Abstract
purpose. To investigate the potential use of amniotic membrane transplantation
(AMT) in the construction of glaucoma filtering blebs.
methods. Twenty-four albino rabbits underwent glaucoma filtration surgery in one
eye. In alternate cases, the conjunctival flap was replaced with AMT.
Postoperative examination data were grouped into three time points. Six
animals with AMT and six filtration surgery-controls were euthanatized
at each of two postoperative time points, and tissue was obtained for
histologic examination. Conjunctival biopsies were explanted for
estimation of fibroblast outgrowth.
results. Bleb formation was observed in all eyes, and amniotic membranes were
epithelialized after 11.2 ± 2.48 (mean ± SD) days.
Throughout the study IOPs were significantly lower in operated than
unoperated fellow eyes. Between postoperative days 11 and 16 (the
middle time point), the percentage IOP reduction in AMT eyes was
significantly greater than in filtration surgery controls
(P = 0.014), though not at other time points.
Filtration surgery survival was significantly longer in the AMT group
(22.3 ± 3.8 days; mean ± SE) than in “No AMT” controls
(14.0 ± 1.6 days; P = 0.035). In tissue
culture, significantly less fibroblast outgrowth occurred from AMT
explants when compared with unoperated conjunctiva
(P = 0.01) between postoperative days 3 and 9 (the
early time point). Amniotic membrane transplants were intact on
histologic examination after 14 days but were associated with
considerable granulomatous inflammation. After 36 days, the ocular
surfaces remained clinically intact, but lysis of AMT was noted
histologically.
conclusions. AMT exhibits potential as an alternative tissue to conjunctiva in the
construction of glaucoma filtration blebs. The healing response as
demonstrated by fibroblast outgrowth is retarded when compared with
conventional conjunctival closure. The improvement in bleb survival
must be weighed against the potential for complications related to
delayed healing. In rabbits, human amniotic membrane elicited a late
xenograft reaction, leading to granulomatous inflammation and
dissolution of the membrane.
The formation of an adequate bleb after filtration surgery
may be compromised by subconjunctival fibrosis, which obstructs aqueous
outflow from the operation site. To combat this effect, antifibrotic
drugs such as 5-fluorouracil
1 2 3 4 5 6 7 8 9 and mitomycin
C
10 11 12 13 14 15 16 17 are commonly used at the time of filtration
surgery. However, these drugs may also influence the integrity of the
conjunctival barrier, resulting in a thin-walled avascular drainage
bleb.
18 19 20 21 The end result is often poor
epithelialization and increased susceptibility to leakage and
hypotony
3 22 23 24 or infection,
25 26 27 28 sometimes months or years after surgery. Conversely, when adjunctive
antifibrotics are not used, drainage surgery in certain patient groups,
such as those who have had previous conjunctival surgery, is more
likely to result in failure than success.
7
Replacement of conjunctiva over the filtration surgery site with a
tissue that heals less aggressively is an alternative approach to the
reduction of subconjunctival healing in glaucoma filtration surgery.
Amniotic membrane transplantation (AMT) has been used recently in the
reconstruction of the ocular surface after pterygium
excision,
29 in nonhealing corneal epithelial
defects,
30 31 32 in cicatrizing conjunctival
disease
33 34 and to retard corneal neovascularization in
patients with limbal stem cell deficiency.
35 Amniotic
membrane exhibits a number of characteristics that might be of benefit
in filtering bleb construction, that is, good epithelialization, good
integration with surrounding tissue, a low-healing response,
suppression of TGF-β activity,
36 poor immunogenicity,
and yet a high hydraulic conductivity.
37 The purpose of
this study was to investigate the potential usefulness of AMT in the
construction of glaucoma filtering blebs in an adapted animal
filtration surgery model.
Postoperatively, each eye received dexamethasone ointment daily
for 3 days. Postoperative examinations were carried out twice weekly
under general anesthesia (xylazine/ketamine as above). The IOP was
measured in each eye on each occasion using a Tonopen XL (Mentor,
Norwell, MA) with readings repeated to obtain a <5% disparity between
the highest and lowest readings. The frequency of aqueous leakage from
conjunctiva/AMT after surgery, and the degree of AMT epithelialization
was examined by staining the bleb surface with 2% fluorescein drops
(Chauvin Pharmaceuticals Ltd., Romford, United Kingdom). The stained
surface was observed for the presence of aqueous leakage and an
epithelial defect during each examination.
Conjunctival and amniotic membrane biopsies were explanted
individually in each well of 12-well tissue culture plates (Costar,
Cambridge, MA) in tissue culture medium containing Dulbecco’s modified
Eagles’s medium/F-12/M-199/Hepes buffer (Gibco, Grand Island, NY) with
20% fetal calf serum (Gibco) as previously described.
39 Each explant was incubated initially in one drop of tissue culture
medium at 37°C in a humidified atmosphere containing 5%
CO
2. After approximately 1 hour, 1 ml of tissue
culture medium was gently added to each well so as not to disturb the
explants.
Explants were examined daily for fibroblast and epithelial cell
outgrowth and photographed using a microscope-mounted 35-mm camera.
After 14 days the monolayers were fixed in 100% methanol and stained
with Gram Crystal Violet (Bacto; Difco Laboratories, Detroit, MI).
After fixation, wells were individually photographed, and a series of
5 × 7-inch black and white prints was produced, each representing
one well. The area of fibroblast outgrowth from each explant was
assessed from its respective photograph by planimetry using a Kurta
1212 Summasketch Tablet Board and Sigmascan software (SPSS Inc.,
Chicago, IL). The magnification factor was calculated from the ratio of
the diameter of a well on photographic print to the actual well
diameter and the actual area of outgrowth expressed in
cm2.
Samples taken for histology were mounted in paraffin, sectioned,
mounted, stained with either periodic acid-Schiff (PAS) or
Masson-Trichrome, and examined by light microscopy.
PAS-stained sections were examined by a masked observer (consultant
ophthalmic pathologist) for the presence and state of amniotic membrane
(whether intact or fragmented), presence of histologically complete
epithelialization, and degree of underlying inflammation.
Primary outcome measures included bleb formation, rate of
epithelialization, frequency of aqueous leakage, degree and duration of
IOP lowering, and the frequency of postoperative complications. The
rate of epithelialization of AMT, as judged by lack of fluorescein
staining by unepithelialized membrane, was calculated as the time
(days) from surgery to complete epithelialization.
IOP lowering in the operated eye was calculated as the percentage
lowering in comparison with the unoperated eye. The time to filtration
surgery failure was defined as the time to normalization of the IOP
when compared with the unoperated eye. Postoperative complications such
as bleb infection, hypotony, and intraocular hemorrhage were also
recorded.
Secondary outcome measures included quantification of fibroblast
outgrowth from AMT biopsies versus conjunctival biopsies in tissue
culture, the qualitative histologic state of the amniotic membrane
postoperatively, and the pattern of subconjunctival/submembrane
inflammatory response.
Bleb Formation, Epithelialization of Amniotic Membranes, and
Aqueous Leakage from Blebs.
Lowering of IOP Relative to Unoperated Eye and Filtration Surgery
Survival.
Complications.
Fibroblast Outgrowth from AMT Biopsies versus Conjunctival Biopsies
in Tissue Culture.
Histologic State of Amniotic Membrane.
The success of glaucoma filtration surgery is often hampered by
excessive fibrosis, which obliterates the subconjunctival space in the
vicinity of the filtration site reducing the area available for aqueous
drainage and absorption.
40 41 42 Antifibrotic drugs inhibit
this activity, thereby improving surgical success, but there is
widespread concern that these drugs result in an increase incidence of
postoperative complications.
28
A different approach to the reduction of postoperative scarring
after glaucoma filtration surgery would be to replace conjunctiva and
tenon’s capsule in the vicinity of the filtration site with a tissue
that has a lower propensity to scar. Suitable candidates should be
biocompatible and nonimmunogenic, as well as semipermeable to water.
These requirements are similar to the biological characteristics of
amniotic membrane. Placental membranes were first reported to assist
healing of exposed body surfaces in 1912.
43 When amniotic
membrane without chorion is used, healing occurs with less scarring
than with primary healing alone. A number of studies have reported
similar findings when amniotic membrane is used in ocular surface
reconstruction.
29 30 33 35 44 These effects are mediated
via an influence on conjunctival epithelial and subconjunctival
fibroblast function, promoting epithelial maturation,
45 46 and downregulating fibrogenic TGF-β signaling and myofibroblast
differentiation.
36 47 Finally, amniotic membrane is also
relatively permeable to water.
37
In this study, a trans-scleral cannula was used to prevent scleral
healing. This extra step prevents scleral closure, which would
otherwise occur at an early stage in the rabbit model. In the absence
of scleral closure as a confounding factor, bleb failure results
largely from subconjunctival fibrosis and bleb survival time can be
used as an index of the subconjunctival healing response.
AMT was observed to form satisfactory drainage blebs. The time to
complete epithelialization of the AMT bleb (11.2 ± 2.5 days,
mean ± SD) was longer than would normally be expected for
conjunctiva, but this did not appear to be of clinical importance and
was also comparable to epithelialization times reported with other uses
of AMT. The two cases of early leakage, both from the limbal edge,
highlight a potential problem when attempting to achieve a watertight
seal from suturing avascular tissue. This was corrected in later cases
by advancing the AMT edge over the limbus onto peripheral cornea and
adding extra sutures. There were no episodes of bleb-related infection,
intraocular hemorrhage, or sequelae of hypotony observed during the
study period. One eye had a low IOP at the time of harvesting. This was
attributable to a leak as discussed above.
AMT filtration procedures were shown to have longer survival than
control procedures in terms of IOP reduction
(Fig. 4) . This achieved
statistical significance at the middle time point
(Table 1) . Exclusion
of the two cases with persistent bleb leakage from the analysis
increased the
P value slightly (
P = 0.027
for absolute reduction and
P = 0.018 for percentage
reduction) but did not affect significance at the 95% confidence
level.
It is interesting that the IOP reduction in the control filtration
group was lower in the middle time point when compared with the
previous and subsequent time points. However, closer examination of the
data revealed that by chance a disproportionate number of early
failures had been harvested at day 14, thereby influencing the IOP
level at the middle (days 11–16) but not the late (days 28–38) time
point. There was no hypertensive phase noted in these animals.
The observation of significantly lower fibroblast outgrowth in tissue
culture from explanted AMT biopsies is a likely explanation for the
improved duration of survival. Previous studies have shown that AMT has
the ability to downregulate fibroblast activity chiefly through an
effect on TGF-β function. We consider this model to represent, as
closely as possible, the effects of subconjunctival fibrosis on
filtration function rather than scleral closure because of prevention
of the latter by the indwelling trans-scleral cannula. Scleral closure
would otherwise occur early in the rabbit.
Histologically, amniotic membranes were found to be intact after 2
postoperative weeks, whereas those examined at the end of the study
period showed significant granulomatous inflammation with giant cell
formation. This finding was also reflected in the significantly higher
fibroblast outgrowth in tissue culture at the late time point from
peri-AMT conjunctival biopsies. We presume the observed inflammatory
response was because of the xenogenic nature of the human AMT used in
rabbits. Similar findings have not been reported in previous studies in
which human to human transplants have been used.
There are two potential areas where we consider that an alternative
low-healing tissue might of use in filtration surgery. In certain first
trabeculectomies where there is deemed to be a high risk of failure or
in repeat trabeculectomies, a low-healing tissue might obviate the need
for antifibrotics. The second is in the revision of leaking or
dysesthetic filtration blebs in patients who have previously undergone
filtration surgery. Conventional revision of such blebs involves
advancement, rotation, or autografting of existing conjunctiva but is
often accompanied by an exaggerated subconjunctival fibroblastic
response and potentially filtration failure with loss of IOP
control.
48 An alternative tissue might be useful where a
muted wound healing response would reduce the risk of filtration
failure after surgical revision. In common with the use of antifibrotic
agents, the indications for the use of such tissue should be balanced
against the potential risk of later leakage and other late bleb-related
complications. The two cases of early leakage may indicate a potential
problem when translating this type of study into clinical practice, and
we have recently reported a problem with late leakage when using AMT in
the repair of leaking glaucoma filtration blebs.
49 An
important difference between these two studies is the prior exposure to
antiproliferatives of the patients in the latter study.
Estimating the potential for late filtration bleb leakage would be
problematic in the rabbit model. The rapid healing response observed in
the rabbit model in this study resulted in survival of only one AMT
bleb and no control blebs at the end of the study period. It is
unlikely that a longer period of observation would provide better
long-term data without further modification of the animal model.
In conclusion, the amniotic membrane transplanted rabbits
epithelialized adequately and demonstrated a longer trabeculectomy
survival in terms of IOP normalization than control animals, with
significantly lower rates of fibroblast outgrowth from the bleb wall in
tissue culture. Two cases were complicated by delayed healing, but
exclusion of these cases did not reduce the significance of the
improvement in bleb survival. These results suggest that amniotic
membrane may be an appropriate tissue to use in selected cases of
glaucoma filtration surgery. However, the disadvantage of a muted
healing response might be a higher risk of leakage after surgery.
Supported by the TFC Frost Charitable Trust, London, and the Iris Fund
for Prevention of Blindness, London, United Kingdom (KB); by National
Institutes of Health, National Eye Institute, Bethesda, Maryland, Grant
EY06819 (SCGT); and by Research to Prevent Blindness, New York, New
York (SCGT and DLB). KB and PTK also receive support from the National
Health Service (NHS) Executive, London, United Kingdom. The views
expressed in this publication are those of the authors and not
necessarily of the NHS Executive.
Submitted for publication September 25, 2000; revised February 14,
2001; accepted March 12, 2001.
Commercial relationships policy: P. SCGT has obtained a patent of the
method of preparation and clinical uses of human amniotic membrane and
has a financial interest in Bio-Tissue, a tissue bank that procures,
processes and distributes amniotic membrane for research and clinical
use.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked“
advertisement” in accordance with 18 U.S.C. §1734
solely to indicate this fact.
Corresponding author: Keith Barton, Moorfields Eye Hospital, 162 City
Road, London EC1V 2PD, UK.
keith1barton@cs.com
Table 1. Percentage versus Absolute Reduction in IOP
Table 1. Percentage versus Absolute Reduction in IOP
Time Point | Absolute Reduction in IOP Relative to Unoperated Eye (mmHg) | | | Reduction in IOP Relative to Unoperated Eye (%) | | |
| Control | AMT | P | Control | AMT | P |
Days 3–9 | 2.32 ± 0.74 | 2.47 ± 1.10 | NS | 17.6 ± 5.8 | 20.8 ± 5.2 | NS |
Days 11–16 | 0.25 ± 0.62 | 2.20 ± 0.50 | 0.023 | 2.00 ± 4.3 | 21.7 ± 6.0 | 0.014 |
Days 17–38 | 2.01 ± 0.62 | 1.29 ± 0.71 | NS | 10.1 ± 4.4 | 8.4 ± 3.5 | NS |
Table 2. Fibroblast Outgrowth from Explants in Tissue Culture
Table 2. Fibroblast Outgrowth from Explants in Tissue Culture
| Biopsy Site* | | | |
| 1, † | 2, ‡ | 3, § | 4, ∥ |
Early | | | | |
No. of biopsies | 24 | 24 | 14 | 12 |
Fibroblast outgrowth (cm2) | 0.205 ± 0.061 | 0.283 ± 0.096 | 0.0 ± 0.0 | 0.331 ± 0.166 |
Significance as compared with site 1, ¶ | | 0.50 | 0.003 | 0.39 |
Late | | | | |
No. of biopsies | 24 | 24 | 12 | 12 |
Fibroblast outgrowth (cm2) | 0.057 ± 0.017 | 0.234 ± 0.076 | 0.032 ± 0.029 | 0.022 ± 0.013 |
Significance compared with site 1, ¶ | | 0.032 | 0.46 | 0.096 |
Overall | | | | |
No. of biopsies | 48 | 48 | 26 | 24 |
Fibroblast outgrowth (cm2) | 0.131 ± 0.033 | 0.258 ± 0.061 | 0.015 ± 0.013 | 0.176 ± 0.088 |
Significance compared with site 1, ¶ | | 0.07 | 0.04 | 0.56 |
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