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Replacing retinal pigment epithelium may be key to success in treatment of AMD
Poor submacular surgery results are largely due to removal of the adjacent retinal pigment epithelium and its basement membrane.
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PESARO, Italy – Combined CNV excision plus RPE replacement may restore good vision in age-related macular degeneration (AMD) patients, according to Marco Zarbin, MD, PhD, chairman of the New Jersey Medical School.
“Visual outcome after surgical excision of choroidal neovascularization (CNV) is usually poor in AMD patients. We believe these poor results are largely due to the removal of the adjacent retinal pigment epithelium (RPE) and the RPE basement membrane. By replacing the iatrogenically removed RPE, the results of CNV excision might be very different in these patients,” Dr. Zarbin said at the “Italiani d’America” meeting here.
AMD is an increasingly diffuse disease and one of the major causes of visual loss among the elderly. Approximately 70% of cases of severe visual loss in AMD are due to growth of subfoveal CNV under the RPE and the retina, with secondary exudative retinal detachment, subretinal hemorrhage and outer retinal degeneration.
Why CNV excision fails
“The rationale for surgical excision of CNVs is that removal of the CNV eliminates the source of bleeding, serous retinal detachment and subretinal fibrosis, all of which can cause retinal atrophy. Moreover, submacular surgery does not depend on a precise delineation of the CNV boundaries, in contrast to laser photocoagulation and photodynamic therapy, and is therefore potentially applicable to a much larger proportion of all patients with AMD-associated CNVs,” Dr. Zarbin said.
However, the results of this type of treatment have been disappointing so far. Among the factors limiting visual recovery, the removal of CNV-associated RPE at surgery may be the most determinant.
“After removal of the retinal pigment epithelium, native RPE cells are unable to repopulate the dissection bed, and this probably results in choriocapillaris and photoreceptor atrophy. That’s why this type of surgery, which has great potential for the treatment of AMD, should be associated with the replacement of the RPE,” he said.
Poor adhesion
RPE transplants in patients who had undergone CNV excision have been attempted in the past, but have failed.
“Allogeneic transplants are always susceptible to rejection, and systemic immune suppression is not usually tolerated by elderly patients for extended periods of time. Alternatively, the use of harvested autologous cells would avoid the problem of immune rejection of RPE grafts, and methods for culturing such cells do exist,” Dr. Zarbin said.
Also, some RPE transplants have been demonstrated to fail independent of immune rejection.
“Histopathological results from immune-suppressed patients and our own observations in vitro have shown that the graft may fail for other reasons than rejection,” he said.
“In particular, it has been demonstrated that if transplanted RPE cells cannot adhere to the dissection bed within 24 hours, they undergo apoptosis. When we transplant RPE cells in vitro into Bruch’s membrane of aged cadaver eyes, however, adhesion is poor.”
As Dr. Zarbin noted, successful RPE transplants in experimental animals have involved transplantation onto normal Bruch’s membrane, in contrast with the situation of AMD patients, where Bruch’s membrane is itself abnormal and, in most cases after CNV excision, lacks native RPE basement membrane.
“We assumed this might be the reason for graft failure in AMD patients: the transplanted cells don’t find a suitable environment for attachment and growth. Consequently, we chose to study RPE attachment to aged submacular Bruch’s membrane specimens in the presence and absence of native RPE basement membrane,” he said.
RPE basement membrane
In experimental studies, human RPE sheets were harvested from adult donor eyes and, following incubation in collagenase, were mechanically fragmented into microaggregates. RPE in microaggregates were seeded onto Bruch’s membrane specimens, with and without RPE basement membrane. The cultures were then incubated, and the percent coverage of the debrided surface by microaggregates was determined by sampling the center of the explants with scanning electron microscopy.
“RPE microaggregate attachment to Bruch’s membrane was significantly greater at all time points in samples with intact basement membrane,” Dr. Zarbin said.
“Based on their morphological appearance, many cells were dying as early as 1 hour following seeding. To increase surface coverage, we seeded four times the above number of cells and incubated the specimens for 1 hour. Coverage on explants lacking RPE basement membrane showed no increase in the number of cells attached to the inner collagenous layer. There was a significant, approximately threefold, increase in the number of cells attached in the presence of basement membrane.”
RPE ingrowth
An alternative to RPE transplantation is to stimulate RPE ingrowth from the edge of the dissection bed.
“Also in this case, AMD seems to create an unfavorable situation. While most in vitro RPE wound healing models are associated with complete wound resurfacing, human pathological studies indicate that RPE ingrowth following CNV excision in AMD patients is incomplete and aberrant,” Dr. Zarbin said.
“We attributed this behavior to the same cause that resulted in graft failure and did a similar type of experiment.”
Explants of RPE-choroid-sclera were excised from beneath the macula of 26 human donors. A 3-mm diameter RPE defect was created gently enough to leave the RPE basement membrane intact. For fellow eyes, RPE defects of the same size were created with the basement membrane removed, exposing the inner collagenous layer.
Organ cultures were maintained in serum-containing media for 10 days. At several time points during the incubation period, resurfacing of the RPE wound was photographed with fluorescence microscopy, using RPE endogenous fluorescence to follow the ingrowth of RPE cells. Organ cultures were fixed at day 10 and studied by scanning electron microscopy (SEM).
“About half of the explants of both groups showed no ingrowth at all. In the remaining specimens with intact basement membrane, more than 50% of the defect was covered by RPE,” Dr. Zarbin said.
“In contrast, although 11 specimens with RPE basement membrane removed showed ingrowth of RPE cells, the coverage of the wound was less than in the other group, and only three defects showed coverage of more than 50% of the surface.
“In summary, there is a rationale for RPE replacement after CNV excision in AMD patients. We hope that through continued experimentation, techniques to resurface RPE defects will be developed. These techniques, in conjunction with CNV excision, may lead to improved vision for patients with subfoveal CNV.”
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(REPRINTED WITH PERMISSION FROM THE BRITISH JOURNAL OF OPHTHALMOLOGY.) | (REPRINTED WITH PERMISSION FROM THE BRITISH JOURNAL OF OPHTHALMOLOGY.) |
(REPRINTED WITH PERMISSION FROM THE BRITISH JOURNAL OF OPHTHALMOLOGY.) |
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(REPRINTED WITH PERMISSION FROM THE BRITISH JOURNAL OF OPHTHALMOLOGY.) | (REPRINTED WITH PERMISSION FROM RETINA.) |
(REPRINTED WITH PERMISSION FROM RETINA.) |
For Your Information:
- Marco A. Zarbin, MD, PhD, can be reached at the Department of Ophthalmology, New Jersey Medical School, 90 Bergen St., 6th Floor, Room 6155, Newark, NJ 07103; (973) 972-2938; fax: (973) 972-2068.