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Purpose To discuss the potential contribution of rod and cone synapses to the loss of visual function in retinal injury and disease. Methods The published literature and the authors' own work were reviewed. Results Retinal detachment is used as a case study of rod spherule and cone pedicle plasticity after injury. Both rod and cone photoreceptors terminals are damaged after detachment although the structural changes observed are only partially overlapping. For second-order neurons, only those associated with rod spherules respond consistently to injury by remodeling. Examination of signaling pathways involved in plasticity of conventional synapses and in neural development has been and may continue to be productive in discovering novel therapeutic targets. Rho kinase (ROCK) inhibition is an example of therapy that may reduce synaptic damage by preserving normal synaptic structure of rod and cone cells. Conclusions We hypothesize that synaptic damage contributes to poor visual restoration after otherwise successful anatomical repair of retinal detachment. A similar situation may exist for patients with degenerative retinal disease. Thus, synaptic structure and function should be routinely studied, as this information may disclose therapeutic strategies to mitigate visual loss. more...

Purpose The RhoA pathway is activated after retinal injury. However, the time of onset and consequences of activation are unknown in vivo. Based on in vitro studies we focused on a period 2 hours after retinal detachment, in pig, an animal whose retina is holangiotic and contains cones. Methods Under anesthesia, retinal detachments were created by subretinal injection of a balanced salt solution. Two hours later, animals were sacrificed and enucleated for GTPase activity assays and quantitative Western blot and confocal microscopy analyses. Results RhoA activity with detachment was increased 1.5-fold compared to that in normal eyes or in eyes that had undergone vitrectomy only. Increased phosphorylation of myosin light chain, a RhoA effector, also occurred. By 2 hours, rod cells had retracted their terminals toward their cell bodies, disrupting the photoreceptor-to-bipolar synapse and producing significant numbers of spherules with SV2 immunolabel in the outer nuclear layer of the retina. In eyes with detachment, distant retina that remained attached also showed significant increases in RhoA activity and synaptic disjunction. Increases in RAC1 activity and glial fibrillary acidic protein (GFAP) were not specific for detachment, and sprouting of bipolar dendrites, reported for longer detachments, was not seen. The RhoA kinase inhibitor Y27632 significantly reduced axonal retraction by rod cells. Conclusions Activation of the RhoA pathway occurs quickly after injury and promotes synaptic damage that can be controlled by RhoA kinase inhibition. We suggest that retinal detachment joins the list of central nervous system injuries, such as stroke and spinal cord injury, that should be considered for rapid therapeutic intervention. more...

Cell-based therapy involving RPE transplantation might preserve or restore vision in AMD patients with evolving atrophy or in patients with other diseases in which vision loss is associated with dysfunctional RPE. Cell transplantation in patients with AMD has been attempted using a number of cell types and preparations, including fetal and adult RPE (autologous and allogeneic), translocated autologous choroid/RPE, and autologous iris pigment epithelium (IPE; see review by Binder1). Transplantation of autologous RPE and IPE is attractive because there is no risk of immune rejection. However, older cells: (1) do not behave as robustly as those from young donors,2–4 (2) may carry AMD-related gene defects or modifications caused by aging,1,5,6 and (3) may not have the ability to perform all the functions necessary to maintain the photoreceptors.5 Because fetal human RPE begin to show morphologic abnormalities after five to six passages, they are not suitable as a “universal” donor source, regardless of the possible immunogenicity of such cells.7 In addition, the supply of RPE from young donors is limited, so it would not be practical to develop a RPE transplant paradigm based on the use of such cells. Embryonic stem cells offer an advantage over fetal or adult RPE because of their ability to undergo large-scale expansion, assuring an abundant supply of well characterized, pathogen-free cells that can be manufactured in a manner compatible with clinical practice.8 Genetic analysis of such cells shows a high degree of similarity to in situ RPE.9 The method to generate RPE derived from human embryonic stem cells (hES-RPE) is reproducible and can be achieved in a manner that does not cause embryo destruction.10 Manipulation of hES-RPE in culture could take advantage of stem cell plasticity to optimize their ability to attach and survive on aged or diseased Bruch's membrane (BM) and to minimize rejection.11 To assess the potential of hES-RPE for cell replacement therapy in AMD patients, we compared the attachment and survival of hES-RPE of different degrees of pigmentation on BM with cultured human fetal RPE (fRPE) whose behavior has been characterized previously on aged and AMD BM.4,12,13 The goals of this study were to determine: (1) whether hES-RPE have the potential to attach and survive on aged BM; (2) whether a characteristic integrin mRNA profile can predict attachment and/or survival; (3) whether hES-RPE and fRPE have similar morphology after attachment to and growth on BM; and (4) whether hES-RPE secrete neurotrophic proteins after attachment and survival on aged human BM. Using the same hES-RPE preparations as in the present study, Lu et al.8 demonstrated long-term safety and functionality of hES-RPE after subretinal injection in rodents. These and other studies using hES-RPE derived in a similar fashion from spontaneously forming pigmented colonies in confluent hES cultures have shown that hES-RPE express RPE-specific genes, phagocytose outer segments, show polarization of Na+/K+ ATPase, and exhibit morphologic features of RPE.8,9,14–16 Therefore, hES-RPE might serve well for RPE replacement therapy in patients with retinal degenerations where the primary cause of vision loss is diseased or missing RPE. Although animal studies show that hES-RPE can survive in the subretinal space and rescue photoreceptors,8,14,16,17 such studies do not always predict the ability of cells to survive on diseased BM in AMD patients.18 Integrins are important for RPE attachment to BM (reviewed by Afshari19), and alpha integrin subunits have an important role in RPE adhesion to this surface.20 In cultured cells, integrin expression can be modulated by the degree of and time at confluence,21 and integrin upregulation through culturing improves RPE and iris pigment epithelium (IPE) adhesion to BM.4,20,22 One criterion by which one might assess hES-RPE as candidates for transplantation in AMD eyes is the presence of an integrin mRNA profile compatible with attachment to BM (e.g., a profile similar to that of cultured fRPE), which show robust attachment to BM by 24 hours after seeding.4,12,20 To determine whether the integrin mRNA profile can predict successful hES-RPE adhesion and survival on aged submacular human BM, we compared integrin mRNA expression and survival on BM of three different batches of hES-RPE of different degrees of pigmentation (harvested at increasing times in culture) with that of fRPE. In addition to replacing lost or diseased RPE cells with cells capable of performing RPE functions, transplanted cells could rescue dying photoreceptors through the secretion of proteins such as neurotrophic factors and cytokines. This idea is supported by in vivo studies showing that non-RPE cells can rescue photoreceptors despite their inability to phagocytose outer segments.23–26 Therefore, even if the cells do not appear to be fully differentiated on BM after transplantation, secretion of neuroprotective growth factors might have a rescue effect on the overlying retina. As a result, in the studies reported here, we also compared growth factor and cytokine secretion into the media overlying BM explants on which hES-RPE and fRPE were seeded. more...

PURPOSE. Cell transplantation has emerged as a possible remedy for degeneration and injury in the central nervous system (CNS). In the retina, photoreceptor transplantation is a potential treatment for retinal degenerative disease. Graft survival has been well documented, but evidence of functional recovery is lacking. A major obstacle to recovery of vision is lack of synapse formation between grafted photoreceptors and host bipolar and horizontal cells. A prior study demonstrated that photoreceptors prepared for transplantation undergo rapid morphologic changes, including retraction of axon terminals toward their cell bodies, away from potential synaptic partners, a phenomenon that may interfere with graft- host synaptic interaction after transplantation. In this study, prevention of retraction of photoreceptor axon terminals was possible by pharmacological intervention. METHODS. Photoreceptor sheets, prepared by vibratome sectioning, and full-thickness retinas, harvested from adult porcine eyes, were maintained in culture and treated with either the cyclic adenosine monophosphate analogue 8-(4-chlorophenylthio)-cyclic 3',5'-adenosine monophosphate (CPT-cAMP), or forskolin, an adenylyl cyclase stimulant, for up to 48 hours. RESULTS. Both CPT-cAMP and forskolin treatments successfully blocked retraction of photoreceptor axon terminals. This effect was not due to cell toxicity and was reversed after removal of treatment, indicating its specificity. CONCLUSIONS. Pharmacological manipulation of photoreceptor axonal plasticity may improve graft- host synaptic interaction after subretinal photoreceptor cell transplantation. more...

PURPOSE To determine whether iris pigment epithelium (IPE) cells can attach to aged submacular human Bruch's membrane and to assess whether IPE cells express the integrin subunits that may be necessary to bind to the known extracellular matrix ligands present in Bruch's membrane. METHODS IPE cells were seeded onto the RPE basement membrane (RPEbm) or inner collagenous layer (ICL) of aged submacular Bruch's membrane as microaggregates or were expanded in culture until enough cells could be obtained for seeding. Cell morphology and the percentage of cell coverage were determined 1 or 7 days after seeding. Messenger RNA was extracted from cultured and uncultured IPE cells and analyzed by RT-PCR. The expression of integrin subunits alpha1 to alpha6 and beta1 mRNA was examined. RESULTS Coverage by uncultured IPE was low on both surfaces at day-1 (RPEbm, 7.9% +/- 4.8%; ICL, 5.0% +/- 2.5%) with few intact cells present. Culturing IPE improved attachment with similar coverage on both surfaces and no significant difference between day-1 (RPEbm, 89.9% +/- 9.1%; ICL, 63.4% +/- 26.5%) and day-7 (RPEbm, 97.8% +/- 2.3%; ICL, 94.7% +/- 6.6%). By day-7, cell morphology and coverage on both surfaces was variable, ranging from few intact cells to a high degree of coverage by flattened cells. All integrin subunits studied were expressed in cultured cells, whereas alpha2, alpha3, and alpha4 showed less or no expression in uncultured cells. CONCLUSIONS Upregulation of integrin mRNA expression may be one explanation for the difference in coverage by cultured versus uncultured IPE cells. The presence of dead, dying, or flattened cells at day 7 indicates that IPE may not survive or differentiate on aged submacular Bruch's membrane. more...

Purpose To investigate systematically the early morphologic changes in axon terminals of adult mammalian rod and cone photoreceptors prepared as a sheet for subretinal transplantation. Methods An in vitro system was designed to maintain adult porcine retinas for up to 48 hours. Photoreceptor sheets, prepared by vibratome sectioning, and full-thickness retinas were cultured at temperatures similar to those in pretransplantation storage (4 degrees C) and after transplantation (37 degrees C). Changes in the outer nuclear and outer plexiform layers were analyzed, using immunohistochemistry, laser scanning confocal microscopy, and image analysis. Results Morphologic changes were observed in photoreceptor sheets as early as 10 minutes after incubation. The most significant change was the retraction of photoreceptor axons and terminals toward their cell bodies. Retraction was temperature dependent, being exacerbated at 37 degrees C compared with 4 degrees C, at its maximum by 24 hours of culture, and present in sheets obtained from both superior and inferior retina. The cause of this movement was not preparation techniques associated with vibratome sectioning or gelatin removal. Retraction was also present in full-thickness neural retina incubated at 37 degrees C. Reduction in outer nuclear layer cell counts and thickness were also evident in these preparations, primarily in photoreceptor sheets. Conclusions Adult photoreceptor sheets, a potential graft preparation for retinal transplantation, show a rapid retraction of axon terminals toward the cell bodies during culture. Although retraction may impede synaptic integration after transplantation, this intrinsic plasticity could be redirected to stimulate graft-host interaction. more...

Purpose To compare retinal pigment epithelium (RPE) resurfacing on the RPE basement membrane and inner collagenous layer (ICL) in human submacular Bruch's membrane explants. Methods Debridements were created in RPE-choroid-sclera explants (mean donor age 71.91 +/- 7.76 years) to create defects exposing the RPE basement membrane (RPEbm(+) defects), the ICL immediately below the RPE basement membrane (superficial ICL, [SICL]) or deeper layers of the ICL (DICL). Eleven pairs of eyes--four pairs with one eye having an RPEbm(+) defect and the fellow eye having an SICL defect and seven pairs with corresponding RPEbm(+) and DICL defects--were observed for 10 days by visualizing RPE ingrowth with 4',6'-diamino-2-phenylindole (DAPI) filters. At day 10, specimens were processed for scanning electron microscopy. Results Resurfacing of localized RPE defects occurred to some degree in all 11 pairs of eyes. No significant difference in the percentage of resurfacing of RPEbm(+) defects (67.35% +/- 18.82%) and SICL defects (64.26% +/- 16.07%) was observed although healing of the SICL showed more variability in the morphology of RPE cells migrating into the defect. Significant differences in healing were observed between pairs with RPEbm(+) defects versus DICL defects (84.07% +/- 15.35% and 54.00% +/- 14.54% resurfacing, respectively). RPE ingrowth into DICL defects exhibited the greatest morphologic variability. Conclusions RPE basement membrane supports RPE resurfacing of localized RPE defects. The deeper portion of the ICL of aged submacular human Bruch's membrane does not support RPE resurfacing to the same extent as does the RPE basement membrane. The poor RPE resurfacing observed in DICL defects mimics the histopathological findings in patients with age-related macular degeneration after excision of choroidal new vessels. more...

PURPOSE. To determine whether transduction with adeno-associated virus encoding green fluorescent protein (AAV-GFP) is useful for labeling transplanted retinal pigment epithelial cells (RPE). METHODS. Transduction was performed by infection of confluent or subconfluent cultured feline RPE or by subretinal injection. Cells transduced in vitro were analyzed to determine label stability over time and label conservation with cell division. RPE transduced in vivo were harvested at 5 weeks for transplantation or immunohistochemical detection. Two cats received subretinal injections of harvested cells and were killed at 3 or 7 days. RESULTS. In vitro transduction of confluent RPE resulted in stable GFP fluorescence for at least 3 months. There was a marked decline in fluorescence after cell division. Nonconfluent transduced cells conserved label after cell division but showed a marked decline in the number of cells, due to cell death. In vivo transduction resulted in a high level of labeling, allowing labeled cells to be harvested and transplanted. Transplanted cells were detected immunohistochemically. Photoreceptor labeling was detected over areas containing a high density of transplanted, labeled RPE derived from cells transduced in vivo. Possible light toxicity to transduced RPE was observed. CONCLUSIONS. AAV-GFP‐labeling of confluent cultured RPE and RPE in situ can be used to identify transplanted RPE, with some reservations. Cell division may cause dilution of the label, and release of cell contents into the subretinal space may cause label transfer to photoreceptors. Exposure to light of transduced cells should be limited. (Invest Ophthalmol Vis Sci. 2003;44:772‐780) DOI:10.1167/iovs.02-0091 more...