Morphologic and clinical effects of subretinal injection of indocyanine green and infracyanine green in rabbits.

PURPOSE: The aim of this study was to determine the effects of subretinal injection of indocyanine green (ICG), infracyanine (IfCG), and balanced salt solution (BSS) in rabbits. METHODS: Ten (10) animals were subjected to a subretinal injection of 0.05% ICG (279 mOsm), 0.5% IfCG (276 mOsm), and BSS (300 mOsm) used as a control. Animals were examined at 6, 12, and 24 h and 14 days following the surgical procedure by indirect binocular ophthalmoscopy, fluorescein angiography (FA), and light and transmission electron microscopy. RESULTS: The subretinal injection of ICG caused damage to all retinal layers and retinal pigment epithelium (RPE) during the entire follow-up. Subretinal injection of IfCG resulted in abnormalities of the photoreceptor outer segments (POSs) during the entire follow-up; however, abnormalities of the photoreceptor inner segments (PISs) and outer nuclear layer (ONL) were observed only 24 h and 14 days after surgery; no RPE damage was observed. FA showed that window defects were more prominent in the subretinal ICG bleb position than the IfCG-related area. BSS caused only abnormalities of the POS layer and no RPE alterations. CONCLUSIONS: Subretinal injection of 0.05% ICG results in more significant retinal damage than 0.5% IfCG. In this model, iodine-free IfCG demonstrates a safer profile than a tenfold lower concentration of ICG, which contains iodine in its composition.     

Subretinal injection of preservative-free triamcinolone acetonide and supernatant vehicle in rabbits: an electron microscopy study

Background

To evaluate the effect of injections of benzyl alcohol (BA)-free triamcinolone acetonide (TA) solution (MTA-PF) and the supernatant vehicle of TA (STA) containing BA into the subretinal space of rabbit eyes.

Methods

Sixteen rabbits underwent vitrectomy and subretinal injection of 0.02 ml of either 40 mg/ml MTA-PF, 40 mg/ml STA, or balanced salt solution (BSS). The animals were examined 6, 12, and 24 hours and 14 days after the procedure by fundus examination and fluorescein angiography (FA), as well as histological studies by light and transmission electron microscopy. The histological injury was classified in four stages: (1) stage 1, photoreceptor outer segment injury, (2) stage 2, stage 1 + photoreceptor inner segment injury, (3) stage 3, stage 2 + outer nuclear layer damage, and (4) stage 4, stage 3 + retinal pigment epithelium (RPE) damage.

Results

FA showed no window defects in areas where MTA-PF, STA, or BSS have been injected. Histological examination revealed that subretinal BSS-injection resulted in stage 1 damage during entire follow-up. Subretinal injection of MTA-PF resulted in damage stage 2 at 24 h and 14 days after surgery. However, at the STA position, stage 3 damage was noted 24 h and 14 days postoperatively. No RPE or choroidal damage was observed.

Conclusions

The histological lesions induced by subretinal STA were more relevant than the damage induced by MTA-PF. The vehicle BA may be involved in these abnormalities. The data indicate that care must be taken when using TA during internal limiting membrane peeling in macular hole surgery, due to the possibility of unintentional subretinal migration and for retinal pharmacotherapy.     

Effects of indocyanine green injection on the retinal surface and into the subretinal space in rabbits

PURPOSE: To evaluate the effects of indocyanine green (ICG) injection on the retinal surface and into the subretinal space of rabbit eyes. METHODS: Twenty-two Dutch-belted rabbits underwent two-port vitrectomy followed by injection of ICG (5 mg/mL) on the retinal surface and into the subretinal space. Balanced salt solution (BSS) was also injected subretinally. The locations where ICG was delivered (both epiretinal and subretinal) were exposed to light from an endoilluminator for 7 minutes. The animals were examined at 1, 7, and 14 days after surgery. The eyes were studied by fluorescein angiography as well as light and electron microscopy. RESULTS: No damage was observed after epiretinal ICG injection, but subretinal ICG injection resulted in damage to the outer nuclear layer, photoreceptor inner and outer segments, and retinal pigment epithelium. This damage was more severe with longer follow-up. Control experiments without ICG, in which balanced salt solution was injected into the subretinal space or light was delivered on the epiretinal surface, demonstrated only damage to the photoreceptor outer segments. CONCLUSION: Subretinal delivery of ICG (5 mg/mL) in rabbits induces retinal pigment epithelium, photoreceptor inner and outer segment, and outer nuclear layer damage. These mechanisms of damage may explain the retinal pigment epithelium changes that are sometimes seen after ICG-assisted internal limiting membrane peeling in humans.     

Biocompatibility of brilliant blue G in a rat model of subretinal injection

PURPOSE: To evaluate the toxicity of brilliant blue G (BBG) compared with those of indocyanine green (ICG) and trypan blue (TB) in a rat model of subretinal injection. METHODS: Retinal detachment was produced by subretinal injection of the dyes. The biocompatibility of BBG (0.25 mg/mL) was evaluated over 2 months and 2 weeks by ophthalmic examinations. The eyes were enucleated and analyzed by light, fluorescence, as well as transmission electron microscopy. Apoptotic cell death was detected by TdT-dUTP terminal nick-end labeling. The results were compared with those for ICG (5 mg/mL) and TB (1 mg/mL). RESULTS: ICG caused retinal degeneration and retinal pigment epithelial (RPE) cell atrophy 2 weeks after subretinal injection. Apoptotic cell death was detected in the inner and outer nuclear layers and the RPE layer, especially the photoreceptors. TB caused less retinal degeneration, mainly in the area detached by the subretinal injection. BBG had no detectable toxic effects after 2 months and 2 weeks. Apoptotic cell death was detected in the ICG and TB groups, mainly in the photoreceptors. CONCLUSIONS: Subretinal injection of the dyes caused retinal cell degeneration at lower concentrations than those reported for intravitreous injection. However, subretinal injection of BBG at 0.25 mg/mL appeared to provide satisfactory biocompatibility.     

Photoreceptor rescue in the RCS rat without pigment epithelium transplantation

Transplantation of normal retinal pigment epithelium (RPE) to the subretinal space has been reported to rescue photoreceptors in the RCS rat. Moreover, the rescue effect was surprisingly large considering the relatively small number of RPE cells transplanted. The reason for this widespread rescue of photoreceptors is not known, nor is the mechanism for outer segment phagocytosis in photoreceptors not apposed to the transplanted RPE cells. This suggests that the rescue effect may not be solely mediated by the transplanted cells. We therefore wished to test whether the transplantation surgery itself might contribute to the rescue of RCS photoreceptors. For these control experiments, we performed the surgery on juvenile RCS rats as described by others for the transplantation of RPE but instead of injecting RPE, we injected saline. We sacrificed the RCS control operates two months following surgery. In the area of the surgery (superior retinal quadrant) the outer nuclear layer (ONL) was up to 8-10 photoreceptor cells thick, while at the extreme inferior margin of the retina the ONL was almost eliminated. To investigate the role of temporary retinal detachment in photoreceptor rescue we repeated the above experiment using our trans-corneal approach to the subretinal space. This procedure results in a large temporary retinal detachment and little or no damage to the choroid and sclera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene therapy for detached retina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor

PURPOSE: To examine the protective effect of glial cell line-derived neurotrophic factor (GDNF) on retinal detachment (RD)-induced photoreceptor damage by using gene delivery. METHODS: Gene delivery to photoreceptors was achieved by subretinal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in the right eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the left eyes of Lewis rats. RD in bilateral eyes was induced with subretinal injection of high-density vitreous substitute in the temporal retina 3 weeks after gene delivery. The synthesis and accumulation of GDNF within the retina was monitored 3 weeks after RD by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. The rescue of photoreceptors was evaluated by monitoring the preservation of the thickness of photoreceptor outer segment (OS) and outer nuclear layer (ONL). Apoptosis in the photoreceptors was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method 2 days after RD. Müller cell activity was checked using the immunohistochemistry with glial fibrillary acidic protein (GFAP) antibody 28 days after RD. RESULTS: Gene delivery was demonstrated by immunohistochemical study. The results of ELISA confirmed that high levels of neurotrophic factors were produced in retinas. Photoreceptor OS degeneration and the gradual shortening of the ONL were noted after RD in all the eyes. However, rAAV-GDNF-treated eyes retained longer OS than rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. ONL was also longer in rAAV-GDNF-treated eyes than in rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. GDNF-treated eyes had statistically less apoptotic cells than control eyes in photoreceptor layer (P = 0.043). Subretinal proliferation of Müller cells was suppressed in the GDNF-treated group, indicating less scar formation. CONCLUSIONS: GDNF is a potential factor that can protect photoreceptors from degeneration. In addition to preserving the OS and ONL structures, GDNF may exert its protective action by preventing the apoptosis of photoreceptors after RD. GDNF gene therapy may be a valuable adjuvant to current treatments in certain complicated forms of RD.     

Xenotransplantation of retinal pigment epithelial cells into RCS rats

PURPOSE: Successful engraftment of retinal pigment epithelial cells (RPE) to treat RPE-related retinopathy will depend, at least in part, on controlling the immune response. In order to understand this process we evaluated the fate of RPE xenografts in the subretinal space, anterior chamber, and subcutis of nonimmunosuppressed Royal College of Surgeons rats. METHODS: Freshly isolated adult porcine RPE cells were used as xenografts and implanted when recipients were 17 to 21 days old. The extent of photoreceptor rescue by subretinal transplants was determined by counting the maximum layers of surviving photoreceptor nuclei in histologic sections. Cellular immune response was evaluated by immunohistochemistry. RESULTS: Compared to non- or sham-injected eyes, subretinal xenografts in RPE-transplanted eyes were able to induce a dramatic rescue effect (P <.01). However, the effect was not absolute and photoreceptor cell degeneration was only delayed. Xenografts both in the anterior chamber and in the subcutaneous tissue led to an inflammatory cellular infiltration. CONCLUSION: RPE xenografts in subcutaneous space and in the anterior chamber are rejected by a delayed but vigorous inflammatory cell infiltration. Subretinal RPE xenografts are protected from a strong cellular rejection, but seem to undergo a slow functional deterioration, reflected by a decline in their capability to rescue adjacent photoreceptors.     

Tomographic features of serous retinal detachment with multilobular dye pooling in acute Vogt-Koyanagi-Harada disease

PURPOSE: To clarify the morphologic background of serous retinal detachment with multilobular dye pooling on fluorescein angiography (FA) in acute Vogt-Koyanagi-Harada (VKH) disease. DESIGN: Retrospective uncontrolled case series. METHODS: setting: Institutional study. study population: Ten eyes of five patients aged 16 to 52 years (average age, 31 years) with VKH disease who presented with a serous retinal detachment. observation procedures: Clinical examination and optical coherence tomography (OCT). main outcome measures: Findings on OCT and FA. RESULTS: All eyes had multilobular dye pooling with a dark rim on late-phase FA. OCT revealed subretinal septa that divided the subretinal space into several compartments. The subretinal septum lay on the retinal pigment epithelium (RPE) in the foveal area, forming the posterior wall of the subfoveal compartment while it detached from the RPE in the perifoveal area, which served as a lateral wall between the subfoveal and perifoveal compartments. The dark rims of each area of subretinal dye pooling corresponded to the subretinal septa. Soon after steroid pulse therapy, the subretinal septa resolved completely in all eyes. CONCLUSION: Subretinal septa caused multilobular dye pooling in serous retinal detachment associated with acute VKH disease. We assume that the subretinal septa are comprised of inflammatory products such as fibrin, which immediately resolve after steroid pulse therapy.     

Subretinal delivery of immunoglobulin G with gold nanoparticles in the rabbit eye

Purpose  To examine the feasibility of subretinal delivery of immunoglobulin G (IgG) adsorbed onto gold nanoparticles (GNPs) and its histologic distribution in the rabbit retina after the injection. Methods  Goat IgG was adsorbed onto GNPs electrostatically. Goat IgG-adsorbed GNPs or buffer with goat IgG was injected into the subretinal space of rabbit eyes and followed up for 3 months by examination of fundus photographs, immunohistochemistry against goat IgG, and transmission electron microscopy (TEM). Human retinal pigment epithelial cells (ARPE-19 cells) were cultured, and cell proliferation with or without GNPs was assayed. Results  At 1 week after the subretinal injection of goat IgG-adsorbed GNPs, retinal degeneration was observed in the outer retina, and goat IgG was immunolabeled in the retinal pigment epithelium (RPE) and the photoreceptor cells. TEM showed GNPs located in the outer segments and in the lysosomes in the RPE at 1 month and no apparent cytotoxicity of the RPE. There were no inhibitory effects of GNPs on proliferation of ARPE-19 cells. Conclusions  Goat IgG was successfully delivered into photoreceptor cells and RPE using GNPs, though retinal degeneration in the outer retina occurred in this model. This might be an alternative drug delivery method to photoreceptors and RPE.     

Subretinal perfluorodecalin toxicity

Background: Subretinal injection of perfluorocarbon liquids (PFCL) can occur during vitreoretinal surgery. The long-term effects of this complication are not well established. Methods: A case report is presented of a patient with retained subretinal perfluorodecalin following retinal detachment repair for a giant retinal tear. Results: In the early postoperative period, the macular retinal pigment epithelium (RPE) became opalescent in appearance and by 2 months postoperatively the patient developed macular RPE atrophy with resulting poor central vision. Conclusions: Toxicity of subretinal perfluorodecalin causing RPE atrophy is proposed. We recommend all traces of PFCL should be removed if possible.     

Cyclosporine treatment of RPE allografts in the rabbit subretinal space

PURPOSE: To determine the effects of systemic cyclosporine A (CsA) on the survival of retinal pigment epithelial (RPE) allografts in the subretinal space in an animal model using atraumatic transplantation surgery. METHODS: Following pars plana vitrectomy, an RPE cell suspension from brown rabbits was injected with a glass micropipette into the subretinal space of 39 albino rabbits. For immunosuppression, 22 rabbits were given an injection of CsA, 20 mg daily intramuscularly, 17 rabbits with RPE grafts were controls. The grafts were monitored by biomicroscopy, color fundus photography, and fluorescein angiography. Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy including immunohistochemistry. RESULTS: After three months, the transplanted RPE cells, in both the CsA group and the controls, formed a monolayer in the subretinal space. Although a few macrophages were encountered, there was no massive cellular infiltration and the photoreceptor layer was well preserved. After six months, however, there was a disruption of grafted RPE cells in both groups, characterized by dispersion of melanin pigment in the subretinal space, and invasion of macrophages with focal photoreceptor damage but no infiltration of lymphocytes in the retina or choroid. No significant differences between the CsA treated and the control eyes were discernible. CONCLUSION: Although the subretinal space has been considered an immunologically privileged site, we found that the survival of RPE allografts was limited. CsA did not prevent RPE allograft destruction in the subretinal space. The transplant seems to be disrupted either by immunological mechanisms that are not inhibited by CsA, or by nonimmunologic events.     

Optimal conditions for long-term photoreceptor cell rescue in RCS rats: the necessity for healthy RPE transplants.

Retinal pigment epithelial (RPE) cells from normal Long-Evans, healthy Royal College of Surgeons (RCS) pigmented congenic (rdy+p+) and RCS pigmented dystrophic rats were transplanted into retinas of RCS non-pigmented dystrophic rats at post-natal days 17 and 26 (P17 and P26). When examined at P60, rescued photoreceptor cells were found in all transplanted groups. In addition, a small, but significant increase in the outer nuclear layer (ONL) thickness was detected in retinas injected with vehicle (sham control); however, the ONL thickness was reduced to control levels by 3-5 months after injections in both the sham controls and the group grafted with RCS pigmented dystrophic RPE cells. Likewise, large volumes of vehicle were injected into the subretinal space resulting in no long lasting beneficial effects. Also, there was no significant difference between the ONL thickness in retinas grafted with RCS congenic RPE and those grafted with Long-Evans RPE. When donor RPE cells from Long-Evans rats at neonatal and adult stages were transplanted into P26 RCS hosts, photoreceptor cell rescue could be found; however, the younger RPE cells affected a better rescue than those derived from adult eyes. In addition, when RPE cells from 6- to 9-day-old Long-Evans rats were transplanted into retinas of RCS dystrophic rats at 10, 17 and 26 days, significant rescue of photoreceptor cells was observed. Of these transplantation times, day 17 appeared to affect the best rescue of photoreceptor cells up to 1 yr. In contrast, little or no rescue was observed in the retinas of those RCS dystrophic rats when RPE cell transplantation was performed at P38, P43 and P48. The photoreceptor cell rescue was also found to be dependent on the concentration of RPE cells injected, which was maximal at 60,000-120,000 cells microliters-1. These findings lead us to conclude that in order to affect long-term, up to 1 yr, rescue of photoreceptor cells in the RCS rat, young, healthy RPE cells are required and transplantation needs to be performed at early stages of the disease process.     

Effects of hemicholinium-3, a photoreceptor and pigment epithelial toxin,on retinal adhesiveness and subretinal fluid absorption

Retinal adhesiveness and subretinal fluid absorption was studied in Dutch rabbit eyes given intravitreal injections of hemicholinium-3 (HC-3) which causes loss of photoreceptor outer segments and retinal pigment epithelial (RPE) damage. After HC-3 administration, some areas of the fundus showed pigmentary changes and others appeared normal. Small, non-rhegmatogenous retinal detachments were made in both areas. Within 2–5 days after HC-3 injection, only in the areas of visible damage, subretinal fluid spread laterally to make very flat retinal detachments, and the fluid absorbed very quickly. At later intervals, absorption was slower than normal, presumably because of scarring and RPE metabolic damage. HC-3 provides an experimental technique for transiently weakening retinal adhesiveness in vivo but its use as a model must account for the effects of both outer segment and RPE damage.     

Retinal degeneration and RPE transplantation in Rpe65(-/-) mice

PURPOSE: To determine whether transplanting normal retinal pigment epithelium (RPE) into the subretinal space influences photoreceptor function and degeneration in Rpe65(-/-) mice. METHODS: RPE cells were isolated from eyes of normal mice and transplanted to the subretinal space of one eye of Rpe65(-/-) mice. The other eye received a subretinal injection of saline or was not touched. Corneal electroretinograms (ERGs) from both eyes were monitored before and after surgery to follow progression of the degeneration. The width of the outer nuclear layer was measured in the area of transplantation and compared with a similar area in control retinas. RESULTS: Transplantation of RPE increased ERG amplitude maximally at 3.7 weeks after surgery. This rescue effect slowly diminished with time. Sham surgery had little effect on the ERG. The width of the outer nuclear layer in the area receiving RPE transplants was slightly greater than in control subjects. Evidence of the presence of RPE transplants in the subretinal space decreased with time after transplantation without signs of inflammation. CONCLUSIONS: Retinal degeneration in the Rpe65(-/-) mice is slowly progressive. Photoreceptor function can be transiently increased for several months and anatomic degeneration slightly reduced in Rpe65(-/-) mice by RPE cell transplantation. Loss of the rescue effect may be due to degeneration of the transplanted RPE.     

Clinicopathologic studies of eyes that were obtained postmortem from four patients who were enrolled in the submacular surgery trials: SST Report No. 16

PURPOSE: To compare the fundus photographic and fluorescein angiographic features with the histologic findings in eyes from patients enrolled in the Submacular Surgery Trials (SST). DESIGN: Clinical trials with clinicopathologic correlation. METHODS: Eyes that were obtained postmortem from patients who participated in the donor program were processed at the SST Pathology Center and examined histologically; the macular regions were reconstructed topographically with two-dimensional cartography. Fundus photographic and fluorescein angiographic features were correlated with the histopathologic and two-dimensional cartographic findings. RESULTS: The eyes from two patients each from the SST Group N and B Trials were studied. The study eye of one patient that had been assigned randomly to observation contained a subretinal fibrovascular scar that corresponded to a histologic growth pattern of a thick, collagenized subretinal component combined with a subretinal pigment epithelium (subRPE) fibrovascular component. The study eye of the other patient who was assigned randomly to observation showed angiographic occult without classic choroidal neovascularization (CNV) that corresponded to subRPE CNV. The study eye of one patient who was assigned randomly to surgery showed an angiographic surgical defect without CNV and histologic retinal pigment epithelium (RPE)/photoreceptor atrophy that was associated with a thin layer of subRPE CNV. The study eye of the other patient who was assigned randomly to surgery showed an angiographic surgical defect with classic CNV that corresponded to histologic RPE/photoreceptor atrophy that was associated with subRPE fibrovascular tissue and subretinal CNV. Both surgical eyes contained linear breaks in Bruch's membrane that included chevron-shaped breaks. CONCLUSION: Four SST study eyes that were examined postmortem contained CNV. The angiographic patterns and histologic features of the CNV support previous correlations of surgically excised CNV.     

Retinal detachment in the cat: the outer nuclear and outer plexiform layers

The retinae of cats were surgically detached for 1/2 hr to 14 months, and the outer nuclear (ONL) and outer plexiform layers (OPL) were studied by light and electron microscopy. The longer the duration or the greater the height of detachment the more likely was the occurrence of cell death. Histologic signs of degeneration were present 1 hr after detachment. The number of photoreceptor nuclei in the ONL decreased significantly by 1 month. Loss of cells in the ONL occurred by necrosis and by the migration of photoreceptor cell bodies into the subretinal space. The OPL degenerated by the necrosis of cell processes and synaptic terminals and by the retraction of the synaptic terminals. By 2 weeks most synaptic terminals were necrotic or in the process of retracting. Photoreceptor synaptic contact with second order neurons was diminished by 30 days and was essentially absent by 50 days. Müller cells proliferated and hypertrophied; their nuclei and cell processes filled the intraretinal spaces left by the degenerating photoreceptors. In addition, Müller cells protruded into the subretinal space and formed multiple layers of cell bodies and processes between the retina and retinal pigment epithelium. By 14 months these subretinal Müller cell processes covered the entire detached retina, and appeared morphologically like an astroglial scar. Similar changes in human retinal detachments may significantly influence the degree of visual recovery after retinal reattachment, especially in retinae detached for more than a few days.     

视网膜视细胞的成片移植

目的 探索用准分子激光切削技术制备视网膜单层细胞植片,经内入路视网膜下腔的单层视细胞成片移植。方法 用准分子激光对大鼠视网膜进行切削,制取单层视细胞植片,此后,按内入路手术方法进行了兔视网膜下腔的异种移植。结果 切削后所得视细胞植片由单层视细胞组成,结构完整,包括外丛状层、外核层和外节层;视细胞植片经明胶包埋后被准确植入宿主视网膜下腔中,移植术后第1,2天宿主观视网膜未能复位,呈脱离状态,移植物没能与视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节也宿主视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节与宿主视网膜色素上皮层相贴,未见明显免疫排异现象。结论 准分子激光制备单层视细胞植片方法简单、可行;初步观察到内入路单层视细胞成片移植后,视细胞植片能够在宿主视网膜下腔中以正常生理位置存活;视网膜下腔为理想的视网膜移植的受位。     

Fundus autofluorescence findings in a mouse model of retinal detachment

Purpose. Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD). Methods. RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4-5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence. Conclusions. In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.     

Assessment of Structure and Function Over a 3-year Period after Gene Transfer in RPE65−/− dogs

Aim: To assess retinal structure and function over a 3-year period in a group of five RPE65−/− dogs treated by unilateral rAAV- mediated subretinal gene transfer. Methods: Post-operative functional follow-ups were performed using simultaneous, bilateral, full-field ERGs. Structure was evaluated by SLO using FL and ICG angiography and by EM. Results: Significant improvement of retinal function was observed through ERGs approximately 4 weeks following surgery. Scotopic b-wave amplitudes peaked 3 months after surgery. Then there was a successive reduction, although greater amplitudes than base-line values were observed at all post-operative time points. A-wave amplitudes increased at a later time than b-wave amplitudes and were sustained throughout the follow-up period. The increased cone function was preserved longer than the rod function. Angiography showed structural changes at the site of injection, corroborated by photoreceptor destruction observed ultrastructurally. Immediately adjacent to the subretinal injection area photoreceptor outer segments appeared normal. Conclusion: Despite local structural alterations at the subretinal injection site, subretinal gene transfer in the RPE65 null mutation dog effectively increases retinal function for at least 3 years after surgery.     

Morphologic observations of retinal pigment epithelial proliferation and neovascularization in the rabbit

Neovascularization and proliferation of the retinal pigment epithelium (RPE) was induced in the rabbit by subretinal injection of vitreous without rupture of Bruch's membrane. New vessels developed between the layer of RPE and photoreceptor outer segments, but were enveloped in proliferating RPE. For this reason they were occult; no fluorescein leakage was visible by angiography. The vessels were identified only by histologic examinations. Endothelial cell budding was the initial stage of vessel development, first seen two weeks after injection. The new vessels grew from the choriocapillaris, penetrated Bruch's membrane, and spread into the subretinal space, despite the absence of subretinal fluid. Fenestrations with diaphragms were found in the endothelial walls during the earliest stages of vessel formation, and were also present in the fully matured vessels. Intermediate junctional complexes were frequently observed among the endothelial cells. During maturation of these plexi, junctions changed from open to putative tight junctions. The mature vessels were ultimately completely enveloped by collagen and RPE cells. Our results show that all new vessels in this animal model have the morphologic characteristics of choriocapillaris. We assume that they leak fluorescein, as does the choriocapillaris, but that the dye has no opportunity to pool in the subretinal space and thus cannot be seen during angiography.