In Vivo Confocal Microscopy Study of Blebs after Filtering ... - Sanotek

In Vivo Confocal Microscopy Study of
Blebs after Filtering Surgery
Antoine Labbé, MD, 1,2 Bénédicte Dupas, MS, 1 Pascale Hamard, MD, PhD, 1 Christophe Baudouin, MD, PhD 1,2,3
Objective: To analyze bleb structure after filtering surgery at the cellular level using a new generation in vivo
confocal microscope.
Design: Observational case series.
Participants: We retrospectively evaluated 17 filtering blebs of 13 patients after trabeculectomy.
Methods: Ophthalmologic examinations included slit-lamp examination, applanation tonometry, and in vivo
confocal microscopy (Heidelberg Retina Tomograph II, Rostock Cornea Module). Eyes were classified into 3
groups: (1) functioning blebs (6 eyes), (2) nonfunctioning blebs (6 eyes), and (3) functioning blebs after application
of mitomycin C (5 eyes). Cellular patterns, morphologic appearance, and functional aspects of functioning and
nonfunctioning blebs were compared in a masked manner.
Main Outcome Measures: In vivo confocal microscopy images were analyzed for number of intraepithelial
microcysts, density of subepithelial connective tissue, presence of blood vessels, or encapsulation.
Results: All functioning blebs had numerous intraepithelial optically-empty microcysts, whereas all nonfunctioning
blebs had none or few. Subepithelial connective tissue was widely spaced in all functioning blebs,
whereas the tissue was dense in 83.3% of nonfunctioning blebs. Functioning blebs with mitomycin C had numerous
microcysts and loosely arranged subepithelial connective tissue as compared with nonfunctioning blebs.
Conclusions: In vivo confocal microscopy study of blebs is an original method that agrees well with ex vivo
histologic examination. The number of microcysts and the density of the subepithelial connective tissue observed
with in vivo confocal microscopy are correlated with bleb function. By providing details of the structures of
filtering blebs at the cellular level, in vivo confocal microscopy constitutes a new promising way to understand
wound healing mechanisms after filtering surgery. Ophthalmology 2005;112:1979–1986 © 2005 by the American
Academy of Ophthalmology.
Trabeculectomy has become the method of choice in
surgical treatment of patients with glaucoma. 1 This surgical
technique is usually associated with an elevation of
the conjunctiva overlying the scleral flap (i.e., the filtering
bleb). The long-term success of filtering surgery is not only
dependent on surgical technique; age, type of glaucoma, 2 ethnic
origin, prior failed filtering surgery or long-term use of
topical medications before surgery 3 are also critical factors.
However, the development of a filtering bleb, determined by
the postoperative wound healing process, is a major factor of
efficiency and long-term success of surgical procedures.
For these reasons, many authors have investigated
Originally received: January 27, 2005.
Accepted: May 29, 2005. Manuscript no. 2005-88.
1
Department of Ophthalmology III, Quinze-Vingts National Ophthalmology
Hospital, Paris, France.
2
INSERM U598, University of Paris 5, Paris, France.
3
Department of Ophthalmology, Ambroise Paré Hospital, APHP, University
of Versailles, Versailles, France.
Supported by Quinze-Vingts National Ophthalmology Hospital, Paris,
France.
Correspondence to Christophe Baudouin, MD, PhD, Service d’Ophtalmologie III,
C.H.N.O. des Quinze-Vingts, 28 rue de Charenton, Paris, 75012 France.
E-mail: baudouin@quinze-vingts.fr.
morphologic criteria of these blebs to correlate clinical
and functional aspects. 4,5 However, in some cases, there
is no correlation between bleb appearance or shape and
intraocular pressure. The reasons for bleb failure, related
to bleb scarring and histological changes within the bleb
tissue, are not easy to study in clinical practice.
In vivo confocal microscopy can provide details of
ocular structures at the cellular level, and it has already
been used in numerous studies of normal and pathologic
corneas. Recent advances in the in vivo confocal microscopy
technique have permitted visualization of peripheral
ocular structures. However, in vivo confocal microscopy
is a relatively new method for investigating conjunctiva. 6
In a preliminary report, we have presented the technique
allowing the description of blebs using in vivo
confocal microscopy. 7 The aim of the present study was
to quantify and to compare, in a masked manner the in
vivo confocal microscopy aspects of functioning and
nonfunctioning blebs, using the new confocal microscope
Heidelberg Retina Tomograph II/Rostock Cornea Module
(Heidelberg Engineering GmbH, Heidelberg, Germany)
to provide a better understanding of filtering
mechanisms and the wound healing process after filtering
surgery.
© 2005 by the American Academy of Ophthalmology ISSN 0161-6420/05/$–see front matter
Published by Elsevier Inc. doi:10.1016/j.ophtha.2005.05.021
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Patients and Methods
Ophthalmology Volume 112, Number 11, November 2005
In this pilot study, we retrospectively evaluated 17 filtering blebs
of 13 patients who had previously undergone trabeculectomy, and
who were followed-up at the Quinze-Vingts National Ophthalmology
Hospital, Paris, France. Six of the patients were female
(46.1%) and 7 were male (53.9%), whose ages ranged from 21 to
79 (meanstandard deviation; 55.516.4 years). There were 10
right eyes (58.9%) and 7 left eyes (41.1%). Of the 17 eyes, 15
(88.2%) had primary open-angle glaucoma and 2 (11.8%) had
juvenile open-angle glaucoma. In 5 cases (29.4%), mitomycin C
(MMC) had been intraoperatively applied for 2 minutes using a
small piece of surgical sponge soaked in 0.2 mg/ml MMC. After
exposure, the conjunctival flap area was immediately irrigated with
250 ml balanced salt solution (BSS Alcon, Fort Worth, TX).
The period between surgery and in vivo confocal microscopy
evaluation ranged from 1 to 228 months (meanstandard deviation;
47.963 months). First, all patients had a slit-lamp examination
and Goldmann applanation tonometry. This study was performed
in compliance with French regulations on biomedical
research, and all individual patients were informed of the aims of
recording these data, and their consent was obtained.
Patients were examined using a new in vivo confocal microscope
(i.e., the Heidelberg Retina Tomograph (HRT) II/Rostock
Cornea Module [Heidelberg Engineering GmbH, Heidelberg, Germany]).
The HRT II is a confocal scanning laser ophthalmoscope
that was initially developed for the analysis of the posterior pole of
the eye, especially the optic nerve head, and it has become a
standard for investigating glaucomatous changes of the optic
disc. 8,9 With the addition of the Rostock Cornea Module, the HRT
II is converted to an in vivo confocal microscope available for
investigating the ocular surface. 10 Before the in vivo confocal
microscopy examination, 1 drop of topical anesthetic (Novesine
0,4% [oxybuprocaïne 0.4 %] MSD-Chibret, Paris, France) and 1
drop of gel tear substitute (Lacrigel, carbomer 0,2%, Europhta,
Monaco) are instilled into the lower conjunctival fornix. The
patient is then seated at an examination table with the head into the
headrest. The patient fixates with the contralateral eye at a small
bright red light. The adjustment of the eye is performed by means
of the live image and under control of a charged-coupled device
color camera (640 480 pixels, RGB, 15 frames/s). The x-y
position of the image and the section depth are controlled manually.
The objective of the microscope is an immersion lens (Olympus,
Hamburg, Germany), magnification 60, covered by a polymethyl
metacrylate cap. The laser source used in the HRT II/
Rostock Cornea Module is a diode laser with a wavelength of 670
nm. Images consist of 384 384 pixels covering an area of 400
m 400 m, with transversal optical resolution of 2 m and
longitudinal optical resolution of 4 m (Heidelberg Engineering).
For all eyes, several confocal microscopic images of the superficial
epithelium and of the subepithelial connective tissue of the
conjunctiva located over the trabeculectomy site were taken. Each
eye was examined for less than 5 minutes and no complications
related to in vivo confocal microscopy evaluation were noted.
Eyes were divided into 3 groups: (1) functioning blebs (6 eyes;
35.3%), (2) nonfunctioning blebs (6 eyes; 35.3%), and (3) functioning
blebs after MMC application (5 eyes; 29.4%). In the
nonfunctioning bleb group (group 2), 2 clinical appearances were
analyzed: (1) flat blebs (5 blebs) and (2)encapsulated blebs (1
bleb).
Surgical success of filtering surgery was defined by the intraoc-
ular pressure (IOP), according to previous studies. 5,11,12 Functioning
blebs were defined as IOP 21 mmHg without antiglaucoma
treatment, and nonfunctioning blebs were defined as IOP 21
mmHg and/or the necessity for an antiglaucoma medication.
To ensure consistency, images were analyzed retrospectively
by a single researcher (CB) who was masked toward clinical
features and IOP control. In vivo confocal microscopy images
were evaluated for conjunctival and corneal epithelium changes,
presence and number of intraepithelial microcysts rated from 0
(none) to 3 (numerous) (Fig 1A), size of microcysts classified as
100 m and 100 m, density of subepithelial connective tissue
rated from 0 (loosely) to 3 (dense) (Fig 1B), presence of blood
vessels, or encapsulation of the bleb.
Statistical values of number of microcysts and density of subepithelial
connective tissue (meanstandard error of the mean
(SEM) were calculated in each group and compared using the
nonparametric Mann–Whitney U test. Probability values less than
0.05 were considered significant.
Results
For group 1, the functioning blebs in vivo confocal microscopy
showed several distinct characteristics. Conjunctival and corneal
epithelial cells were perfectly seen and had normal appearance
compared with normal eyes 13 or patterns found in areas distant
from the blebs (Fig 2 [available at http://aaojournal.org]). Between
normal epithelial cells, numerous optically clear spaces filled with
fluid were seen corresponding to microcysts as observed with the
slit lamp (Fig 3A). These microcysts were particularly numerous in
functioning blebs, with all the functioning blebs having a number
of microcysts rated 2 or 3 (meanSEM, 2.50.22; P 0.0001, as
compared with nonfunctioning blebs, meanSEM, 0.6670.21),
and were predominantly found in the conjunctiva adjacent to the
limbus. The size of the microcysts was between 10 and 150 m in
83.3% of the functioning blebs. Subepithelial connective tissue
images showed a loosely arranged tissue, with all the functioning
blebs having a density of connective tissue rated 0 or 1
(meanSEM, 0.6670.21; P 0.001, as compared with nonfunctioning
blebs, meanSEM, 2.50.34). This tissue was widely
spaced and contained clear spaces (Fig 3B). No blood vessel was
clearly seen in the subepithelial connective tissue.
Regarding group 2, that of the nonfunctioning blebs, conjunctival
and corneal cells were again perfectly seen with normal
appearance. Very few optical clear spaces corresponding to microcysts
were observed between superficial conjunctival cells
(meanSEM, 0.6670.21; P 0.0001, as compared with functioning
blebs, meanSEM, 2.50.22), and all the nonfunctioning
blebs had microcysts rated 0 or 1 (Fig 4A). Three (50%) of the
nonfunctioning blebs showed few microcysts containing optically
dense material (Fig 4B). Within the clinically encapsulated nonfunctioning
bleb, dense fibrotic tissue evocative of encapsulation
was observed (Fig 4C). Conversely, an encapsulation-like pattern
was observed with in vivo confocal microscopy in 1 clinically flat
nonfunctioning bleb. In an encapsulated bleb, microcysts were
observed mostly in the peripheral area of the bleb. The subepithelial
tissue of nonfunctioning blebs showed a dense, connective
tissue with few or no clear spaces, with 83.3% of these blebs
having a density that was rated 2 or 3 (meanSEM, 2.50.34;, P
0.001, as compared with functioning blebs, meanSEM,
3
Figure 1. In vivo confocal microscopy images of blebs (400 m400 m). A, Rating of microcysts in the conjunctival epithelium: 0, 1, 2, or 3. B, Rating
of density of subepithelial connective tissue: 0, 1, 2, or 3.
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Labbé et al In Vivo Confocal Microscopy and Filtering Blebs
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Figure 3. In vivo confocal microscopy images of functioning blebs (400 m400 m). A–D, Examples of numerous microcysts in the conjunctival
epithelium. E–H, Examples of subepithelial connective tissue widely spaced, containing clear spaces.
0.6670.21) (Fig 4D). Blood vessels were seen in the subepithelial
connective tissue of 4 (66.7%) nonfunctioning blebs (Fig 4D).
Regarding group 3, the functioning blebs with MMC, we
observed numerous clear spaces corresponding to large confluent
microcysts (Fig 5A) between morphologically normal conjunctival
epithelial cells. Four of 5 of these blebs had a number of microcysts
rated 2 or 3 (meanSEM, 2.20.22; P 0.001, as compared
with nonfunctioning blebs, meanSEM, 0.6670.21).
These microcysts had different sizes, ranging from 10 to 300 m.
Some of these microcysts showed hyper-reflective microdots in the
superficial epithelium layer (Fig 5A) in 3 blebs. The nature of these
hyper-reflective dots remains unknown, but they might have been
necrotic epithelial cells or inflammatory cells. The subepithelial
tissue was characterized by loosely arranged connective tissue
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Ophthalmology Volume 112, Number 11, November 2005
with numerous large clear spaces (Fig 5B), and 4 of 5 of these
blebs had a density of subepithelial tissue rated 0 or 1
(meanSEM, 0.80.37; P 0.0084, as compared with nonfunctioning
blebs, meanSEM, 2.50.34). In one of these blebs, an
encapsulation was observed with in vivo confocal microscopy. No
blood vessel was observed in the subepithelial tissue of functioning
blebs with MMC. These results and bleb confocal microscopy
image characteristics are shown in Table 1.
Discussion
The long-term success of trabeculectomy is mainly dependent
on the development of a functioning bleb. The forma-

Labbé et al In Vivo Confocal Microscopy and Filtering Blebs
Figure 4. In vivo confocal microscopy images of nonfunctioning blebs (400 m400 m). A–C, Examples of blebs having none or few microcysts in
the conjunctival epithelium. D, E, Examples of microcysts containing optically dense material. F, Encapsulation observed at the periphery of the bleb.
G, H, Examples of dense subepithelial connective tissue. I, Blood vessels in the subepithelial tissue.
tion and the maintenance of this functioning bleb, with
regard to wound healing and conjunctival scarring, are
therefore of primary importance. For these reasons, many
authors have presented classifications of these blebs to
correlate the morphologic criteria observed biomicroscopically
with the outcome of these blebs. Picht and Grehn 11,12
classified the developing, filtering bleb, showing that favorable
bleb development was characterized by microcysts of
the conjunctiva, paucity of vessels, diffuse bleb, and moderate
elevation of the bleb. In contrast they observed that
unfavorable bleb development was characterized by increased
vascularization, cork screw vessels, encapsulation
of the bleb and high-domed appearance. Because in some
cases the appearance of the bleb is not correlated to IOP,
and because the reason of failure is often unclear, some
authors have looked for new in vivo evaluation techniques
such as ultrasound biomicroscopy 14–16 to understand bleb
failure mechanisms. However, ultrasound biomicroscopy
evaluation or morphologic criteria observed biomicroscopically
can only indirectly observe and cannot analyze histological
changes explaining bleb failure.
The principle of confocal microscocopy was first described
by Minsky in 1957 17 The technological advances of
the past 40 years, have led to the development of in vivo
1979.e5

Figure 5. In vivo confocal microscopy images of functioning blebs with mitomycin C (MMC) (400 m400 m). A, B, Examples of large, confluent,
and numerous microcysts. C, D, Microcysts containing hyper-reflective microdots. E, F, Examples of loosely arranged connective tissue.
confocal microscopy for observation of the human cornea
under normal 6,18 or pathological conditions. 19–27 More recently,
the conjunctival epithelium and stroma could also be
observed at the cellular level using a new-generation in vivo
confocal microscope. 6
In a preliminary report, we described the first patterns of
blebs after filtering surgery using this new technique of in
vivo confocal microscopy. 7 In the present study, we further
analyzed these findings more precisely and compared the
different types of blebs observed after trabeculectomy. The
purpose of this study was to correlate the images obtained
with in vivo confocal microscopy, with regard to the number
of microcysts, the subepithelial tissue density, and the
presence of encapsulation or vessels, with the morphological
and functional aspects of these blebs.
Functioning blebs (group 1) showed a normal conjunctival
epithelium with numerous microcysts and a subepithelial
tissue arranged loosely and hypo-reflective, with a high
number of optically clear spaces. In contrast, nonfunctioning
blebs (group 2) showed none or very few microcysts, and
in this case, the subepithelial tissue was hyper-reflective, with
dense collagenous connective tissue and blood vessels.
For Powers et al, 28 who studied functioning blebs using
light and electron microscopy, the subepithelial connective
tissue was loosely arranged with widely spaced collagen.
These authors also described clear spaces in the superficial
substantia. In a study of blebs using light and electron
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Ophthalmology Volume 112, Number 11, November 2005
microscopy, Addicks et al 29 showed that nonfunctioning
blebs had dense collagenous connective tissue. For these
authors, functioning blebs had looser subepithelial connective
tissue with histologically clear spaces corresponding to
mycrocysts. Our results are in good consistency with ex
vivo histological analyses of functioning and nonfunctioning
blebs previously described. The presence and number of
these microcysts are assumed to be a positive predictive
factor for functioning blebs, and interestingly these microcysts
were observed in this study as early as the first month
after surgery. The morphologic analysis of functioning
blebs frequently showed these microcysts. 5,11,30 The in vivo
confocal microscopic examination of functioning blebs
seems to add strength to the argument that these microcysts
are channels for the passage of aqueous humor. 30
It has been well known for some time that the subepithelial
connective tissue of the blebs has a major influence
on IOP control. Fibrotic scarring in the subconjunctival
space and excessive extracellular matrix production has
been previously observed in nonfunctioning blebs. 29–31 In
this study, in vivo confocal microscopy images of functioning
blebs showing loosely arranged connective tissue and
nonfunctioning blebs showing dense subepithelial tissue
were well consistent with previous histological studies. 28,29
Mitomycin C is applied during filtering surgery to
reduce the risk of bleb failure. 32 By inhibiting cell proliferation,
MMC prevents an excessive healing response

Labbé et al In Vivo Confocal Microscopy and Filtering Blebs
Table 1. Individual Data and In Vivo Confocal Microscopy Characteristics
In Vivo Confocal Microscopy Evaluation
Microcysts Connective tissue
Follow-up Classification of
No. Eye Period (mos) Blebs Quantity (0–3) Size* Characteristics of Microcysts Density (0–3) Encapsulation Vessels
1 R 15 F 3 E 1 N N
2 R 228 F 3 E 1 N N
3 L 60 F 2 E 1 N N
4 R 5 F 2 E 1 N N
5 R 96 F 2 E 0 N N
6 R 27 F 3 E 0 N N
7 R 30 Non F, En 1 E 3 Y N
8 L 28 Non F 1 D 1 N N
9 L 6 Non F 1 D 2 N Y
10 L 120 Non F 0 — — 3 N Y
11 L 144 Non F 0 — — 3 Y Y
12 R 43 Non F 1 D 3 N Y
13 R 6 F with MMC 3 E, HMD 0 N N
14 R 2 F with MMC 2 E, HMD 1 N N
15 L 2 F with MMC 3 E, HMD 0 N N
16 R 1 F with MMC 1 E 2 Y N
17 L 1 F with MMC 2 E 1 N N
*Size of microcysts: 100 m; 100 m.
D dense; E empty; F functioning bleb; F with MMC functioning bleb with mitomycin C; HMD Hyper-reflective microdots; L left; N
no; Non F nonfunctioning bleb; Non F, En nonfunctioning clinically encapsulated bleb, R right; Y yes.
and scarring, and thus enhances the success of the procedure.
Shields et al 33 have used light and electron microscopy
to analyze an excised bleb after trabeculectomy
with MMC; they found numerous microcysts within the
epithelium and a loosely arranged connective tissue within
the subepithelium. Necrotic nuclei and cytoplasmic vacuoles
were also observed in the epithelium of these blebs using
electron microscopy. 29 The hyper-reflective microdots observed
with in vivo confocal microscopy between and
within microcysts would likely correspond to these necrotic
nuclei. In our study, in vivo confocal microscopy images of
functioning blebs with MMC well agreed with previous ex
vivo analyses.
In vivo confocal microscopy evaluation of blebs after
filtering surgery thus allowed us to study, at a cellular
level, the conjunctival wall of these blebs. By providing
in vivo high-resolution images of these structures, we
obtained results similar to ex vivo microscopic analyses.
However, we observed a nonfunctioning bleb, according
to clinical criteria, with a loosely arranged connective
tissue, and one functioning bleb with MMC that had few
microcysts. These few cases probably demonstrate the
limits of this evaluation. Only a limited part of the
conjunctiva covering the bleb is examined at a time, and
one cannot make conclusions from such focal examination.
Nevertheless, in vivo confocal microscopy evaluation
is now providing new insight into the analysis of filtering
blebs. This new method is rapid and noninvasive and it
allows clinicians and researchers to visualize in vivo
functioning or nonfunctioning blebs at the cellular level.
Using this technique we can observe directly the histological
processes that correlate with filtration or failure.
By studying the wound healing mechanisms after filter-
ing surgery in vivo, this technique will permit a better
understanding of filtration failure. Clinicians, with images
at a cellular level, would be able to predict the
outcome of these blebs and eventually provide specific
treatments to enhance success rates of their surgical
procedures.
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Labbé et al In Vivo Confocal Microscopy and Filtering Blebs
Figure 2. In vivo confocal microscopy images of (A) normal conjunctival and (B) corneal cells (400400 m).
1979.e9