, 172, 175, 184 72, 73, 74, 79, 81, 86, 87, 88, 89, 94, 98, 101,
axonal degeneration, 153 102, 103, 105, 106, 107, 108, 110, 111, 115, 120,
axons, 62, 90, 103 121, 125, 126, 127, 128, 129, 130, 131, 132, 134,
135, 137, 138, 140, 143, 144, 145, 146, 147, 157,
158, 160, 161, 162, 163, 165, 166, 168, 170, 171,
B 172, 173, 174, 177, 179, 181, 182, 183, 184, 185,
186, 187, 191, 192, 195, 197, 198, 200, 201, 202
bacteria, xi, 149, 150
brain abnormalities, 161
bacterial infection, 151
brain activity, 163, 183
bacterial pathogens, 57
brain damage, 103
basal forebrain, 186
brain functions, ix, xi, 59, 60, 137, 143
basal ganglia, 68
brain stem, 65, 71, 73, 105
behavioral change, 68, 72
brain tumor, 130, 131, 135
behavioral models, 185
brainstem, 99
behavioral sensitization, 96
branching, 90, 103
behaviors, vii, xi, 3, 9, 16, 137, 138, 141, 191, 193,
breakdown, 23, 66, 129, 151
202
breast cancer, 130, 199
beneficial effect, viii, ix, 22, 43, 44, 45, 47, 83, 84,
bulimia, 193, 195
86, 88, 95, 128, 129, 159, 163, 165, 167, 168
benefits, 24, 47, 57, 129
benzodiazepine, 138 C
bias, 28, 44, 46
biceps femoris, 45 Ca2+, 7, 85, 99, 146
bilateral, 33, 37, 39, 67, 80, 115, 124, 153 calcium, 128, 130, 134, 135, 160
bile, 150 caloric intake, 175
bioelectricity, 26 caloric restriction, 111, 175
biological activity(s), ix, 83, 84 cancer, 19, 134, 152, 162, 170
biological systems, 158 capillary, 23, 132
Index 205
carcinogen, 200 cochlear implant, x, 113, 114, 115, 116, 121, 122,
carcinoid tumor, 156 123, 124
carcinoma, 156 coding, 46
cardiovascular system, 88 coenzyme, 153
case study(s), 39, 56, 57, 167 cognition, 69, 158, 163, 165, 180, 181, 186, 187,
CBD, ix, 59, 65, 66, 67, 68, 72, 73 188, 191
cDNA, 17 cognitive ability, 87, 189
cell biology, 126 cognitive deficit(s), ix, 59, 160, 163
cell body, 71 cognitive domains, 165
cell culture, 132 cognitive dysfunction, 74, 145, 154, 159
cell fate, 106 cognitive function, 65, 139, 147, 154, 156, 163, 165,
cell line(s), 10, 16, 20, 127, 128, 129, 132, 140, 171 168, 186, 189, 190, 192
central nervous system (CNS), vii, ix, x, 2, 4, 7, 16, cognitive impairment, 107, 114, 121, 152, 154, 156,
77, 79, 83, 84, 85, 87, 88, 89, 92, 95, 97, 98, 102, 159, 160, 165, 201
103, 108, 125, 128, 130, 138, 139, 140, 146, 158, cognitive performance, 161, 163, 186
165, 182, 188, 191, 196 cognitive skills, 64
cerebellum, 71, 73, 146 cognitive system, 64
cerebral blood flow, 78 cognitive tasks, 164
cerebral cortex, 60, 61, 63, 68, 71, 72, 75, 79, 104 cognitive therapy, 108
cerebral edema, 126, 134 collagen, 26
cerebral palsy, 121 colorectal cancer, 130
cerebrospinal fluid, 166, 174 combination therapy, 129
cervical spondylosis, 154 communication, 114, 119, 121, 124
challenges, 22, 47 communication skills, 114
chemical, 14, 43, 84, 85, 87, 88, 97, 104 community, 55, 108, 150, 194
chemical reactions, 104 comorbidity, 172, 194
chemical structures, 85 complex interactions, xi, 157
chemokines, 129 compliance, 14, 55, 117, 155
children, x, 113, 114, 115, 116, 117, 118, 119, 120, complications, 22, 41, 52, 115
121, 122, 123, 124, 194 composition, 66, 84, 162
China, 85 compounds, ix, 83, 84, 139, 170, 179, 182
Chinese medicine, 84, 97 compulsive behavior, 187
cholesterol, 138, 140, 146 computed tomography, 102, 105
chronic fatigue syndrome, 154 conception, 105
circulation, 65, 133, 150, 166 conditioned stimulus, 173
citalopram, 199 conditioning, 12, 36, 173, 183, 192
classes, 119, 151, 178 conductivity, 129
classification, 78 configuration, 43
claudin-5, 126, 127, 129, 130, 132, 133 conflict, 145
cleavage, 138, 146, 174 conflict of interest, 145
clinical application, 97, 109 connectivity, 62, 120, 174
clinical assessment, 179 consciousness, 152
clinical depression, 157, 166 consensus, 27, 46, 76, 78, 180
clinical diagnosis, 80 consent, 117
clinical neurophysiology, 115 consolidation, 60, 103, 173, 178
clinical presentation, 175 contamination, 170
clinical symptoms, 68, 70 continuous data, viii, 22
clinical syndrome, 70, 71 contraceptives, 177
clinical trials, vii, viii, 1, 13, 22, 44, 54, 55, 188 control condition, 91, 92, 93
closure, 32 control group, viii, x, 22, 29, 31, 32, 33, 40, 41, 43,
clozapine, 162 44, 47, 113, 116, 117
cobalamin, xi, 149, 150, 153, 154, 155 controlled studies, 29, 40, 167
controlled trials, 27, 40, 43, 47, 55
206 Index
controversial, 64, 67, 164 dementia with Lewy bodies (DLB), ix, 59, 65, 66,
coronary artery disease, 155 67, 69, 71, 72, 73, 74, 78, 80
corpus callosum, 61 demyelination, 73, 152, 153
correlation, 75, 81, 154, 162, 164, 174, 175 dendrites, 62, 90, 92, 103
cortex, 60, 61, 64, 67, 68, 71, 72, 80, 90, 103, 104, dephosphorylation, 133
105, 107, 108, 109, 119, 126, 138, 160, 173, 178, depolarization, 7, 12, 17, 91
188 deposition, 66, 74
cortical neurons, 182 deposits, ix, 59, 65, 67, 69, 70, 73, 74
corticobasal degeneration, ix, 59, 65, 67, 76, 77, 78, depression, 5, 12, 18, 19, 102, 103, 110, 139, 145,
79, 81 152, 154, 156, 166, 167, 168, 180, 182, 184, 185,
corticotropin, 193, 194 186, 189, 191, 195, 196, 198, 199, 200, 201
cortisol, 166, 173, 175, 193 depressive symptoms, 154, 159, 167, 168, 178, 180
Creutzfeldt-Jakob disease, ix, 59, 65, 66, 67, 74, 75, deprivation, x, 114, 115, 116, 117, 118, 119, 120,
81 121, 122, 134
critical analysis, 192 derivatives, 88, 96
critical period, 103, 107, 177 dermis, 23
Cross-Modal Plasticity, 113, 122, 123 destruction, 63
CSF, 193 detection, 28, 64, 97, 103, 116, 185
Cuba, 113, 117 developing brain, 106
culture, 139, 142, 143 dexamethasone, vii, x, xi, 125, 126, 127, 128, 129,
culture media, 142 130, 131, 134, 135, 137, 139, 147
culture medium, 139, 142, 143 Diabetes, 15, 180
cures, vii, ix, 83, 84, 87, 95 diabetic neuropathy, 13, 14, 15
cycling, 158, 185 Diagnostic and Statistical Manual of Mental
cyclooxygenase, 17, 18 Disorders, 159
cytochrome, 138, 140, 146 diagnostic criteria, 75, 169
cytokines, vii, viii, 2, 4, 9, 10, 128 diarrhea, 151
cytoplasm, 65, 69, 71 diastolic blood pressure, 25
cytoskeleton, 126, 128 diet, xi, 149, 150, 151, 152, 155
diffusion, 120, 194
dimerization, 126
D disability, 107, 121, 122, 165, 172
discharges, 10, 12
DART, 99
discrimination, 98, 116, 121
data analysis, 29
disease progression, 154
data gathering, 65
diseases, 11, 65, 75, 84, 95
database, 26, 27, 190
disorder, 66, 68, 69, 70, 72, 73, 80, 146, 151, 158,
deaf-blind children, x, 113, 114, 115, 116, 117, 118,
159, 160, 165, 169, 171, 172, 175, 178, 179, 182,
119, 120, 121, 122, 123
184, 185, 188, 190, 191, 192, 194, 195, 196, 198
deaf-blindness, vii, x, 113, 114, 115, 119, 120, 121,
distribution, vii, x, 5, 25, 34, 45, 52, 54, 56, 57, 60,
122
69, 70, 71, 72, 74, 79, 94, 95, 113, 114, 115, 116,
debridement, 24
117, 118, 119, 120, 122, 133, 179
declarative memory, 60
diversity, 3, 96, 97
decubitus ulcer, 27, 54
DNA, 26, 55, 65, 66, 126, 149
deficiency(s), xi, 13, 147, 149, 150, 151, 152, 153,
donors, 79
154, 155, 156, 176, 187
dopamine, 86, 88, 89, 106, 160, 162, 173
deficit, ix, xi, 83, 149, 152
dopaminergic, 107, 159, 160, 161, 162, 187
deformation, 23
dorsal horn, vii, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16,
degenerative dementia, 76
18, 20
degradation, 97, 129, 134, 166
dorsolateral prefrontal cortex, 163
delusions, 154, 159
dosage, 155
dementia, ix, xi, 59, 60, 65, 66, 67, 68, 69, 70, 71,
Down syndrome, 79
72, 73, 74, 75, 77, 78, 79, 80, 81, 139, 149, 152,
down-regulation, 4, 11, 130, 144
154, 156, 158, 159, 201
Index 207
drainage, 23 environmental stimuli, 108

dressings, 23, 24, 55 enzyme(s), xi, 86, 137, 138, 143, 144, 146, 147, 150,
drug abuse, 88 158, 165, 175
drugs, 14, 98, 100, 107, 137, 160 epidemiologic, 180
duodenum, 150 epidemiology, 182, 185
dyspepsia, 151 epidermis, 23, 25
dysphoria, 168 epinephrine, 173
epithelial cells, 133
epithelium, 89
E equilibrium, 104
erythrocytes, 152
eating disorders, 158, 159, 181, 183, 189, 192
EST, 31
ECM, 128
estrogen, xii, 127, 133, 157, 158, 161, 162, 163, 165,
edema, 104, 127, 129, 130, 131
166, 168, 170, 172, 173, 174, 176, 178, 180, 181,
EEG, 164
182, 183, 185, 186, 187, 188, 189, 190, 191, 192,
elderly population, 199
193, 194, 195, 196, 197, 198, 199, 200, 201
electric current, 32
ethyl acetate, 139
electric field, 54, 57
etiology, 13, 194
electrical fields, 25
evidence, viii, x, xi, 2, 4, 5, 6, 8, 10, 12, 14, 16, 21,
electrical resistance, 127
25, 26, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 41,
electrical stimulation, vii, 1, 16, 22, 24, 25, 26, 27,
44, 45, 46, 47, 54, 55, 57, 71, 75, 84, 92, 109,
32, 36, 38, 42, 52, 53, 54, 55, 56, 57, 120
114, 115, 119, 137, 140, 151, 152, 154, 157, 158,
electroacupuncture, vii, viii, 2, 15, 16, 17, 18, 19, 20
160, 161, 165, 166, 167, 168, 169, 170, 173, 174,
electrodes, viii, 22, 25, 31, 32, 33, 34, 35, 36, 37, 38,
175, 176, 177, 178, 181, 190, 193
39, 40, 42, 43, 45, 115
evoked potential, vii, x, 5, 113, 114, 115, 118, 122,
electroencephalography, 154
123
electrolyte, 175
evolution, 133
electromagnetic, 56
excitability, 2, 88, 89, 91, 92, 96, 104, 108
electromagnetic fields, 56
excitation, 44, 94, 103
emission, 80, 102, 105, 181
excitatory synapses, 103
emotional experience, 2
excitotoxicity, 186
emotional responses, 161
executive function, 160
emotional state, 122
exercise, 25, 36, 57, 181
encephalomyelitis, 128, 134
exercise program, 57
encoding, 146, 170, 175, 176, 198
experimental autoimmune encephalomyelitis (EAE),
encouragement, 145
128, 134
endocrine, viii, 2, 158, 172, 173, 176, 177, 179
experimental design, 119
endocrine system, viii, 2, 176
explicit knowledge, 109
endocrinology, 130, 134, 193
exposure, 128, 132, 140, 144, 147, 167, 172, 173,
endometrial hyperplasia, 162, 190
178, 183, 199
endoscopy, 152
extensor, 152
endothelial cells, 126, 127, 129, 130, 131, 132, 134,
external validity, 28
135
extinction, 173, 178, 182, 185, 192, 201
endothelium, 133, 134
extraction, 29
endotoxemia, 19
extravasation, 135
energy, 107
exudate, 24
engineering, 55
enlargement, 71, 139
entorhinal cortex (EC), vii, viii, ix, 59, 60, 61, 62, F
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
77, 78, 188 facial expression, 185
environment(s), 64, 107, 109, 111, 119, 121, 122, families, 114
129, 130, 172, 194 family physician, 187
environmental factors, xi, 157, 158 far right, 118
208 Index
fascia, 61, 81 gene expression, xi, 11, 17, 104, 126, 133, 137, 139,
fat, xi, 23, 137, 141, 200 141, 142, 144, 145, 147, 168, 193, 202
fear, 170, 172, 173, 174, 179, 182, 183, 184, 185, gene targeting, 133
188, 192, 199, 201 generalized anxiety disorder, 169, 170, 171, 187
fear response, 173, 179, 199 genes, x, 11, 18, 105, 106, 125, 126, 127, 128, 132,
feces, 150 140, 141, 142, 143, 144, 146, 147, 160, 176, 177,
feedback inhibition, 90 178, 184, 194
feelings, 152, 172 genetic background, 182
female rat, 110, 111, 161, 162, 167, 169, 185, 186, genetic factors, 69, 105, 178
197, 201, 202 genetic predisposition, 166
fertilization, 159 genetics, 176, 185, 188, 194
fiber(s), vii, 1, 4, 5, 7, 10, 12, 15, 16, 18, 61, 62, 63, genotype, 77, 170, 174
90, 103 genus, 84, 98
fibroblast growth factor, 110 Germany, 30, 33, 125
fibroblast proliferation, 44 gestation, 105
fibroblasts, 54, 55 ginseng, ix, 83, 84, 85, 86, 87, 88, 95, 96, 97, 98, 99,
filament, ix, 59, 65, 66, 67, 71, 75, 76 100
flashbacks, 172 ginsenosides, vii, ix, 83, 84, 85, 86, 87, 88, 89, 95,
flexibility, 45 97, 98, 99
fluid, 193 gland, 196
fluoxetine, 167 glia, 19, 20, 109, 110
folate, 153 glial cells, 8, 9, 65, 138, 140
folic acid, 152 glioma, xi, 137, 141, 144, 147
food, 149, 151, 175, 177 globus, 73
Food and Drug Administration, 16 glucocorticoid(s), x, xi, 106, 107, 110, 125, 126, 128,
food intake, 175 130, 131, 132, 133, 134, 137, 138, 139, 143, 144,
force, 20, 44 146, 147, 166, 168, 184, 194, 198, 199, 201
forebrain, 143, 193 glucocorticoid receptor, x, 125, 126, 130, 131, 132,
formation, 23, 25, 54, 60, 61, 62, 63, 65, 67, 68, 70, 134, 138, 146, 184, 198, 201
71, 72, 74, 77, 103, 128, 129, 155 glucose, 134, 175
foundations, 196 glutamate, 6, 8, 89, 90, 91, 92, 93, 94, 95, 96, 99,
France, 109 103, 106, 139, 160, 162, 168, 193
freezing, 167 gluteus maximus, 34, 35, 37, 45
frontal cortex, 73, 160, 161, 194 glycoproteins, 103
frontal lobe, 71, 73, 78 gonadotropin-releasing hormone, 176
fruits, 98, 150 gonads, 158, 167
Functional Magnetic Resonance Imaging (fMRI), gray matter, 61, 72
105, 115, 185 growth, 74, 102, 106, 110, 130
fungi, xi, 149 growth factor, 106, 110
guidelines, viii, 21, 24, 26, 27, 28, 78
G
H
GABA, 6, 8, 14, 19, 86, 89, 91, 92, 95, 96, 139, 160
gadolinium, 128, 133 hallucinations, 69, 154, 159
gait, 68 hamstring, 33, 35, 39, 45, 57
gamma globulin, 19 head injury, 74, 78, 134
ganglion, 6, 7, 12, 16, 20 head trauma, 74
gastrectomy, 151 healing, viii, 21, 22, 24, 25, 28, 29, 31, 32, 33, 39,
gastrin, 152 40, 43, 44, 47, 53, 54, 55, 57
gastrinoma, 151 health, xi, 20, 23, 26, 28, 33, 45, 46, 47, 52, 55, 114,
gastritis, 151, 153 121, 131, 147, 157, 184, 185
gel, 188 health care, 55, 185
gender differences, 161, 180 health condition, 114
Index 209
hearing impairment, 124 ileum, 150

hearing loss, 116, 121 imbalances, 200
heart rate, 152 immune activation, 131
heat shock protein, 126, 130 immune response, 11
hematocrit, 152 immune system, vii, 2, 4, 20, 87, 126
hematology, 155 immunomodulation, 100
hemoglobin, 56, 152 immunomodulatory, 85
herbal medicine, 99 immunoprecipitation, 127
heritability, 160, 188 immunoreactivity, 8, 9, 78, 184, 193
heterogeneity, 29, 40, 75, 80 impairments, 88
heterozygote, 177 implants, 45, 122, 123, 124, 194
high-risk populations, 175 improvements, 161
hippocampal formation, 60, 61, 62, 63, 65, 67, 68, impulses, 5
70, 71, 72, 77 in situ hybridization, 146
hippocampal neural activity, vii, xi, 137, 138, 141 in utero, 178
hippocampus, ix, 59, 60, 64, 66, 67, 74, 76, 79, 80, in vitro, x, 54, 79, 86, 87, 88, 95, 97, 125, 129, 130,
81, 87, 96, 99, 101, 105, 106, 110, 111, 138, 159, 131, 132, 134
160, 163, 165, 170, 173, 174, 179, 181, 184, 194, in vivo, x, 47, 86, 88, 89, 94, 95, 98, 125, 130, 172
196, 198, 199, 200, 201, 202 indirect bilirubin, 153
histamine, 151 individual differences, 186
HIV, 13, 55 individuality, 121
HIV/AIDS, 55 individualization, 3
homeostasis, ix, x, 101, 108, 125, 159, 179 individuals, 13, 22, 23, 24, 41, 53, 54, 56, 66, 108,
homocysteine, 150, 152, 154, 155 150, 160, 163, 199
hormone(s), vii, viii, xi, 2, 111, 127, 137, 145, 147, induction, 6, 103, 127, 140, 141, 142, 144, 146, 179
157, 158, 159, 162, 164, 165, 166, 167, 169, 170, infants, 124
172, 173, 175, 176, 177, 178, 179, 180, 181, 183, infection, 24
184, 186, 187, 192, 194 inflammation, 9, 10, 17, 19, 88, 128, 138
hormone levels, 159, 165, 166, 179, 180 inflammatory mediators, 11
hospitalization, 175 inflammatory responses, 131
human body, x, 25, 125, 126 information processing, 67, 120, 160
human brain, 74, 127, 134, 193 ingredients, 85, 86, 95, 99
human health, 108, 146 inheritance, 158
hydrocortisone, 128, 131, 132 inhibition, 10, 12, 16, 18, 19, 60, 63, 64, 65, 89, 95,
hydrolysis, 99 97, 98, 103, 106, 111, 119, 131, 133, 147, 169,
hyperactivity, 166, 168 172, 188
hyperarousal, 172 inhibitor, 127, 129, 132, 197
hyperplasia, 153 initiation, 9, 65
hypersensitivity, 9, 17, 18, 20, 104 injections, 154
hypersomnia, 166 injur(ies), viii, 3, 4, 9, 10, 15, 16, 17, 18, 19, 20, 21,
hypophosphatemia, 175 22, 23, 25, 26, 30, 37, 38, 43, 52, 53, 56, 99, 105,
hypothalamo-pituitary-adrenal axis (HPA axis), 138, 108, 129, 132, 154, 186
166, 167, 169, 186, 194, 197 insanity, 159
hypothalamus, 110, 159, 162, 168, 176, 177, 183, insertion, 115
190, 191, 200, 201 insulin resistance, 177, 184
hypoxia, 196 integration, ix, 59, 60, 123
integrins, 103
integrity, 127, 128, 129, 131, 133
I intensive care unit, 153
intercellular adhesion molecule (ICAM), 128
IASP, 2
interface, 25, 34, 36, 37, 38, 44, 45, 52, 54, 56, 60,
iatrogenic, 69
63, 126
IDA, 7
internal validity, 28
identification, 97, 99, 116, 121, 195
210 Index
interneurons, 88, 89, 160, 163, 189, 201 locomotor, ix, 83, 88, 89, 94, 95, 99
intervention, x, 13, 27, 28, 29, 34, 37, 42, 45, 47, locus, 62, 65
101, 107, 108, 169, 174 longitudinal study, 52, 174, 189, 194
intestinal malabsorption, 155 LTD, 5, 6, 12, 103
intestine, 150 luciferase, 172
intracellular calcium, 103 lung cancer, 130
intracranial pressure, 134 lymphocytes, 198
ion channels, 2, 6, 85, 88 lymphoma, 154
ion transport, 25
ionization, 97
ions, 25 M
ipsilateral, 9
macrocytosis, 155
irritability, 152, 168
macrophages, 9, 19
ischemia, x, 23, 125
magnetic resonance imaging (MRI), 34, 102, 105,
isolation, 172
120, 128, 133, 154, 156, 194
isozymes, 143
magnetoencephalography, 102, 105
magnitude, 2, 8
J major depression, 159, 188, 194, 196
major depressive disorder, 158, 192
Japan, 81, 116, 137, 145 malabsorption, 151, 153
jejunum, 150 malignancy, 13
Jordan, 185, 202 maltreatment, 179
management, 14, 19, 23, 45, 46, 53, 56, 131, 134
mania, 154
K manic, 159, 197
manipulation, 14, 87
K+, 85
mass, 88, 97, 99, 200
keratinocyte(s), 44, 57
mass spectrometry, 88, 97, 99
kidney, 4, 16
matrix, 8, 17, 19, 128, 133
matrix metalloproteinase, 8, 19, 133
L measurement(s), 27, 29, 35, 80, 152, 199
media, 175
laminar, 62, 95 median, vii, x, 70, 113, 117, 119, 120
language development, 116 mediation, 17, 171
language processing, 120 medical, 52, 84, 151, 175, 194, 197
latency, 117, 121, 122, 123, 124 Medicare, 81
LC-MS, 88 medicine, ix, x, 14, 52, 83, 84, 98, 125, 131, 134,
learning, ix, 83, 84, 87, 88, 95, 96, 102, 103, 104, 181, 190, 195, 196, 199
107, 108, 111, 158, 163, 173, 178, 200, 201 MEG, 102, 105, 120
learning process, 107 melanoma, 130
left hemisphere, 104, 120 melatonin, 129, 134
legs, 152 memory, ix, 59, 60, 63, 64, 65, 67, 68, 76, 80, 83, 84,
lesions, 66, 68, 70, 71, 72, 76, 80, 81, 128, 133, 134, 87, 88, 95, 96, 97, 99, 103, 104, 108, 146, 152,
154, 188 154, 158, 159, 163, 165, 170, 173, 174, 183, 185,
leucocyte(s), 129, 152 188, 190, 192, 194, 197, 201
life sciences, 134 memory loss, 68
lifetime, 159, 172, 188 memory performance, 88, 192, 197
ligand, 6, 17, 85, 126, 150, 159, 183 menopause, 158, 165, 184
light, ix, 79, 97, 101, 107, 108, 171, 173 menstruation, 176
light scattering, 97 mental disorder, 165
limbic system, 159 mental health, 195
liquid chromatography, 88, 97, 100 mental illness, 159, 161, 165, 189, 190, 194
localization, 14, 93 mental impairment, 69
Index 211
messenger ribonucleic acid (mRNAs), 9, 127, 139, multiple sclerosis, x, 86, 125, 128, 129, 132, 133,
144, 146, 161, 162, 168, 169, 181, 182, 194, 198, 134, 137, 154
199, 201 multipotent, 105
meta-analysis, viii, 22, 29, 40, 47, 54, 180, 181, 190 muscarinic receptor, 11
metabolism, 86, 126, 130, 143, 147, 175 muscle atrophy, 24
metabolites, 85, 152, 155, 174, 184 muscle contraction, 33, 44, 46
metalloproteinase, 132 muscles, 25, 33, 34, 35, 36, 38, 39, 42, 45, 46, 56, 57
metastasis, 134 mutation, 171, 195, 202
methamphetamine, 88, 99 myelin, 71, 153
methodology, 14, 46, 121, 122, 150 myocardium, 147
methylprednisolone, 129, 134 myoclonus, 68
mice, 7, 16, 87, 88, 89, 94, 96, 97, 98, 99, 111, 126, mythology, 46
127, 129, 132, 146, 147, 164, 167, 168, 170, 171,
172, 173, 177, 179, 181, 183, 184, 187, 188, 189,
191, 193, 200 N
microcirculation, 23, 52
Na+, 85, 89
micrograms, 150
National Academy of Sciences, 181, 189, 191, 193,
microorganisms, 149
198, 199
midbrain, 73, 160, 162
National Health and Nutrition Examination Survey,
migration, 26, 44, 54, 102, 133, 163
150
military, 182, 195
National Institutes of Health, 3, 16
mineralocorticoid, 126, 138, 166
necrosis, 23
Ming dynasty, 84
negative effects, 162
Ministry of Education, 145
neocortex, 79
mitochondria, 138, 146, 162
neoplasm, 154
mitogen, 11, 16, 17, 19
nerve, vii, viii, x, 3, 4, 5, 9, 10, 12, 13, 15, 16, 17, 18,
MMP(s), 8, 9, 127, 128
20, 22, 27, 36, 45, 56, 88, 102, 103, 113, 117,
MMP-2, 8, 9
119, 120, 154
MMP-9, 8, 9
nerve growth factor, 102, 103
model system, 52
nervous system, 2, 8, 66, 83, 102, 133, 138, 145,
modelling, 172
146, 153, 155
models, vii, 2, 3, 4, 8, 9, 16, 17, 25, 44, 47, 86, 87,
Netherlands, 30
98, 106, 127, 128, 130, 133, 145, 167, 198, 201
neural connection, 103
molecular biology, xi, 157
neural network(s), 63, 104, 119
molecular pathology, 80
neural system, 181
molecules, xi, 125, 126, 127, 134, 140
neurobiology, 96, 108, 188
monoamine oxidase inhibitors, 166
neurodegeneration, 66
monolayer, 127
neurodegenerative disease(s), vii, ix, 59, 60, 65, 74,
mood disorder, 147, 154, 165, 178, 196, 199
76, 83, 84, 86, 87, 95, 145
morbidity, 175, 197
neurodegenerative disorders, ix, 59, 60, 65, 67, 71,
morphine, 18, 88, 96
73, 87, 98
morphogenesis, 105
neurofibrillary tangles, 65, 74, 78, 79, 80
morphology, 60, 75, 79, 121
neurogenesis, vii, ix, 87, 101, 105, 106, 107, 108,
morphometric, 78
109, 110, 111, 159, 168
mortality, 72, 155, 175
neuroglial cells, vii, 2, 4
mortality rate, 175
neuroimaging, 102, 105
motor activity, 65
neuroinflammation, 8, 87, 100, 133, 146, 194
motor control, 104, 108
neurological disease, 80, 84
motor neuron disease, 70, 78
neurological disorders, vii, ix, xi, 83, 84, 86, 87, 95,
motor task, 104
98, 134, 149
movement disorders, 80
neurologist, 107
mucosa, 105, 150, 153
neuromodulation, vii, viii, ix, 2, 101
multiple regression analysis, 195
neuronal apoptosis, 165
212 Index
neuronal cells, 144, 194 nucleus, 61, 65, 68, 71, 72, 73, 126, 159, 166, 167,
neuronal circuits, 88 170, 173, 187, 191, 194, 200, 201
neuronal density, 87 nursing care, 31
neuronal electrical stimulation, vii, 1 nutrients, 23
neuronal ensembles, vii, 1 nutrition, 24
neuronal intermediate filament inclusion disease
(NIFID), ix, 59, 65, 66, 67, 71, 73, 75, 76
neuronal systems, 4 O
neurons, vii, ix, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16,
obesity, 177
17, 18, 19, 20, 60, 61, 64, 65, 68, 71, 72, 73, 74,
obsessive-compulsive disorder (OCD), 169, 170, 180
78, 79, 88, 89, 90, 92, 93, 94, 95, 97, 99, 101,
Occipital lobe, 120
103, 104, 106, 108, 110, 111, 126, 138, 160, 162,
occipital regions, 120
164, 166, 168, 169, 186, 187, 190, 191, 194, 198,
occludin, 126, 127, 128, 129, 130, 132
200, 202
ocotillol, ix, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
neuropathic pain, vii, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
94, 95, 96, 97, 99
13, 14, 15, 16, 17, 18, 19, 20
oedema, 26
neuropathy, xi, 13, 16, 19, 149, 153, 154
olanzapine, 199
neuropeptide hormones, vii, viii, 2
old age, 111
neuroplastic changes, vii, x, 107, 113, 115, 120, 122
oleanolic acid, ix, 83, 85, 86, 95
neuroplasticity, vii, ix, 101, 102, 103, 104, 105, 107,
olfactory nerve, 89, 90, 92, 98
108, 109, 111, 114, 115, 120, 124
oligodendrocytes, 68, 71, 126
neuroprotection, 179, 182, 186, 197
omeprazole, 151
neuroprotective agents, 86
opioids, 2, 7, 17
neuropsychological tests, 154
optic nerve, xi, 149, 153, 155
neuroscience, vii, 131, 134, 170, 180, 183, 190, 192,
organ, 55, 159, 177
200, 201
oscillation, 164
neurosecretory, 182
oscillatory activity, 160
neuroses, 190
osteoporosis, 162, 176, 185
neurosteroid biosynthesis, vii, xi, 137, 139, 140, 141,
outpatients, 196
144, 145, 146, 147
ovariectomy, 163, 181, 197
neurotoxicity, 88, 99
ovaries, 158
neurotransmission, 69, 71, 86, 139
overlap, 74, 75
neurotransmitter(s), 2, 6, 8, 20, 86, 104, 106, 139,
oxidative stress, 86, 165
146, 159, 166, 168
oxygen, 23, 24, 37, 38, 44, 52, 54, 56, 134
neurotrophic factors, 104
neutral, 173
neutral stimulus, 173 P
New Zealand, 182, 190, 193
nicotine, 85 Pacific, 53, 190
Nigeria, 30, 31 paclitaxel, 6, 7, 16, 18
nitric oxide, 8, 9, 15, 88, 96 pain, vii, 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
nitric oxide synthase, 8, 15 16, 18, 19, 20, 46, 47
NMDA receptors, 9, 92, 99, 162 pain management, 14
nociceptive activation, vii, 1, 12 palliative, 55
noradrenergic system, 106 pallor, 152
norepinephrine, 110, 166, 173 pancytopenia, vii, xi, 149, 151, 156
normal development, 121 panic attack, 169
North America, 85, 97 panic disorder, 169, 183
novel stimuli, 65 parallel, 104, 172
NPC, 105, 106 paralysis, 22
NSAIDs, 2 parasites, 151
nuclear receptors, 145 parenchyma, 110, 126, 129, 130
nuclei, 62, 65, 71, 182 parents, 117, 119
Index 213
paresthesias, 152 polypeptide, 174

parkinsonism, 68, 69, 70, 71 population, viii, 21, 24, 26, 27, 35, 43, 47, 66, 69, 70,
participants, 28, 32, 36, 37, 40, 41, 42, 44 79, 90, 106, 151
parvalbumin, 160, 201, 202 positive macrophages, 129
pathogenesis, viii, 2, 12, 14, 65, 133, 161, 175, 183 positron, 102, 105, 181
pathology, ix, xi, 59, 60, 65, 66, 67, 68, 71, 73, 74, positron emission tomography (PET), 102, 105, 120
75, 76, 77, 78, 79, 133, 157 post-extinction, 173
pathophysiology, xi, 157, 158, 160, 164, 165, 166, postpartum depression, 167, 179, 183
168, 174, 176, 178, 179 posttraumatic stress, 146, 182, 185, 188, 190, 192,
pathway(s), vii, x, 1, 2, 4, 5, 8, 11, 12, 14, 23, 61, 62, 195, 196, 198
63, 67, 68, 69, 70, 71, 73, 104, 105, 107, 120, post-traumatic stress disorder (PTSD), 157, 159, 172,
125, 133, 138, 154, 159, 163, 173, 174 173, 174, 178, 182, 188, 191, 193
PCP, 162 potassium, 25, 130, 135
PCR, 139, 141, 200 prefrontal cortex, 62, 65, 96, 159, 160, 172, 191, 201
pelvic floor, 45 premenstrual syndrome, 167
pelvis, 31 preparation, 45
peptic ulcer disease, 151 pressure gradient, 34, 35, 37
peptide, 7, 17, 186 pressure sore, 23, 27, 52, 53, 54, 56, 57
perceptual processing, 164 pressure ulcer, vii, viii, 21, 22, 23, 24, 25, 26, 33, 44,
perforant path, ix, 59, 60, 65, 67, 68, 72, 73 52, 53, 54, 55, 57
perfusion, 36, 44 prevention, vii, viii, 16, 21, 22, 24, 26, 27, 30, 42,
pericytes, 126 44, 45, 47, 52, 53, 54, 56, 57, 130, 134, 201
peripheral nervous system, 138 prevention and treatment, vii, viii, 21, 22, 24, 26, 27,
peripheral neuropathy, 15, 18, 20, 152, 154, 155 47, 52, 53
permeability, 88, 127, 130, 131, 133, 135 primary brain tumor, 130
pernicious anemia, 151, 152, 154, 155 primary visual cortex, 61, 124
personality, 68, 160 primate, 16, 159
personality disorder, 160 probands, 173, 174, 176, 195
phantom limb pain, 13 probe, viii, 22, 33, 39, 52
pharmaceutical, 133 problem solving, 160
pharmacological agents, 158 professionals, 26, 47, 52, 108
pharmacological treatment, 2, 160 progenitor cell(s), 87, 105, 106, 109, 147
pharmacology, 97, 182, 188, 200, 201 progesterone, xi, 137, 138, 140, 141, 142, 158
phenotype(s), xi, 71, 78, 157, 162, 170, 171, 177, progestins, 183
178, 197 prognosis, 130
phobic anxiety, 170 progressive supranuclear palsy (PSP), ix, 59, 65, 66,
phosphorylation, 7, 9, 133 67, 68, 73, 75, 76, 77, 78, 79, 80
physical therapy, 107 pro-inflammatory, 9, 10, 128
physiology, 132, 133, 172 prolactin, 180
phytomedicine, 97 proliferation, 87, 106, 107, 110, 111, 200
pineal gland, 129 promoter, 127, 132, 133
placebo, 13, 54, 134, 162, 167, 183, 188, 194, 195, propagation, 76
199 prophylaxis, 56
plasma levels, 4 propranolol, 7
plasma membrane, 126 prostaglandins, 9
plasticity, x, 18, 20, 101, 103, 104, 105, 107, 108, proteasome, 129, 133
109, 111, 114, 115, 122, 123, 124, 163 protective factors, 106
platelets, 152 protective role, 160, 161, 165, 167, 168
PM, 54, 57, 123 protein kinases, 19, 104
polarity, 44, 47, 57 proteins, ix, x, xi, 4, 11, 60, 65, 69, 71, 72, 74, 85,
polymerase chain reaction, 146 88, 89, 99, 103, 125, 126, 128, 130, 131, 141,
polymorphism(s), 161, 163, 166, 170, 171, 174, 176, 142, 149, 150, 160, 187
177, 185, 187, 192, 193, 194, 196, 198, 199, 200 proton pump inhibitors, 151
214 Index
protopanaxadiol, ix, 83, 85, 86, 95 rehabilitation, 23, 32, 52, 55, 107, 109, 153
protopanaxatriol, ix, 83, 85, 86, 95 rehabilitation program, 107
PSA, 110 relapses, 128
psychiatric disorder(s), 152, 172, 175, 191, 193 reliability, 28, 78, 188
psychiatric illness, xi, 157, 158, 159, 178, 179 relief, 7, 8, 14, 15, 24, 44, 57
psychiatrist, 159 remission, 166
psychiatry, 170, 180, 181, 183, 184, 185, 187, 188, remyelination, 183
189, 190, 192, 193, 194, 195, 196, 199, 200, 201 repair, 23, 24, 25, 105, 108, 111
psychobiology, 182, 183, 185 replication, 170, 177
psychological stress, 174, 189 repression, 138
psychology, 190 reproduction, 158, 193
psychopathology, 161, 162, 181, 183, 186 requirement(s), 14, 27, 150, 155
psychopharmacology, 198 resection, 3, 155
psychosis, 152, 154, 159, 162, 163, 181, 182, 193 residues, 9, 104
psychosomatic, 185, 194 resistance, 25
psychostimulants, 202 response, ix, x, 2, 6, 13, 16, 37, 39, 44, 45, 54, 65,
psychotic disorders, vii, xi, 157 66, 73, 74, 87, 92, 95, 101, 104, 107, 111, 116,
psychotic symptoms, 161 117, 118, 119, 123, 126, 128, 132, 137, 138, 153,
psychotropic drugs, 109 154, 160, 166, 167, 168, 169, 171, 174, 177, 184,
PTEN, 54 188, 189, 190, 191, 198, 201, 202
puberty, 158, 159, 166 restrictions, 27, 46
PVA, 102, 105 retina, 127
pyramidal cells, 62, 90 retrograde amnesia, 63
pyrimidine, 15 risk(s), xi, 24, 25, 28, 43, 44, 52, 53, 57, 72, 151,
152, 155, 156, 157, 162, 168, 170, 172, 173, 174,
175, 176, 178, 179, 180, 183, 194, 198, 201
Q risk factors, 52, 53, 172, 175, 179, 194
rodents, 3, 11, 18, 85, 166, 168, 170, 172
quadriceps, 33, 35, 37, 39, 42
root(s), viii, 4, 6, 7, 9, 10, 12, 16, 19, 20, 22, 27, 36,
quality of life, 23, 47, 107, 108
45, 56, 84, 85, 87, 99
rotations, 88
R
S
radiation, 154
radical formation, 86, 88
sacrum, 22
reactions, 41
safety, 57
reactivity, 166, 167, 168, 169, 173, 174, 175
saponin, 97
receptive field, 18
sarcoidosis, 154
receptor(s), vii, xi, 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12,
SARS, 36, 42, 45
15, 16, 17, 18, 19, 20, 74, 81, 89, 90, 91, 92, 93,
Schilling test, 151, 152
94, 95, 96, 103, 106, 110, 130, 131, 133, 134,
schizophrenia, 145, 157, 159, 160, 161, 162, 163,
137, 138, 139, 141, 143, 144, 146, 150, 151, 158,
164, 165, 168, 178, 179, 180, 181, 182, 183, 185,
160, 161, 162, 163, 165, 166, 168, 170, 171, 172,
186, 187, 188, 189, 190, 191, 192, 194, 195, 196,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182,
197, 198, 199, 201, 202
183, 185, 186, 187, 188, 189, 190, 191, 192, 193,
sclerosis, 132, 133
194, 195, 196, 198, 199, 200, 201, 202
secrete, 74
recognition, 47, 116, 118, 185, 190, 200
secretion, 150, 163, 166, 169, 176, 182
recognition phase, 118
seed, 74
recovery, 108, 109, 129, 132, 153, 161, 162, 176
selective estrogen receptor modulator, xii, 157, 162,
recurrence, 53
165, 190, 200
redistribution, 44, 128
selectivity, 64
reflexes, 152, 153
sensation(s), 2, 13, 153
regeneration, 18, 104, 129
sensitivity, 134, 152, 167
Index 215
sensitization, 2, 4, 8, 11, 12, 15, 19 species, 84, 85, 97, 102, 158
sensorineural hearing loss, 114 speech, 68, 123, 152
sensory data, ix, 59, 60, 68 speech perception, 123
sensory impairments, 121 spinal cord, vii, viii, xi, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
sensory symptoms, 15 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 24, 27, 52,
sensory system(s), 64, 114, 119, 121 53, 54, 55, 56, 57, 97, 138, 149, 153, 154, 156
SEP-N20, vii, x, 113, 114, 115, 116, 117, 118, 119, spinal cord degeneration, 156
120, 122 spinal cord injury, vii, 13, 22, 24, 52, 53, 54, 55, 56,
septum, 62, 65, 110 57
serine, 133 spine, 37, 38, 42, 46
serotonin, 85, 102, 106, 110, 162, 166, 168, 170, spleen, 4
180, 181, 188, 193, 197 sprouting, 10, 103, 104, 159, 188
sertraline, 189 stabilization, 126, 134, 153
serum, 4, 9, 132, 150, 151, 152, 154, 155, 164, 168, standardization, 3
174, 176, 177, 181, 189, 190, 192, 193 starvation, 23, 175, 176, 177, 179
severe stress, 172 state(s), 6, 8, 15, 18, 20, 64, 65, 67, 103, 126, 151,
sex, vii, xi, 157, 158, 159, 160, 162, 164, 166, 167, 154, 164, 181, 189
169, 170, 172, 173, 175, 176, 177, 178, 179, 183, statistics, 29, 52
186, 192 stem cells, 87, 98, 105, 109, 111
sex differences, xi, 157, 158, 166, 169, 183, 186 steroids, 106, 111, 133, 138, 139, 142, 179, 181, 188,
sex hormones, 158, 164, 166, 167, 179, 186 191
sex steroid, 158, 175 stimulant, 106
sham, 29, 31, 32, 40, 41, 43, 44 stimulation, vii, viii, x, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11,
shape, 54, 95, 178 12, 13, 14, 16, 18, 19, 21, 22, 24, 25, 26, 27, 28,
sheep, 128, 132 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44,
shock, 167 45, 46, 47, 52, 53, 54, 55, 56, 57, 102, 105, 107,
short-term memory, 66 108, 110, 113, 114, 116, 117, 119, 120, 138
showing, 4, 11, 62, 63, 92, 94, 117, 167, 169 stimulus, vii, 1, 3, 5, 12, 13, 16, 41, 43, 65, 117, 119,
side chain, 140 120, 123
side effects, xii, 2, 14, 130, 157, 166 stomach, 150
signal transduction, 11 storage, 64
signaling pathway, 11, 86, 99 stress, xi, 9, 85, 107, 109, 111, 126, 137, 138, 145,
signalling, 131, 160, 162, 163, 165, 170, 171, 173, 158, 160, 166, 167, 168, 169, 172, 174, 175, 182,
174, 179, 196 183, 184, 185, 190, 191, 193, 194, 195, 197, 200
signals, 6, 10, 12, 14, 54, 117 stress response, 166, 174
signs, xi, 7, 71, 73, 74, 149, 153, 154, 155 stressors, 167
simulation, 40 striatum, 71, 110, 162
skeletal muscle, 23 strictures, 151
skin, vii, 1, 3, 5, 23, 25, 27, 31, 32, 33, 36, 39, 40, stroke, 109, 129
41, 43, 44, 45, 52, 54, 55 structural changes, 103
social care, 23 structure, vii, 4, 61, 62, 63, 85, 88, 98, 102, 126, 133,
social interaction, 167 176
social phobia, 171 subacute, vii, xi, 149, 152, 153
sodium, 25, 43 subacute combined spinal cord degeneration, vii, xi,
solution, 93 149
somata, 92 subcortical nuclei, 73
Spain, 101, 149 subgroups, 85
spastic, xi, 149, 153 substance abuse, 166
spatial ability, 165 substrate(s), 104, 105, 192
spatial learning, 98 suicide, 175
spatial location, 60 sulfate, 191
spatial memory, 60, 146, 163, 178, 201 superimposition, 5
specialists, 46 suppression, 16, 145, 173
216 Index
surface area, 38 therapy, xi, 54, 55, 56, 107, 108, 114, 121, 125, 128,
surgical intervention, 23 129, 130, 133, 134, 151, 153, 155, 161, 170, 178,
survival, x, 19, 70, 81, 87, 96, 101, 106, 110 181, 182, 184, 190, 194
susceptibility, 167, 168, 172, 175, 184, 192, 193 threonine, 9, 133
symmetry, 104 thrombosis, 133
symptomology, xi, 79, 157, 169, 171, 178 TIMP, 127, 128, 132
symptoms, xi, 7, 13, 66, 69, 70, 72, 73, 74, 139, 149, TIMP-1, 127, 128, 132
151, 153, 154, 155, 158, 159, 160, 161, 162, 163, TIMP-3, 127, 132
164, 167, 168, 174, 178, 180, 181, 182, 185, 188, tissue, 2, 9, 19, 23, 25, 38, 42, 43, 44, 45, 46, 52, 53,
189, 190, 191, 195, 197, 198, 199 54, 56, 67, 73, 162
synapse, 90 tissue perfusion, 26
synaptic efficacy, vii, viii, 2, 9, 12 TLR2, 9
synaptic plasticity, 20, 87, 99, 103, 104, 107, 159, TLR4, 9, 99
163, 173, 189, 190, 197 TNF, 4, 9
synaptic transmission, 5, 12, 91, 92, 103, 173 TNF-α, 4, 9
synaptogenesis, 103 tobacco, 193
synchronization, 160 tonic, 84, 98
syndrome, ix, 59, 65, 66, 67, 70, 73, 76, 77, 78, 79, toxicity, 79, 186
80, 81, 169, 175, 183, 184, 198 Traditional Chinese Medicine, vii, 1, 3
synthesis, xi, 8, 26, 55, 95, 96, 106, 137, 138, 139, trafficking, 182
140, 141, 142, 144, 145, 149, 153, 166, 170, 193 training, 3, 103, 108
syringomyelia, 154 trait anxiety, 171
systematic review, 3, 22, 24, 26, 43, 46, 53, 54, 55, traits, 169, 170, 171, 194
57, 122, 190 trajectory, 201
transactions, 55
transcription, 126, 127, 159, 163, 172, 194
T transcription factors, 126, 127
transducer, 126
T cell(s), 128, 133, 134
translocation, 126
tactile stimuli, 119, 120
transmission, 5, 6, 8, 9, 12, 20, 65, 69, 80, 89, 92, 95,
tangles, 66, 68, 78
104, 133, 177, 191
target, x, 30, 85, 88, 125, 127, 131, 132, 133, 134,
transplantation, 55
140, 143, 147, 159, 160, 166, 169, 178
transport, 79, 80, 109, 138, 151, 154
target population, 30
trauma, 23, 27, 172, 179
tau, 65, 66, 68, 72, 73, 74, 76, 77, 78, 80
traumatic brain injury (TBI), 87, 96, 129, 130, 134
taxonomy, 15
traumatic events, 172
techniques, 105, 108, 115
treatment, vii, viii, x, xi, 1, 3, 4, 7, 10, 11, 13, 14, 15,
TEM, 130
16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29,
temporal lobe, viii, 59, 60, 64, 65, 66, 70, 74, 75
30, 31, 32, 33, 34, 39, 40, 41, 43, 44, 47, 52, 53,
tendon, 152
54, 55, 56, 84, 88, 110, 114, 125, 126, 127, 128,
tension, 37, 38, 52, 54, 56
129, 139, 149, 153, 154, 155, 156, 157, 158, 160,
terminals, 16, 62, 69, 71, 92, 93, 98
161, 162, 163, 164, 165, 166, 167, 168, 169, 174,
testing, 152, 171
176, 178, 179, 180, 181, 182, 183, 184, 185, 186,
testis, 139, 158
189, 190, 191, 192, 196, 197, 198, 199, 201
testosterone, 158, 164, 165, 168, 171, 174, 175, 177,
tremor, 72
180, 181, 184, 185, 186, 188, 189, 190, 191, 192,
trial, 26, 28, 29, 39, 40, 41, 134, 181, 183, 188, 189,
193, 195, 197, 198, 200
195, 199, 201
thalamus, 105, 110
triceps, 45
therapeutic agents, 129, 178
tricyclic antidepressant(s), 2, 167
therapeutic approaches, 161
triggers, 166
therapeutic effect(s), 84
trypsin, 150
therapeutic interventions, 24, 53
tryptophan, 182
therapeutic use, 179
tumor(s), 9, 98, 126, 127, 130, 135
therapeutics, 179, 188
Index 217
tumor cells, 130 visual system, 61, 109

tumor necrosis factor, 9 vitamin A, 140
tumorigenesis, 200 vitamin B1, vii, xi, 149, 150, 151, 152, 153, 154,
twins, 183 155, 156
tyrosine, 9, 11, 133, 187 vitamin B12, vii, xi, 149, 150, 151, 152, 153, 154,
155, 156
Vitamin B12, xi, 149, 150, 151, 152, 153, 154, 155,
U 156
vitamin B12 deficiency, vii, xi, 149, 150, 151, 152,
ubiquitin, 69, 70, 72, 78
153, 154, 155, 156
ulcer, vii, viii, 21, 22, 23, 24, 25, 27, 28, 29, 31, 33,
vitamin D, xi, 11, 137, 140, 146, 147
39, 40, 43, 47, 52, 53, 55, 57, 151
vitamin E, 154, 156
ultrasound, 24
vitamins, xi, 137, 141, 145
United Kingdom, 22, 23, 30, 53, 81
vulnerability, ix, 60, 73, 172, 174, 178, 190
United States (USA), 22, 23, 30, 31, 33, 34, 35, 36,
37, 38, 45, 54, 81, 85, 96, 146, 152, 181, 189,
193, 198 W
unmasking, 101, 102, 103, 104
urinary bladder, 45 water, 85, 155
urine, 150 weight gain, 176, 177
weight loss, 195
white matter, 9, 72, 81, 154, 194
V withdrawal, 28
working memory, 60, 65, 161, 164, 188
vagus nerve, 4
World Health Organization (WHO,), 3, 20, 185
variations, xi, 69, 149
wound healing, 25, 26, 32, 33, 43, 53, 54, 55, 56
vascular cell adhesion molecule (VCAM), 128
vasculature, 25, 128
vasodilation, 25 Y
vasomotor, 23
vastus lateralis, 46 young women, 184, 185, 189
VEGF, 106
ventricle, 61, 110
vessels, 23, 44, 130 Z
vibration, 12
zinc, 11
vision, 116
Zollinger-Ellison syndrome, 151
visual area, 61
visual processing, 61

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Horizons in Neuroscience Research, Volume 24

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HORIZONS IN NEUROSCIENCE RESEARCH

Additional books in this series can be found on Nova’s website

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NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

ISBN:  H%RRN

Published by Nova Science Publishers, Inc. † New York

Chapter 8 Dexamethasone Suppresses Neurosteroid Biosynthesis in Human

Salvador Quiroz-González1, Erika Elizabeth Rodríguez-Torres2

or pathological conditions, such as neuropathic pain. Experimental studies performed in

Keywords: electroacupuncture, neuropathic pain, cytokines, spinal cord, non-painful sensory

BEHAVIORAL RESPONSES AND EA IN EXPERIMENTAL

SPINAL MECHANISMS OF ELECTROACUPUNCTURE RELATED TO

Modified from Quiroz-Gonzalez et al., 2014b.

NEUROTRANSMITTERS AND THEIR RECEPTORS

adrenoceptor antagonist) or propranolol (a β-adrenoceptor antagonist), but not prazosin (an

NEUROGLIA AND NEUROIMMUNE MODULATION

SPINAL ADRENERGIC AND SEROTONERGIC SYSTEMS

NON-PAINFUL SENSORY INPUTS IN THE DEVELOPMENT OF

Modified from Quiroz-Gonzalez et al., 2014b.

CLINICAL EFFICACY OF EA STIMULATION ON NEUROPATHIC PAIN

WHAT ARE THE EVIDENCE ABOUT ELECTRICAL

Liang Q. Liu1,∗, Dr, MBBS, PhD, Julie Moody2, MSc,

were accepted. Any outcome measuring effectiveness of PU prevention and treatment

common sites for PU development [9]. An estimated 36 to 50 percent of PU incidence results

Definition and Etiology of PU

A PU is otherwise and perhaps more commonly known as a pressure sore. According to

Impact of PU on People Living with SCI

Current PU Management in SCI

PU Treatment Options in SCI

PU Prevention Strategies in SCI

Electrical Stimulation for PU Management

Regan et al. (2009) conducted a systematic review of preventive and therapeutic

Rationale of Conducting the Systematic Review

Search Methods for Identification of Studies

Review of Therapeutic Studies

Review of Preventive Studies

In addition, for pooled analysis of therapeutic controlled trials, higher heterogeneities

APPENDIX II. WEEKLY HEALING RATE USING PULSED CURRENT VERSUS

[70] Karba R, Semrov D, Vodovnik L, Benko H, Savrin R. Direct Current electrical

STRUCTURE AND FUNCTION

Pathological differences among disorders could reflect either differential

ANATOMY OF THE ENTORHINAL

CC = Corpus callosum, CG = Cingulate gyrus, EC = Entorhinal cortex, FP = Frontal pole, PHG =

Figure 1. Anatomy of the entorhinal cortex and related gyri.

ANATOMICAL CONNECTIONS OF THE ENTORHINAL

FUNCTIONS OF THE ENTORHINAL

Figure 4. Coronal section of the anterior hippocampus and adjacent structures.

ROLE OF ENTORHINAL CORTEX IN NEURODEGENERATIVE DISEASE

Alzheimer’s Disease (AD)

Table 1. Pathological lesions and their molecular composition

Disorder Pathology Molecular constituent

Hippocampal pathway affected

Argyrophilic Grain Disease (AGD)

Corticobasal Degeneration (CBD)

Creutzfeldt-Jakob Disease (CJD)

Armstrong 2006). Patients develop a rapidly progressive dementia, which is sometimes

Dementia with Lewy Bodies (DLB)

Down’s Syndrome (DS)

Frontotemporal Lobar Degeneration (FTLD)

FTLD is the second commonest form of cortical dementia of early-onset after AD

Multiple System Atrophy (MSA)

Neuronal Intermediate Filament Inclusion Disease (NIFID)

Neuronal intermediate filament inclusion disease (NIFID) is a disease of early-onset with

ISBN: H%RRN