An immunohistochemical study of regenerating newt retinas.

Light-microscopical examination was carried out to investigate the emergence and development of several classes of immunoreactive cells in regenerating retinas of the adult newt (Triturus pyrrhogaster) after total retinal ablation. Immunoreactive proliferating cell nuclear antigen (ir-PCNA, a marker for replicating cells) was present in nuclei of all neuroblasts in the early mono-layered to several-layered stages (15-20 days after retinal ablation; days 15-20), but was lost progressively in an intermediate-to-central/peripheral order as cells and layers increased (days 20-25). Cells, which had lost ir-PCNA, began to separate to form the outer nuclear, inner nuclear and ganglion cell layers around days 25-30 (the cell separation stage). Finally, the location of ir-PCNA was restricted to a band of neuroblast cells at the retinal margin (days 30-35) as seen in intact adult retinas. Visinin-immunoreactive (ir) cells, mainly destined to be cones, appeared first singly or as clusters at the most distal layer in the intermediate region of retinas multi-layered with PCNA-ir neuroblasts, which was followed by appearance of opsin-ir rod outer segments and tyrosine hydroxylase-ir amacrine cells around the cell separation stage. Shortly later, cells respectively immunoreactive to glutamic acid decarboxylase, neuropeptide Y, serotonin, glucagon, glutamine synthetase, glial fibrillary acidic protein, substance P and protein kinase C were found to emerge also in an intermediate-to-central/peripheral sequence. Some of the glucagon-ir cells appeared to be of an interplexiform type.     

Photoreceptor transplantation: anatomic, electrophysiologic, and behavioral evidence for the functional reconstruction of retinas lacking photoreceptors.

We have investigated the possibility of using transplantation of immature or mature rodent photoreceptors as well as mature human photoreceptors to reconstruct retinas in which photoreceptor degeneration is either inherited or environmentally induced. To this end, we have devised methods for isolating and transplanting the outer nuclear layer (ONL) (e.g., the photoreceptor layer) to the subretinal space of mature rodents. In addition we found that if portions of the inner retina are transplanted along with the intact photoreceptor sheet, photoreceptor organization is better maintained. In ultrastructural studies of the reconstructed retina an outer plexiform-like layer (OPL) is visible at the interface of the transplanted ONL and the host inner nuclear layer, with invaginating ribbon synapses characteristic of those formed by rod photoreceptors evident within this OPL. Ribbon synapses are found only rarely in unreconstructed retina. These results suggest that synaptic connections between transplanted photoreceptors and host cells may be made. Evidence for the potential recovery of function following photoreceptor transplantation is found in visually evoked cortical responses and behavioral responses (pupillary reflex) to light stimulation of the reconstructed eye. These findings suggest the possibility that neural transplantation can reconstruct a sensory end organ--in this case the retina--to restore evoked activity and an appropriate behavioral response to sensory stimulation.     

Long-term induction of c-jun mRNA and jun protein in rabbit retinal ganglion cells following axotomy or colchicine treatment

The expression of the c-jun, c-fos, and NGFI-A genes was studied in the rabbit retina after optic nerve crush (ONC) or an intravitreal injection of colchicine. By Northern blotting, the basal expression of c-fos and NGFI-A mRNAs were undetectable, whereas c-jun mRNA showed a low basal expression in shamoperated control retinas. Very few or no Jun- or Fosimmunoreactive nuclei were seen in control retinas. From 1 to 95 days after ONC a marked induction of JUN- but not FOS-immunoreactive neurons was seen in the ganglion cell layer peaking at 3 and 7 days. Jun-positive neurons also accumulated immunoreactive phosphorylated neurofilaments, indicating that they were ganglion cells. Northern blots demonstrated that retinal levels of c-jun mRNA, but not of c-fos or NGFI-A mRNAs, were increased 3 and 7 days after ONC. An intravitreal injection of colchicine also induced Jun-immunoreactivity within 24 hr in most of the neurons in the ganglion cell layer, but not in the inner nuclear and outer nuclear layers. The results indicate that axonal damage induces a specific pattern of IEG expression including a long-term induction of the c-jun gene in CNS neurons. © 1993 Wiley-Liss, Inc.     

Migration and synaptogenesis of cone photoreceptors in the developing mouse retina

Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated. J. Comp. Neurol. 388:47–63, 1997. © 1997 Wiley-Liss, Inc.     

Sodium nitroprusside selectively induces apoptotic cell death in the outer retina of the rat.

Sodium nitroprusside (SNP), an NO donor, was studied for its effects on apoptosis in rat retinal neurons. TUNEL-positive cells were observed in the outer nuclear layer (ONL), but not in the inner retina after SNP treatment. Inner retinal neurons died by necrosis. No photoreceptor cells were found in the ONL after seven days. Immunoblotting confirmed that neurnal NO synthase expression increased up to 5 days (approximately 170% of control levels), and then declined by 7 days, suggesting that NO induces apoptosis in the ONL, and that inner retinal neurons die by necrosis due to glutamate from damaged photoreceptors.     

Photoreceptor plasticity in reaggregates of embryonic chick retina: rods depend on proximal cones and on tissue organization

Plasticity of photoreceptors and their integration into epithelial structures homologous to an outer nuclear layer (ONL), was investigated in embryonic chick retinal cell reaggregates by immunohistochemistry using an antibody specific for red plus green cones (RG-cones) and an antibody for rods. If reaggregates are raised in the presence of pigmented epithelium (RPE), completely reconstructed, stratified retinal spheres are produced, where all rods and cones are integrated into an outer laminar ONL, similar to a normal retina. In the absence of RPE, 'rosetted' spheres form which contain internal rosettes homologous to an ONL. Only a minor fraction of cones and rods of 'rosetted' spheres are located within rosettes, while a larger fraction is diffusely displaced in nonorganized areas, thus, not contributing to an ONL-like epithelium. In both types of spheres, the total percentage of RG-cones was similar to the in vivo retina, indicating that expression of cones is autonomous. Following cones, after about one day, rods developed only within already existing RG-cone clusters. Thereby, the ratio of rods to RG-cones increases as the tissue organization decreases: for stratified spheres this ratio is, 0.50 (1 rod/2 cones; similar to mature retina); for rosettes, 0.74 (3 rods/4 cones) and for nonorganized areas, 1.09 (1 rod/1 cone) -- a higher ratio under our conditions has never been detected. Thus, rod expression depends strictly on the presence of nearby cones; their relative numbers are distinctively adjusted according to the cytoarchitecture of the tissue environment. The biomedical implications of these findings are briefly discussed.     

Investigation of the migration path for new rod photoreceptors in the adult cichlid fish retina

In the retina of adult teleost, precursor cells divide in the outer nuclear layer and give rise to new rod photoreceptors. These new rods migrate from the outer limiting membrane to the inner edge of the outer nuclear layer (ONL) before differentiating. In order to understand which cues these cells use during migration and insertion at the appropriate location we combined cell-specific stains in the retina of the cichlid fish Haplochromis burtoni, viewed with confocal laserscan microscopy: Dividing cells were labeled with bromodeoxyuridine (BrdU), Müller glial cells, cone photoreceptors, and horizontal cells were detected by specific antibodies. During the migration phase (24 to 48 h after BrdU uptake) up to 46% of BrdU-labeled cells were spindle shaped and radially oriented. Most of them were in direct proximity to Müller cell processes. Four days after BrdU-uptake, most labeled cells (91%) were found in the inner portion of the ONL and displayed a spherical shape. This marks the end of the movement of the new rods. At this stage, the labeled cells showed no preference to lie near glial fibers but were often found close to the pedicles of double cones. The leading edge of migrating cells reached into the outer plexiform layer (OPL) but not further than processes of horizontal cells. This is beyond the location of mature rods. We hypothesize that the cells are repelled in the OPL and insert back in the ONL to differentiate as rods.     

Optic nerve crush modulates proliferation of rod precursor cells in the goldfish retina

Tritiated thymidine ([3H]TdR) autoradiography revealed a correlation between the rate of cell proliferation of rod precursor cells in the outer nuclear layer (ONL) of the goldfish retina and the postoperative interval after crush of the optic nerve (ONC). Ten days after unilateral ONC there were more labeled nuclei in the ONL of the nerve-crushed retina than in the intact, contralateral retina of the same fish one day after single bilateral intraocular injections of [3H]TdR. From 15 to 25 days after ONC, however, fewer labeled nuclei were found in the ONL of nerve-crushed retinae than in controls, illustrating a decrease in the number of cells entering the S-phase of the cell cycle; by 35 days the differences had disappeared, demonstrating that cell birth recovered to normal. When examined one month after [3H]TdR injection, fewer labeled cells were present in the nerve-crushed retina at all postoperative intervals. Examination of the numbers of labeled cells at various postoperative periods following bilateral ONC, when one retina was examined one day and the other retina was examined one month after [3H]TdR administration revealed that the ratios of labeled cells between the two retinae varied as a function of time after ONC. Therefore, optic nerve crush appears to enhance the proportion of initially labeled cells in the ONL that are either fated to undergo further cell generation cycles or to die.     

Timing and location of rhodopsin expression in newly born rod photoreceptors in the adult teleost retina

Labeling of newly divided retinal cells with bromodeoxyuridine (BrdU) and a rhodopsin mRNA probe revealed that rhodopsin is first expressed by new rod photoreceptors 2 days after cell birth in an adult cichlid fish. Most new cells that expressed rhodopsin had nuclei located in the vitreal half of the outer nuclear layer (ONL), lending further support to the hypothesis that movement from scleral to vitreal ONL is associated with rod differentiation.     

Sensitivity of ERG components from dark-adapted goldfish retinas treated with APB

The electroretinogram (ERG) of the dark-adapted goldfish was examined before and after intravitreal injection of DL-2-amino-4-phosphonobutyric acid (APB). APB abolished the b-wave and decreased absolute sensitivity of the remaining waveform, which was composed of a vitreal-negative component followed by a vitreal-positive component. The sensitivity, time course and amplitude of these components differed from ERGs obtained from animals treated with sodium aspartate. Spectral sensitivity of both post-APB components closely resembled that of the normal dark-adapted b-wave. The results suggest that APB does not act selectively on any particular class of photoreceptors or photoreceptor pathways in the dark-adapted goldfish retina.     

Neuroprotective effect of atrial natriuretic peptide against NMDA-induced neurotoxicity in the rat retina

Atrial natriuretic peptide (ANP) can regulate aqueous humor production in the eye and has recently been suggested to play some functional roles in the retina. It has also been reported that ANP increases tyrosine hydroxylase (TH) mRNA levels and intracellular dopamine levels in PC12 cells. The effect of ANP on TH levels and the role of ANP in retinal excitotoxicity remain unknown. In this study, we investigated the effects of ANP on TH expression and dopamine levels in rat retina after intravitreal injection of NMDA. Immunohistochemistry localized natriuretic peptide receptor-A (NPRA) in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) in the rat retina. Quantitative real-time PCR and Western blot analysis showed a dramatic reduction in retinal TH levels 5 days after NMDA injection, while ANP, at a concentration of 10(-4) M, ameliorated this reduction in TH mRNA and TH protein levels. High-performance liquid chromatography (HPLC) analysis showed that NMDA reduced dopamine levels in the retina, and that ANP attenuated this reduction. Moreover, morphological analysis showed that ANP ameliorated NMDA-induced neurotoxicity through NPRA. The ameliorative effect of ANP was inhibited by a dopamine D(1) receptor antagonist. These results suggest that ANP may have a neuroprotective effect through possible involvement of dopamine induction.     

Retinal growth and cell addition during embryogenesis in the teleost, Haplochromis burtoni.

Neurogenesis of the developing embryonic retina is described for the African cichlid fish, Haplochromis burtoni, from 4 days post fertilization until all cell phenotypes are generated (day 7). Cell addition and differentiation both begin at the same absolute location which later becomes the central retina. As observed in most other vertebrates, cones and ganglion cells differentiate first, followed by amacrine and bipolar cells. Rod photoreceptors, which are added late, differentiate last. Changes in retinal thickness, retinal stretching, cell size, and cell density were measured during development. From day 4 through 7, there is an increase in retinal thickness largely due to the expansion of the inner plexiform layer (IPL) and outer nuclear layer (ONL). The inner nuclear layer (INL) decreases in thickness and there is a transient decrease in the density of cells in the scleral portion of the INL. Cells increase in size in the ganglion cell layer (GCL) and the vitread INL, decrease in size in the sclerad INL, and remain the same in the ONL. Changes in the density of the cell layers were observed: the density of ONL cells increased, the density of GCL cells decreased, and INL cells increased then decreased. From day 4 to day 6, eye growth is entirely due to cell addition because no retinal stretching was observed in the ONL or the horizontal layer. During this same developmental period, the pattern and rate of neurogenesis were measured in the differentiated portion of the retina by means of 3H-thymidine labeling. A small number of cell divisions within the differentiated INL precede the onset of cell divisions in the ONL. The number of 3H-thymidine labeled cells within the INL increases at a low rate consistent with an asymmetric pattern of cell division characteristic of stem cells. In contrast, cell divisions in the ONL increase exponentially, consistent with a symmetric pattern of cell division characteristic of progenitor cells. Double-label experiments (3H-thymidine and a rod specific opsin antibody) show that some of the symmetrically dividing cells in the ONL express the rod specific opsin within 2 days, suggesting that these dividing cells are rod progenitors. Although we do not hae conclusive evidence, these developmental processes support the hypothesis that stem cells within the INL could be the source of rod precursors in the embryonic teleost retina.     

Postnatal neurogenesis in the kitten retina.

Postnatal neurogenesis in the kitten retina was studied using 3H-thymidine radioautography. Kittens were injected with 3H-thymidine at 1 day, 10 days, 3 weeks or 4 weeks after birth and allowed to survive until 14 weeks of age. Labeled neuronal nuclei were not found in the ganglion cell layer in any of the retinas, but they were seen in the other nuclear layers of the same retinas. In retinas from kittens injected at one day after birth, the peripheral 80% of the length of the retina (in sections cut parallel to the dorsoventral meridian) contained labeled nuclei; the central 20%, around the optic disc, contained no labeled nuclei. Near the ora serrata most nuclei in both inner and outer nuclear layers were labeled. Away from the ora serrata the proportion of labeled to unlabeled nuclei gradually decreased. Labeled nuclei extended farther centrally in the the inner than the outer nuclear layer. The same pattern of labeling was repeated in retinas from kittens injected at ten days after birth, but fewer nuclei were labeled, and the central, unlabeled region around the optic disc was longer--55% of the length of the retina. Only a few nuclei near the ora serrata were labeled in retinas from kittens injected at three weeks after birth, and no labeled neurons were found in kittens injected at four weeks. From these results we conclude that all of the ganglion cells in the kitten retina are present by one day after birth, as are all of the other neurons in the central retina. In peripheral regions of the inner and outer nuclear layers, proliferation of cells destined to become neurons continues up to three weeks after birth.     

Rat retinal microglial cells under normal conditions, after optic nerve section, and after optic nerve section and intravitreal injection of trophic factors or macrophage inhibitory factor

Retinal microglial cells may have a role in both degeneration and neuroprotection of retinal ganglion cells (RGC) after optic nerve (ON) section. We have used NDPase enzymohistochemistry to label adult rat retinal microglial cells and have studied these cells under normal conditions, after left ON section, and after left ON section and eye puncture or intravitreal injection of different substances: vehicle, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), or macrophage inhibitory factor (MIF). Resident microglial cells are present in four layers in the adult rat retina: the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Left ON section induces microglial activation in the ipsilateral and contralateral retina as manifested by stronger staining intensity in both retinas and increased microglial cell densities in the NFL, IPL, and GCL of the ipsilateral retina. Left ON section followed by left eye puncture or intravitreal injection increases microglial cell density in both retinas and induces changes in the microglial cells of the ipsilateral retina that vary depending on the substance injected: BDNF injections delay microglial activation, possibly through retinal ganglion cell neuroprotection, whereas NT3 partially inhibits microglial activation in the NFL; MIF injections have no clear effects on microglial activation. In conclusion, retinal microglial cells become activated after an ON section and react more intensely when the eye is also punctured or injected, and this response may be altered by using neurotrophic factors, although the effects of MIF are less clear.     

Regression of retinal capillaries following N‐methyl‐D‐aspartate‐induced neurotoxicity in the neonatal rat retina

Degeneration of retinal capillaries occurs following N‐methyl‐D ‐aspartate (NMDA)‐induced retinal neurotoxicity, and the degree of capillary degeneration decreases in an age‐dependent manner. To determine the role of vascular endothelial growth factor (VEGF) in the high susceptibility of capillaries to neuronal damage during the early postnatal stage, this study compares the vascular regression patterns between NMDA‐treated retinas and retinas treated with N‐[2‐chloro‐4‐{(6,7‐dimethoxy‐4‐quinazolinyl)oxy}phenyl]‐N′‐propylurea (KRN633), a VEGF receptor tyrosine kinase inhibitor, in neonatal rats. Two days after a single intravitreal injection of NMDA (200 nmol/eye) on postnatal day (P) 7, substantial retinal neuron loss and delayed expansion of the retinal vascular bed were observed. The reduction in the capillary density in the central retina reached statistical significance 4 days after NMDA treatment. In retinas of rats injected subcutaneously with KRN633 (10 mg/kg) on P7 and P8, simplified vasculature attributable to capillary regression and prevention of endothelial cell growth were seen on P9, whereas no visible changes in the morphology of the retinal layers were observed. The degree of capillary degeneration in NMDA‐treated retinas was less than that in KRN633‐treated retinas. No apparent changes in immunoreactivities for VEGF were found 2 days after NMDA treatment. These results indicate that neuronal cell loss in the retina precedes retinal capillary degeneration following NMDA treatment, and VEGF‐dependent immature capillaries might be more susceptible to NMDA‐induced neuronal damage. © 2014 Wiley Periodicals, Inc.     

Intraocular injection of folate antagonist methotrexate induces neuronal differentiation of embryonic stem cells transplanted in the adult mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuronal cells. However, the integrated ES cells also have a tumorigenic effect just because they have the ability for multipotential differentiation to various types of tissues. Thus, control of neoplastic potentials of ES cells is very important for the treatment of degenerative or injured diseases. Mouse ES cells carrying the sequence for the green fluorescent protein (GFP) gene were transplanted into adult mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. One week after the ES cell injection, folate antagonist methotrexate (MTX) was injected intravitreally. Eyes were retrieved 4 weeks after ES cell transplantation for histologic analyses. Conventional histological analysis was performed by hematoxylin and eosin staining with the use of paraffin-embedded sections. Neuronal differentiation and teratogenic potential of ES cells were demonstrated by immunohistochemistry. The proliferative activity of transplanted cells was detected by mitotic index, proliferating cell nuclear antigen index and AgNOR count. The incorporation of transplanted ES cells in MTX-treated and non-treated retinas at 4 weeks after transplantation was observed in 8/16 eyes (50%) and 8/16 eyes (50%), respectively. Transplanted ES cells in MTX-treated retina showed increased neuronal differentiation and decreased expression of teratogenic markers, compared with ES cells in non-treated retina. The proliferative activity of transplanted ES cells in MTX-treated retina was lower than that in non-treated retina. These results suggest that intravitreal MTX treatment following transplantation can induce neuronal differentiation in the transplanted ES cells and decrease their proliferative activity.     

Retinal cell and photoreceptor transplantation between adult New Zealand red rabbit retinas.

Adult New Zealand Red (NZR) rabbits were used to determine if the techniques developed in the rat to transplant sheets of photoreceptors could be adapted to larger eyes of the rabbit. Adult donor NZR rabbit retina was cut into small strips, oriented, and drawn up in a small and flat polyethylene tube. By transscleral approach, donor retina was correctly oriented and gently placed into the subretinal space of host retinas that were previously ablated of photoreceptors by intravitreal injection of hemoglobin. Two weeks after surgery, rabbits were terminated and eyes processed for histology. Morphologic evaluation indicated that photoreceptors were transplantable between adult rabbits by adaptation of previously developed methods and that they were well maintained for 2 weeks. Transplanted photoreceptors also appeared to retain nearly normal inner and outer segment (I & OS) morphology and anti-opsin reactivity. Finally, inclusion of the inner retina also appeared to help maintain the correct I & OS apposition to the host retinal pigmented epithelium.     

Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina

The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore-forming subunit of the L-type calcium channel, Ca(v)1.4. The loss of the electroretinogram b-wave in these mice suggests a severe reduction in transmission between photoreceptors and second-order neurons in the retina and supports a central role for the Ca(v)1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Ca(v)1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre- and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre- and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites.     

Photoreceptor cell rescue at early and late RPE-cell transplantation periods during retinal disease in RCS dystrophic rats

Normal retinal pigment epithelial (RPE) cells were transplanted into retinas of Royal College of Surgeons (RCS) dystrophic rats at different stages of the retinal disease process. RPE-cell transplantation at 10, 17 and 26 days resulted in rescue of photoreceptor cells, such that at 4 months the outer nuclear layer (ONL) was 8-10 cells in thickness as shown in retinas of age-matched control rats. Of these transplantation times, day 17 appeared to affect the best rescue of photoreceptor cells. Nongrafted retinas of 4 month-old RCS dystrophic rats exhibited scattered PRC's, most prevalent in the peripheral retina. In addition, a small, but significant increase in the ONL thickness was detected in vehicle-injected retinas (sham control) of 17 day-old RCS dystrophic rats at 2 months; however, at 3 months, the ONL thickness was reduced to control levels. A normal distribution of (Na+ + K+)-ATPase immunostain was demonstrated beneath grafted RPE cells in retinas of 4 month-old RCS dystrophic rats. Dense immunostaining was shown along rescued photoreceptor cell inner segments (IS), within the inner (IPL) and outer (OPL) plexiform layers and on plasmalemma of cell bodies in the inner nuclear layer (INL). In nongrafted retinas of age-matched RCS dystrophic rats, immunostaining for (Na+ + K+)-ATPase was observed only in the INL and IPL. Under RPE-cells transplants in retinas of 4 month-old RCS dystrophic rats, opsin immunostaining was detected along both rescued photoreceptor cell inner and outer (OS) segments and on plasmalemma of ONL cell bodies. However, immunostaining for opsin was restricted to a debris zone in nongrafted retinas of age-matched RCS dystrophic rats.(ABSTRACT TRUNCATED AT 250 WORDS)