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doi:10.1111/jgh.12748

O R I G I N A L A RT I C L E

Confocal laser endomicroscopy: A new gold standard for

the assessment of mucosal healing in ulcerative colitis
Vincent Mac,*,1 Amrita Ahluwalia,,1 Emmanuel Coron,* Marc Le Rhun,* Arnaud Boureille,*
Cline Bossard, Jean-Franois Mosnier, Tamara Matysiak-Budnik* and Andrzej S Tarnawski
*Institut des Maladies de lAppareil Digestif, CIC INSERM 04 et Service dHpato-Gastroentrologie, and Service dAnatomie Pathologique et
EA-4273 BIOMETADYS, CHU de Nantes, France; and VALBHS and the University of California, Irvine, California, USA

Key words
angiogenesis, confocal laser endomicroscopy
(CLE), COX2, mitochondrial DNA (mtDNA)
mutation, mucosal healing, ulcerative colitis.
Correspondence
Andrzej S Tarnawski, Veterans Administration
Long Beach Healthcare System, and the
University of California, Irvine, 5901 E. 7th
Street (09/151), Long Beach, CA 90822-5201,
USA. Email: atarnawski@yahoo.com;
astarnaw@uci.edu
1

These authors contributed equally.

Supportive foundations: Southern California

Institute for Research and Education, and the
VA Merit Review grant to Tarnawski, AS.
Conflict of interest: The funding for this study
was provided by Southern California Institute
for Research and Education, and the VA Merit
Review Award to AST.

Abstract
Background and Aim: Endoscopic assessment of mucosal healing in ulcerative colitis
(UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key
prognostic indicator. While regular endoscopy evaluates appearance of the mucosal
surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000 magnification during ongoing endoscopy. Our aims were to determine
using CLE whether endoscopically normal appearing colonic mucosa in patients with UC
in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and
to identify the mechanisms.
Methods: Twelve patients (six controls and six with UC-IR) underwent colonoscopy
using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of
colonic mucosa and conventional mucosal biopsies were obtained and evaluated using
image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood
microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA
(mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP).
Results: In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic
mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2,
69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa).
Conclusions: (i) Endoscopically normal appearing colonic mucosa of patients with
UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent
inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may
serve as a new gold standard for the assessment of mucosal healing in UC.

Introduction
During the second International Shimoda Symposium in 1998, we
reported that the subepithelial mucosa of macroscopically healed
gastric ulcer displays impaired and disorganized restoration of
glandular and vascular structures and remains histologically and
ultrastructurally abnormal.1 We postulated that these abnormalities
may interfere with oxygenation, nutrient supply, and with mucosal
defense, and therefore could be the basis for ulcer recurrence.1
These studies were basis for formulating a new concept of quality
of gastric ulcer healing.1,2 Arakawa and coworkers demonstrated
the relevance of this concept to healing of human gastric ulcers,3,4
and more recently showed that this concept also applies to ulcers
in inflammatory bowel diseases (IBD).4

Endoscopic assessment of colonic mucosal healing in ulcerative colitis (UC) and Crohns disease is increasingly accepted as
an important measure of disease activity, therapeutic goal, a key
prognostic indicator, and an endpoint in clinical trials.514 The
main contention supporting importance of mucosal healing in
UC is that achieving mucosal healing may reduce or prevent
relapses and complications, and improve quality of life. These
issues were extensively reviewed and discussed in several recent
publications.514
Indeed, complete mucosal healing is associated with sustained
clinical remission and reduces the risk of colectomy in patients
with UC.1114 Mucosal healing in UC is defined mainly by endoscopy, based on macroscopic appearance of mucosal surface
and the absence of ulcerations, vascular congestion, erythema,

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swelling, and nodularity during endoscopic examination.1114 The

quality of mucosal healing assessed by endoscopy has been proposed as the main predictor of recurrence in UC.1214 Subsequent
studies utilizing magnifying chromoendoscopy and narrow band
imaging further improved endoscopic assessment of mucosa in
UC and IBD.4,15 However, even in patients with UC and endoscopically healed colonic mucosa, the disease recurs in a significant proportion of patients, suggesting that macroscopic
healing assessed endoscopically is not an equivalent of a completely normal mucosa. This is not surprising since a standard
endoscopy visualizes appearance of mucosal surface only, and
subepithelial abnormalities cannot be detected.
Confocal laser endomicroscopy (CLE) is a new state-of-the-art
technology that enables, real-time, in vivo visualization of subepithelial mucosal structures and cells at 1000 magnification (virtual
biopsy) during ongoing endoscopy.15 Several studies have shown
that CLE images of colonic mucosa correspond well to a standard
histology and might even replace conventional histological
diagnosis.1521 CLE has been shown to reliably assess activity of
the disease in inflammatory bowel diseases, both in UC1517 and in
Crohns disease,18 and to predict relapse by the detection of cell
shedding and mucosal barrier loss.19 CLE has also been used for
the detection of neoplastic lesions in patients with UC.20 CLE with
intravenous fluorescein infusion allows a direct visualization of
blood vessels in the mucosa, which are filled with fluorescein, and
therefore may be used for evaluation of the mucosal vasculature in
vivo.21 Most of the studies concerning CLE evaluation of UC
included patients with active disease1519 and only one clinical CLE
imaging report focused exclusively on UC patients during remission.22 However, none of these studies examined in depth and
quantitatively colonic mucosal crypts regeneration, regeneration
of blood microvesselstheir number, size, and characteristics nor
did they measure and quantify in real-time and in vivo microvascular permeability, vessel size, and inflammatory cell infiltration.
None of these studies did examine the expression COX2 and
mitochondrial DNA (mtDNA) mutation. The aim of this pilot
study was to test feasibility of in vivo, real-time measurements of
mucosal microvascular permeability and to determine quantitatively using CLE whether and to what extent endoscopically
normal appearing colonic mucosa of patients with UC in remission
(UC-IR) has regenerated mucosal structures both epithelial and
vasculature, resolved inflammation, and to identify the underlying
mechanisms.
Since COX2-generated prostaglandins may stimulate crypt
regeneration and re-epithelialization via transactivating epidermal
growth factor (EGF) receptor23,24 and promote neovascularization,
we examined the expression of COX2 in colonic mucosa of
control and UC-IR patients. In addition, we also examined whether
regenerated crypts of colonic mucosa have somatic mitochondrial
DNA mutations. The latter may have significant implications for
disease recurrence and carcinogenesis.2527

Methods
These studies were approved by the Ethics Committee CHU de
Nantes, France. All patients gave their written, informed consent
to participate in the study in accordance with the Treaty of Helsinki. Patients included in this study were adult aged 3672, with
long-term UC (> 10 years after diagnosis) in complete clinical
86

remission, referred for surveillance colonoscopy to the Department of Gastroenterology of the University Hospital of Nantes
from February 2008 until March 2010. Additionally, control
patients in whom colonoscopy was indicated for other reasons
(colorectal cancer screening, anemia) were also studied. For all
the patients, the following information was collected: demographic data, duration of the disease, clinical disease activity
index (CAI), and current treatment.
Colonoscopy and CLE procedure. Colonoscopy combined with CLE (confocal endomicroscope, Pentax, Tokyo, Japan)
was performed in all the patients. In this endomicroscope, a confocal laser microscope is integrated into the distal tip of a conventional
video endoscope. After intravenous (i.v.) infusion of 5 mL of 10%
fluorescein over 10 min, confocal imaging of colonic mucosa was
performed in a standardized manner, and virtual biopsies were
obtained after gently placing the distal end of the confocal laser
endoscope at the mucosal surface. In addition, conventional biopsies were obtained at adjacent areas for histological examination. In
each patient, at least 10 virtual biopsies and four conventional
biopsies were obtained. All procedures were performed by the same
experienced endoscopist. Endoscopic evaluation of the entire
colonic mucosa was performed using white light followed by indigo
carmine chromoendoscopy. The presence of macroscopic lesions as
well as endoscopic signs of inflammation was recorded.
Analysis of CLE images. Coded CLE images were analyzed by three investigators (AST, TMB, and AA) who were
blinded to the results of standard histology. For each patient, 10
CLE images obtained from colon were analyzed, and the following parameters were examined:
1. Distribution, pattern, and CLE features of crypt regeneration in
CLE images, including measurements of major (MA) and
minor (MI) crypt lumen axis and MA/MI ratio, using the Image
J system (National Institutes of Health NIH), as described
previously.28 This method allows quantitative analysis of
colonic pit and crypt structure and the detection of residual
inflammation.28
2. CLE features of mucosal microvessel: shape, size (maximal
width), and characteristics, for example, elongation and tortuosity were assessed and measured (maximal width) using the
Image J system (NIH).
3. Distribution and intensity of fluorescence inside and outside the
mucosal microvessels. It was measured using the Image J
system in 10 standardized mucosal areas and expressed as
fluorescence signal intensity (FSI) on a scale from 0255,
which is the measure of microvascular permeability. FSI was
measured inside the vessel and outside the vessel lamina
propria in six standardized fields per image on 10 separate
images, and the mean values were calculated for each patient.
The FSI gradient between vessels and lamina propria was
calculated; a lower FSI gradient indicates increased fluorescein
leakage into the extra vascular space and reflects increased
vascular permeability. To assure appropriate, standardized
comparisons, the vascular permeability measurements were
performed on CLE images obtained always at 810 min after
initiation of the i.v. infusion of fluorescein.

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4. CLE features of inflammation. The number of inflammatory

cells (black dots) in five standardized areas of lamina propria
per each CLE image in normal controls and UC-IR patients
were counted.
Histology and immunohistochemistry. Four oriented
mucosal biopsies from each patient were fixed in 10% formalin
and embedded in paraffin. The sections were stained with HE and
used for conventional histology. Unstained sections were used for
immunohistochemistry. Histological activity of the disease was
evaluated according to the Geboes score.29 The immunostaining
for COX2 and mitochondrial DNA encoded cytochrome C oxidase
subunit 1 (CCO) was performed using paraffin-embedded mucosal
sections. Tissue sections were de-paraffinized and incubated with
specific primary antibodies for COX2 (1:100; sc-1745 goat
polyclonal, Santa Cruz Biotechnology, CA) and mitochondrial
DNA-encoded cytochrome c oxidize subunit 1 (CCO) (1:100;
#459600 mouse monoclonal antibody, Invitrogen, CA) overnight
at 4C. After washing, the sections were then incubated with a
biotin-conjugated appropriate anti-goat or antimouse secondary
antibodies (1:500; E0466, Dako, CA) at room temperature for
30 min and finally with peroxidase-conjugated streptavidin
(1:500; P0397, Dako, CA) at room temperature for 30 min. Peroxidase activity was detected with AEC substrate-chromogen
(K3464, Dako, CA). Immunostaining was evaluated independently by two investigators using a Nikon Optiphot microscope.
The staining signal intensity was quantified using MetaMorph 7.0
(Molecular Devices, Downington, PA) in five randomly selected
fields and expressed as arbitrary units.
Statistical analysis. The quantitative values for crypt lumen
axis and size of microvessels were expressed as median (MinMax) and the comparison between UC patients and control
patients was performed using a nonparametric MannWhitney
test. The differences were considered significant for P value less
than 0.05. FSI gradient between outside and inside the
microvessels, and COX2 and CCO staining intensity are expressed
as the mean standard deviation (SD). Students t-test was used to
determine statistical significance, and a P value less than 0.05 was
considered statistically significant.

Results
Patients and regular endoscopy. Six patients with
UC-IR and six control patients were included. Demographic and
clinical characteristics of all the patients are presented in Table 1.
All patients with UC-IR had a macroscopically normal colonic
mucosa on regular endoscopy, except 1 with minimal inflammation. All control patients had endoscopically normal mucosal
appearance.
Analysis of CLE images. In all control patients, detailed
evaluation CLE images showed that colonic mucosal crypts had
regular distribution pattern and had mostly round, regular lumen
(Fig. 1c,d). In patients with UC-IR, crypts had distorted distribution pattern, with increased spaces between crypts indicating
expansion of the lamina propria, and the crypt lumina were irregu-

Confocal laser endomicroscopy & colitis

Table 1

Demographic and clinical data of all patients

Age (years), median (min-max)

Sex (male/female)
Duration of the disease (years),
median (min-max)
Clinical activity index, median
(min-max)
Current treatment

UC (n = 6)

Control (n = 6)

57 (3672)
4/2
18 (1526)

59 (4667)
4/2

2.5 (17)

Imurel 3
5-ASA 1
Dipentum 1
or Combined 1

5-ASA + Corticoids + Methotrexate.

UC, ulcerative colitis.

Figure 1 Representative images of (a) standard endoscopy, (b) standard histology, and (c and d) confocal laser endomicroscopy (CLE) of
normal colonic mucosa in a control patient. CLE shows a normal
mucosal structure, with regular, round crypts (*), surrounded by normal
size microvessels filled with fluorescein (arrows).

lar (Fig. 2c,d). The quantitative analysis of pit structure, performed

similar as in our previous study in relation to Crohns colitis,28
demonstrated that the median major: minor (MA/MI) crypt lumen
axis ratio was significantly higher in UC-IR patients (3.02, range:
1.984.1) than in control patients (1.45, range: 1.21.7)
(P < 0.001), indicating impaired and distorted crypt regeneration
of colonic mucosa in UC-IR patients.
Expression of COX2 in colonic mucosa of control patients was
absent or minimal and limited to few mononuclear cells in the
lamina propria; COX2 expression was absent or minimal in epithelial cells lining mucosal surface and regenerated crypts
(Fig. 3a). In contrast, in UC-IR patients COX2 expression was
significantly increased in both mononuclear inflammatory cells in

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Figure 2 Representative images of colonic mucosa in patient with

ulcerative colitis in remission (UC-IR). (a) Endoscopic view shows
normal appearing mucosa. (b) Standard histology shows deformed
crypts and enlarged blood microvessels between crypts (arrows). In
patients with UC-IR (c and d), despite a normal macroscopic picture of
colonic mucosa at endoscopy, CLE shows distinct abnormalities of
mucosal structures: irregular and distorted crypts (*), with increased
spaces between the crypts reflecting persistent inflammation, impaired
crypt regeneration, and leakage of fluorescein into the extravascular
space (arrows).

the lamina propria (which were significantly increased in number)

as well in the epithelial cells lining mucosal surface and regenerated crypts (Fig. 3b,c). COX2 expression was 6.9-fold higher in
epithelial cells of colonic crypts in UC-IR patients versus normal
controls (P < 0.001) (Fig. 3d).
The mtDNA mutation was assessed using immunostaining for
mitochondrial DNA-encoded cytochrome c oxidase (CCO) subunit
1 in normal (Fig. 4a) and UC-IR patients (Fig. 4b,c). In colonic
mucosa of UC-IR patients, CCO expression (signal intensity) was
significantly decreased (1.6-fold) compared to control patients
(P < 0.001) (Fig. 4d). Furthermore, the number of CCO-deficient
colonic crypts (crypts with mtDNA mutations) was significantly
increased patients with UC-IR (69% vs 7% in normal controls)
(Fig. 4e).
CLE image analysis showed signs of inflammation infiltration
of the mucosa with inflammatory cells (Fig. 5) in all six patients
with UC, and this result correlated with the results of standard
histology (Table 2). In UC-IR patients, the number of inflammatory cells in the lamina propria was significantly increased (6.6fold, P < 0.001) compared to control patients (Fig. 5).
CLE assessment of the mucosal blood microvessels demonstrated that in control patients, mucosal blood microvessels were
regular, thin, and distributed regularly around the normal crypts
88

Figure 3 Expression of COX2 in colonic mucosa in control patients (a)

and in patients with ulcerative colitis in remission (UC-IR) (b and c).
COX2 expression (brown staining) in colonic mucosa of control patients
is limited to few mononuclear cells in the lamina propria (arrowheads).
In contrast, in UC-IR patients, COX2 expression is significantly
increased not only in mononuclear inflammatory cells in the lamina
propria (arrowheads), which are significantly increased in number but
also in the epithelial cells (arrows) lining mucosal surface and regenerated crypts. Quantitative analysis of COX2 expression (d) demonstrates
that COX2 expression in colonic mucosa of patients with UC-IR is
significantly increased (6.9 fold) compared to control patients
(P < 0.001). , Inflammatory Cells; , Epithelial Cells.

(Fig. 1c,d). In contrast, in colonic mucosa of UC-IR patients,

mucosal blood microvessels were increased in number, and were
tortuous and enlarged reflecting their abnormal regeneration and
pathological angiogenesis. (Fig. 6ce). The median width of the
vessels was significantly larger in UC patients (17 m, range:
1622 m) than in control patients (8 m, range 610 m)
(P < 0.001).
In vivo assessment of mucosal microvessels permeability using CLE. The FSI gradient between fluorescein
fluorescence inside and outside the vessels (lamina propria) in
colonic mucosa was used as a measure of vascular permeability. In
UC-IR patients, this gradient was significantly lower than in
control patients, indicating an increased vascular permeability in
UC-IR patients (Fig. 7).

Discussion
The present study shows that in patients with UC-IR, despite
normally appearing colonic mucosa at regular endoscopy, distinct

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Confocal laser endomicroscopy & colitis

Figure 4 Mitochondrial DNA (mtDNA) mutations in colonic mucosa of control and patients with ulcerative colitis in remission (UC-IR) visualized using
immunostaining for expression of mtDNA-encoded cytochrome C oxidase (CCO) subunit 1. MtDNA mutations result in deficiency of CCO enzyme.
Brown staining in epithelial cells represents expression in colonic mucosa of CCO enzyme and reflects normal, nonmutated protein. Some epithelial
cells lining crypts lack positive (brown) stain reflecting mtDNA mutations. In control patients, (a) all epithelial cells lining crypts have positive (brown)
staining. In patients with UC in remission (UC-IR) (b, c), crypts lacking CCO staining are marked with *. (d) Quantitative analysis of CCO expression
(signal intensity) in colonic mucosa of UC-IR patients was significantly decreased (1.6 fold) compared to control patients (P < 0.001). (e) Quantitative
analysis of mtDNA mutations (which result in CCO deficiency) showed that in colonic mucosa of patients with UC-IR, mtDNA mutations were
significantly increased; 69% of the colonic crypts in UC-IR patients showed mtDNA mutations, while only 7% of the colonic crypts in normal controls
showed mtDNA mutations.

Table 2 CLE and histological evaluation of colonic mucosa in patients

with UC in remission and in control patients

CLE diagnosis

Histological grade of
disease activity

Normal
Moderate inflammation
Severe inflammation
Grade 0
Grade 1
Grade 4

UC
(n = 6)

Control
(n = 6)

0
4
2
0
5
1

6
0
0
6
0
0

According to the score of Goebes et al. [29].

CLE, confocal laser endomicroscopy; UC, ulcerative colitis.

Figure 5 Confocal laser endomicroscopy (CLE) features of inflammation. CLE shows increased number of infiltrating inflammatory cells
(black dotsindicated by arrows) in expanded lamina propria of colonic
mucosa in patient with ulcerative colitis in remission (UC-IR). The
number of infiltrating mononuclear cells is 6.6-fold increased in colonic
mucosa of UC-IR patients versus control patients.

abnormalities of mucosal epithelial structures and microvessels are

present. Importantly, these abnormalities were demonstrated by
CLE and diagnosed in vivo and in real time. They include the
presence of irregular, distorted crypt pattern, with deformed crypt
lumina indicating impaired and distorted crypt regeneration, and

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Figure 6 (ac). Confocal laser endomicroscopy images showing abnormal, enlarged and tortuous vessels in colonic mucosa of patients with
ulcerative colitis in remission (UC-IR). Distorted and irregular-shaped
crypts in colonic mucosa of UC-IR patients are surrounded by abnormal,
tortuous, enlarged mucosal blood microvessels (indicated by arrows),
reflecting their abnormal regeneration and pathological angiogenesis.
Compared to normal mucosa of control patients, the vessels in colonic
mucosa in UC-IR patients were significantly wider versus normal control
patients (P < 0.001).

Figure 7 Colonic mucosal microvascular permeability determined by

measurement of fluorescein fluorescence signal intensity (FSI) inside
and outside the vessels (lamina propria) of colonic mucosa in patients
with ulcerative colitis in remission (UC-IR) and in control patients. g.:
gradient between FSI inside the vessels and in lamina propria outside
the vessels. A significantly lower (fourfold) FSI gradient between
vessels and lamina propria in UC-IR versus normal controls (P < 0.001)
indicates increased leakage of fluorescein from the vessels and reflects
increased vascular permeability. , FSI inside the vessels; , FSI in
lamina propria outside the vessels.

enlarged spaces between the crypts likely reflecting the presence of

inflammatory infiltrates, expanded lamina propria and thus persistent inflammation. The mucosal vessels are enlarged and tortuous,
and the mean size of the vessels is significantly increased, all
reflecting pathological angiogenesis in patients with UC-IR.
These findings are important and indicate that the evaluation
criteria of mucosal healing in UC using regular endoscopy, currently accepted and widely used in clinical studies814 may not be
optimal, and that CLE by allowing evaluation of subepithelial
mucosal structures represents a more precise and in depth method
for such evaluation. This has been suggested by some previous
studies also indicating that in a subset of patients with UC-IR or
Crohns disease with complete endoscopic remission, some
changes in crypt and microvessel architecture are present.1618,22
90

Gheorghe and coworkers found in UC patients with a normal

endoscopic appearance of colonic mucosa subtle changes in crypt
architecture in form of focal distortions of crypts and the presence
of fluorescein leakage in the luminal openings of the crypts.22
However, they did not quantify crypts structure deformation.
Regarding the process of colonic crypt regeneration, Wright
et al. demonstrated that during healing of ulceration in human
gastrointestinal tract, there is development of a novel cell lineage,
which forms buds at the ulcer margin that expands through proliferation into tubules then ramifying to and ultimately forming new
crypts.30 That study showed that this novel cell lineage secretes
epidermal growth factor (EGF), which stimulates proliferation of
these cells and induces crypt regeneration and ulcer healing.30 Our
previous study demonstrated that the same process occurs during
experimental gastric ulcer healing; regenerated gastric glands are
deformed and all cells lining these glands strongly express EGF
receptor.31
In the present study, we found increased COX2 expression in
epithelial cells of regenerating colonic crypts. This may represent
a novel mechanism underlying crypt regeneration since COX2generated prostaglandins are able to transactivate EGF receptor, as
shown in our previous study23 and via this mechanism stimulate
epithelial cell proliferation and migration.23,24 Moreover, COX2generated prostaglandin E2 may induce angiogenesis, new blood
vessel formation.3234
Another entirely novel finding of this study is that a significant
proportion (69%) of regenerated mucosal crypts in UC-IR patients
has mutated mtDNA as evidenced by a lack of cytochrome c
oxidase expression. Cytochrome c oxidase reduces oxygen to
water and is central to oxidative phosphorylation and the generation of ATP.35 Somatic mtDNA mutations resulting in CCO deficiency are present in normal colon and in colon cancer.26 Taylor
et al. showed that in some colonic crypt cells, the mitochondrial
CCO genes are mutated resulting in decreased CCO enzyme.27
That study showed that such colon crypt cells clonally expand to
occupy first part and then the whole crypt by a process known as
monoclonal conversion.27 Greaves et al. showed that these mutations spread in the normal human colon by crypt fission.26 Mutation
of mtDNA and CCO deficiency can occur in noncancerous tissues
and may induce ulcer recurrence in UC-IR via a similar mechanism
and may also have implications for carcinogenesis in UC.
In our study, all patients with clinically quiescent UC and macroscopically normal colonic mucosa (UC-IR) had distinct abnormalities not only in colonic mucosal epithelial crypts but also in
mucosal blood microvessels. The mucosal vessels were enlarged
and tortuous, and the mean size of the vessels is significantly
increased, all these reflecting pathological angiogenesis. Angiogenesis plays an important role not only in normal tissue injury
healing, but as shown, more recently also in inflammation.36,37
Vascular endothelial growth factor-A (VEGF-A) and its receptor
(VEGFR2) are considered important mediators of pathological
angiogenesis.36 In active IBD, increased levels of VEGF-A and
VEGFR2 in colonic mucosa have been demonstrated.38 This
finding confirmed the contention that abnormal angiogenesis is not
only a consequence of inflammation but also may be a driving
force inducing and promoting inflammatory process. It is thus
conceivable that persistent pathological angiogenesis in spite of
macroscopically healed mucosa may contribute to the disease
recurrence in UC.

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In this study, we also demonstrated impaired function of endothelial barrier function of mucosal blood vessels in UC-IR
patients, reflected by increased microvascular permeability to
plasma containing fluorescein. For these studies, we used measurements of extravasation of fluorescein reflected by fluorescence
gradient between outside and inside the vessels on CLE images as
a marker of in vivo microvascular permeability. In our previous
study, we have demonstrated that after i.v. passage, fluorescein
crosses the endothelial microvascular barrier and penetrates into
the extravascular space as well into the epithelial cells.39 The
increased microvascular permeability in colonic mucosa of UC-IR
patients may be an important mechanism for sustained inflammation by enabling the flux of inflammatory cells into extravascular
space.40,41
This pilot study was aimed to test the feasibility of using CLE to
quantify crypt regeneration and deformation, vessel size, vascular
permeability and to use standard biopsy specimens to determine
quantitatively the expression of COX2 and mtDNA mutation. In
this regard, these aims were fully achieved. Naturally, limitation of
such pilot study is that because of a small patient number, it cannot
specifically evaluate the effect of treatment, predict recurrence, etc.
However, once the feasibility of CLE quantitative assessment of
mucosal healing in UC has been established, future studies can
answer these questions.
In summary, this study shows that in patients with UC in remission and with normal endoscopic appearance of colonic mucosa,
CLE allows in vivo, real-time detection of structural and microvascular changes of the colonic mucosa, not detectable by standard
endoscopy, which may contribute to the recurrence of the disease.
In addition, CLE allows in vivo measurements of mucosal microvascular permeability and molecular imaging. CLE might be thus
considered as the technique of choice and a new gold standard to
evaluate mucosal healing in UC patients.

References
1 Tarnawski A, Hollander D, Krause WJ, Dabros W, Stachura J,
Gergely H. Healed experimental gastric ulcers remain
histologically and ultrastructurally abnormal. J. Clin. Gastroenterol.
1990; 12 (Suppl. 1): S13947.
2 Tarnawski A, Stachura J, Krause WJ, Douglass TG, Gergely H.
Quality of gastric ulcer healing: a new, emerging concept. J. Clin.
Gastroenterol. 1991; 13 (Suppl. 1): S427.
3 Arakawa T, Kobayashi K. Quality of ulcer healinga new concept
to rank healed peptic ulcers. Gastroenterol. Jpn. 1993; 28 (Suppl. 5):
15862.
4 Arakawa T, Watanabe T, Tanigawa T, Tominaga K, Fujiwara Y,
Morimoto K. Quality of ulcer healing in gastrointestinal tract: Its
pathophysiology and clinical relevance. World J. Gastroenterol.
2012; 18: 481122.
5 Lichtenstein GR, Rutgeerts P. Importance of mucosal healing in
ulcerative colitis. Inflamm. Bowel Dis. 2010; 16: 33846.
6 Mazzuoli S, Guglielmi FW, Antonelli E, Salemme M, Bassotti G,
Villanacci V. Definition and evaluation of mucosal healing in clinical
practice. Dig. Liver Dis. 2013; 45: 96977.
7 Peyrin-Biroulet L, Bressenot A, Kampman W. Histologic Remission:
The Ultimate Therapeutic Goal in Ulcerative Colitis? Clin.
Gastroenterol. Hepatol. 2014; 12: 92934.e2.
8 Yokoyama K, Kobayashi K, Mukae M, Sada M, Koizumi W.
Clinical Study of the Relation between Mucosal Healing and

Confocal laser endomicroscopy & colitis

10

11

12
13

14

15

16

17

18

19

20

21

22

23

24

25

26

Long-Term Outcomes in Ulcerative Colitis. Gastroenterol. Res.

Pract. 2013; doi: 10.1155/2013/192794.
Seidelin JB, Coskun M, Nielsen OH. Mucosal healing in ulcerative
colitis: pathophysiology and pharmacology. Adv. Clin. Chem. 2013;
59: 10123.
Dave M, Loftus EV Jr. Mucosal healing in inflammatory bowel
disease-a true paradigm of success? Gastroenterol. Hepatol. (N Y)
2012; 8: 2938.
Pineton de Chambrun G, Peyrin-Biroulet L, Lmann M, Colombel
JF. Clinical implications of mucosal healing for the management of
IBD. Nat. Rev. Gastroenterol. Hepatol. 2010; 7: 1529.
Neurath MF, Travis SP. Mucosal healing in inflammatory bowel
diseases: a systematic review. Gut 2012; 61: 161935.
Rutgeerts P, Sandborn WJ, Feagan BG, Reinisch W, Olson A,
Johanns J et al. Infliximab for induction and maintenance therapy for
ulcerative colitis. N. Engl. J. Med. 2005; 353: 246276.
Colombel JF, Rutgeerts P, Reinisch W, Esser D, Wang Y, Lang Y
et al. Early mucosal healing with infliximab is associated with
improved long-term clinical outcomes in ulcerative colitis.
Gastroenterology 2011; 141: 1194201.
Kiesslich R, Goetz M, Lammersdorf K, Schneider C, Burg J, Stolte
M et al. Chromoscopy-guided endomicroscopy increases the
diagnostic yield of intraepithelial neoplasia in ulcerative colitis.
Gastroenterology 2007; 132: 87482.
Watanabe O, Ando T, Maeda O, Hasegawa M, Ishikawa D, Ishiguro
K et al. Confocal endomicroscopy in patients with ulcerative colitis.
J. Gastroenterol. Hepatol. 2008; 23 (Suppl 2): S28690.
Li CQ, Xie XJ, Yu T, Gu XM, Zuo XL, Zhou CJ et al.
Classification of inflammation activity in ulcerative colitis by
confocal laser endomicroscopy. Am. J. Gastroenterol. 2010; 105:
13916.
Neumann H, Vieth M, Atreya R, Grauer M, Siebler J, Bernatik T
et al. Assessment of Crohns disease activity by confocal laser
endomicroscopy. Inflamm. Bowel. Dis. 2012; 18: 22619.
Kiesslich R, Duckworth CA, Moussata D, Gloeckner A, Lim LG,
Goetz M et al. Local barrier dysfunction identified by confocal laser
endomicroscopy predicts relapse in inflammatory bowel disease. Gut
2012; 61: 114653.
Kiesslich R, Burg J, Vieth M, Gnaendiger J, Enders M, Delaney P
et al. Confocal laser endoscopy for diagnosing intraepithelial
neoplasias and colorectal cancer in vivo. Gastroenterology 2004;
127: 70613.
Gheonea DI, Crtna T, Ciurea T, Popescu C, Badarau A, Saftoiu A.
Confocal laser endomicroscopy and immunoendoscopy for real-time
assessment of vascularization in gastrointestinal malignancies. World
J. Gastroenterol. 2011; 17: 217.
Gheorghe C, Cotruta B, Iacob R, Becheanu G, Dumbrava M,
Gheorghe L. Endomicroscopy for assessing mucosal healing in
patients with ulcerative colitis. J. Gastrointestin. Liver Dis. 2011; 20:
4236.
Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS.
Prostaglandin E2 transactivates EGF receptor: a novel mechanism
for promoting colon cancer growth and gastrointestinal hypertrophy.
Nat. Med. 2002; 8: 28993.
Pai R, Nakamura T, Moon WS, Tarnawski AS. Prostaglandins
promote colon cancer cell invasion; signaling by cross-talk between
two distinct growth factor receptors. FASEB J. 2003; 17: 16407.
Ussakli CH, Ebaee A, Binkley J, Brentnall TA, Emond MJ,
Rabinovitch PS et al. Mitochondria and tumor progression in
ulcerative colitis. J. Natl. Cancer Inst. 2013; 105: 123948.
Greaves LC, Preston SL, Tadrous PJ, Taylor RW, Barron MJ, Oukrif
D et al. Mitochondrial DNA mutations are established in human
colonic stem cells, and mutated clones expand by crypt fission. Proc.
Natl. Acad. Sci. U.S.A. 2006; 103: 7149.

Journal of Gastroenterology and Hepatology 2015; 30 (Suppl. 1): 8592

2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

91

Confocal laser endomicroscopy & colitis

V Mac et al.

27 Taylor RW, Barron MJ, Borthwick GM, Gospel A, Chinnery PF,

Samuels DC et al. Mitochondrial DNA mutations in human colonic
crypt stem cells. J. Clin. Invest. 2003; 112: 135160.
28 Musquer N, Coquenlorge S, Bourreille A, Aubert P,
Matysiak-Budnik T, des Varannes SB et al. Probe-based confocal
laser endomicroscopy: a new method for quantitative analysis of pit
structure in healthy and Crohns disease patients. Dig. Liver Dis.
2013; 45: 48792.
29 Geboes K, Riddell R, Ost A, Jensfelt B, Persson T, Lfberg R. A
reproducible grading scale for histological assessment of
inflammation in ulcerative colitis. Gut 2000; 47: 4049.
30 Wright NA, Pike C, Elia G. Induction of a novel
epidermal growth factor-secreting cell lineage by mucosal
ulceration in human gastrointestinal stem cells. Nature 1990; 343:
825.
31 Tarnawski A, Stachura J, Durbin T, Sarfeh IJ, Gergely H. Increased
expression of epidermal growth factor receptor during gastric ulcer
healing in rats. Gastroenterology 1992; 102: 6958.
32 Harada S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA
et al. Induction of vascular endothelial growth factor expression by
prostaglandin E2 and E1 in osteoblasts. J. Clin. Invest. 1994; 93:
24906.
33 Wang D, Wang H, Brown J, Daikoku T, Ning W, Shi Q et al.
CXCL1 induced by prostaglandin E2 promotes angiogenesis in
colorectal cancer. J. Exp. Med. 2006; 203: 94151.
34 Pai R, Szabo IL, Soreghan BA, Atay S, Kawanaka H, Tarnawski AS.
PGE(2) stimulates VEGF expression in endothelial cells via

92

35
36

37

38

39

40

41

ERK2/JNK1 signaling pathways. Biochem. Biophys. Res. Commun.

2001; 286: 9238.
Capaldi RA. Structure and function of cytochrome c oxidase. Annu.
Rev. Biochem. 1990; 59: 56996.
Danese S. Inflammation and the mucosal microcirculation in
inflammatory bowel disease: the ebb and flow. Curr. Opin.
Gastroenterol. 2007; 23: 3849.
Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L,
Senger DR. Vascular permeability factor/vascular endothelial growth
factor: an important mediator of angiogenesis in malignancy and
inflammation. Int. Arch. Allergy Immunol. 1995; 107: 2335.
Scaldaferri F, Vetrano S, Sans M, Arena V, Straface G, Stigliano E
et al. VEGF-A links angiogenesis and inflammation in inflammatory
bowel disease pathogen-esis. Gastroenterology 2009; 136: 58595.
Coron E, Mosnier JF, Ahluwalia A, Le Rhun M, Galmiche JP,
Tarnawski AS, Matysiak-Budnik T. Colonic mucosal biopsies
obtained during confocal endomicroscopy are pre-stained with
fluorescein in vivo and are suitable for histologic evaluation.
Endoscopy 2012; 44: 14853.
Munjal C, Tyagi N, Lominadze D, Tyagi SC. Matrix
metalloproteinase-9 in homocysteine-induced intestinal
microvascular endothelial paracellular and transcellular permeability.
J. Cell. Biochem. 2012; 113: 115969.
Bardin N, Reumaux D, Geboes K, Colombel JF, Blot-Chabaud M,
Sampol J et al. Increased expression of CD146, a new marker of the
endothelial junction in active inflammatory bowel disease. Inflamm.
Bowel Dis. 2006; 12: 1621.

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