Royal College Of Surgeons Retina Research Articles

Microglia Inhibition Delays Retinal Degeneration Due to MerTK Phagocytosis Receptor Deficiency.

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive loss of rod followed by cone photoreceptors. An especially early onset form of RP with blindness in teenage years is caused by mutations in mertk, the gene encoding the clearance phagocytosis receptor Mer tyrosine kinase (MerTK). The cause for blindness in mutant MerTK-associated RP (mutMerTK-RP) is the failure of retinal pigment epithelial cells in diurnal phagocytosis of spent photoreceptor outer segment debris. However, the early onset and very fast progression of degeneration in mutMerTK-RP remains unexplained. Here, we explored the role of microglia in the Royal College of Surgeons (RCS) rat model of mutMerTK-RP. We found elevated levels of inflammatory cytokines and CD68 microglia activation marker, and more ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia in RCS retina when compared to wild-type retina as early as postnatal day 14 (P14). Strikingly, renewal of photoreceptor outer segments in P14 wild-type rat retina is still immature with low levels of RPE phagocytosis implying that at this early age lack of this process in RCS rats is unlikely to distress photoreceptors. Although the total number of Iba-1 positive retinal microglia remains constant from P14 to P30, we observed increasing numbers of microglia in the outer retina from P20 implying migration to the outer retina before onset of photoreceptor cell death at ~P25. Iba-1 and CD68 levels also increase in the retina during this time period suggesting microglia activation. To determine whether microglia affect the degenerative process, we suppressed retinal microglia in vivo using tamoxifen or a combination of tamoxifen and liposomal clodronate. Treatments partly prevented elevation of Iba-1 and CD68 and relocalization of microglia. Moreover, treatments led to partial but significant retention of photoreceptor viability and photoreceptor function. We conclude that loss of the phagocytosis receptor MerTK causes microglia activation and relocalization in the retina before lack of RPE phagocytosis causes overt retinal degeneration, and that microglia activities accelerate loss of photoreceptors in mutMerTK-RP. These results suggest that therapies targeting microglia may delay onset and slow the progression of this blinding disease.

Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity.

Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by Mertk loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of-Mertk in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy.SIGNIFICANCE STATEMENT Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking Mertk), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of Mertk function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.

Long-term Characterization of Retinal Degeneration in Royal College of Surgeons Rats Using Spectral-Domain Optical Coherence Tomography.

PurposeProspective treatments for age-related macular degeneration and inherited retinal degenerations are commonly evaluated in the Royal College of Surgeons (RCS) rat before translation into clinical application. Historically, retinal thickness obtained through postmortem anatomic assessments has been a key outcome measure; however, utility of this measurement is limited because it precludes the ability to perform longitudinal studies. To overcome this limitation, the present study was designed to provide a baseline longitudinal quantification of retinal thickness in the RCS rat by using spectral-domain optical coherence tomography (SD-OCT).MethodsHorizontal and vertical linear SD-OCT scans centered on the optic nerve were captured from Long-Evans control rats at P30, P60, P90 and from RCS rats between P17 and P90. Total retina (TR), outer nuclear layer+ (ONL+), inner nuclear layer (INL), and retinal pigment epithelium (RPE) thicknesses were quantified. Histologic sections of RCS retina obtained from P21 to P60 were compared to SD-OCT images.ResultsIn RCS rats, TR and ONL+ thickness decreased significantly as compared to Long-Evans controls. Changes in INL and RPE thickness were not significantly different between control and RCS retinas. From P30 to P90 a subretinal hyperreflective layer (HRL) was observed and quantified in RCS rats. After correlation with histology, the HRL was identified as disorganized outer segments and the location of accumulated debris.ConclusionsRetinal layer thickness can be quantified longitudinally throughout the course of retinal degeneration in the RCS rat by using SD-OCT. Thickness measurements obtained with SD-OCT were consistent with previous anatomic thickness assessments. This study provides baseline data for future longitudinal assessment of therapeutic agents in the RCS rat.

NGF/anti-VEGF combined exposure protects RCS retinal cells and photoreceptors that underwent a local worsening of inflammation.

Our previous study highlighted the potential nerve growth factor (NGF) effect on damaged photoreceptors from a rat model of spontaneous Retinitis Pigmentosa (RP). Herein, we tested the combined NGF/anti-vascular endothelial growth factor (αVEGF) effect on cultured retinal cells isolated from Royal College of Surgeons (RCS) rats receiving an intravitreal VEGF injection (iv-VEGF) to exacerbate retinal inflammation/neovascularization. RCS (n = 75) rats were equally grouped as untreated (n = 25), iv-saline (single saline intravitreal injection; n = 25) and iv-VEGF (single VEGF intravitreal injection; n = 25). Morphological and biochemical analysis or in vitro stimulations with the biomolecular investigation were carried out on explanted retinas. Isolated retinal cells were treated with NGF and αVEGF, either alone or in combination, for 6days and cells were harvested for morphological and biomolecular analyses. Infiltrating inflammatory cells were detected in iv-VEGF exposed RCS retinas, indicative of exacerbated inflammation and neovascularization. In cell cultures, NGF/αVEGF significantly increased retinal cell survival as well as rhodopsin expression and neurite outgrowth in photoreceptors. Particularly, NGF/αVEGF upregulated Bcl-2 mRNA, downregulated Bax mRNA, upregulated trkANGFR mRNA and finally upregulated both NGF mRNA and protein. These data confirm and extend our previous findings on NGF-photoreceptor crosstalk, highlighting that the NGF/αVEGF combination might be an interesting approach for improving neuroprotection of RCS retinal cells and likewise photoreceptors in the presence of neovascularization. Further studies are required to translate this in vitro approach into clinical practice.

Immunosuppressive Treatment Can Alter Visual Performance in the Royal College of Surgeons Rat.

Immunosuppression is frequently employed to enhance survival of xenografted human cells as part of translational proof-of-concept studies. However, the potential effects of this treatment are easily overlooked. As part of baseline testing in the dark-eyed variant of the dystrophic Royal College of Surgeons (RCS) rat, we documented the time course of retinal degenerative changes versus Long Evans controls using bright field retinal imaging, fluorescein angiography, and histology and examined the impact of immunosuppression on visual function. Rats received either no treatment or systemic immunosuppression with oral cyclosporine A and injectable dexamethasone and subsequently underwent functional evaluation by optomotor response testing and electroretinography (ERG) at multiple intervals from P45 to P180. Immunosuppressed RCS animals demonstrated poorer performance on functional tests than age-matched untreated rats during the earlier stages of degeneration, including significantly lower spatial acuities on optomotor threshold testing and significantly lower b-wave amplitudes on scotopic and photopic ERGs. Retinal imaging documented the progression of degenerative changes in the RCS fundus and histologic evaluation of the RCS retina confirmed progressive thinning of the outer nuclear layer. A standard regimen of cyclosporine A plus dexamethasone, administered to RCS rats, results in demonstrable systemic side effects and depressed scores on behavioral and electrophysiological testing at time points before P90. The source of the functional impairment was not identified. This finding has implications for the interpretation of data generated using this commonly used translational model.

Blood Flow Magnetic Resonance Imaging of Retinal Degeneration

This study aims to investigate quantitative basal blood flow as well as hypercapnia- and hyperoxia-induced blood flow changes in the retinas of the Royal College of Surgeons (RCS) rats with spontaneous retinal degeneration, and to compare with those of normal rat retinas. Experiments were performed on male RCS rats at post-natal days P90 (n=4) and P220 (n=5), and on age-matched controls at P90 (n=7) and P220 (n=6). Hyperoxic (100% O(2)) and hypercapnic (5% CO(2), 21% O(2), balance N(2)) challenges were used to modulate blood flow. Quantitative baseline blood flow, and hypercapnia- and hyperoxia-induced blood flow changes in the retinas were imaged using continuous arterial spin labeling MRI at 90 x 90 x 1500 microm. In the normal rat retinas, basal blood flow of the whole-retina was 5.5 mL/gram per min, significantly higher than those reported in the brain (approximately 1 mL/gram per min). Hyperoxia decreased blood flow due to vasoconstriction and hypercapnia increased blood flow due to vasodilation in the normal retinas. In the RCS rat retinas, basal blood flow was diminished significantly (P<0.05). Interestingly, absolute hyperoxia- and hypercapnia-induced blood flow changes in the RCS retinas were not statistically different from those in the normal retinas (P>0.05). However, blood flow percent changes in RCS retinas were significantly larger than in normal retinas due to lower basal blood flow in the RCS retinas. Retinal degeneration markedly reduces basal blood flow but does not appear to impair vascular reactivity. These data also suggest caution when interpreting relative stimulus-evoked functional MRI changes in diseased states where basal parameters are significantly perturbed. Quantitative blood flow MRI may serve as a valuable tool to study the retina without depth limitation.

Photopic ERG Negative Response from Amacrine Cell Signaling in RCS Rat Retinal Degeneration

The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity, which declines more slowly than the b-wave with degeneration. The absence of this response in the P23H rat indicates that the inner retinal cone pathway pathology is different in the two models. A relative increase in Kir4.1 channels on Müller cells of RCS retina may contribute to the enhanced negative ERG response in the RCS rat.

Genetically induced retinal degeneration leads to changes in metabotropic glutamate receptor expression

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [ 35S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.

Prolonged survival of the phosphorylated form of rhodopsin during dark adaptation of Royal College Surgeons rat

To study rhodopsin (Rho) phosphorylation and dephosphorylation in Royal College of Surgeons (RCS) rat retina, specific antibodies toward major Rho phosphorylation sites in vivo, 334Ser or 338Ser, were prepared by immunization of authentic phosphorylated peptides in rabbit. Enzyme-linked immunosorbent assay identified that the raised antibodies exclusively recognized either the phosphorylated 334Ser or 338Ser site. In immunofluorescence labeling, both antibodies recognized photoreceptor outer segments in light-adapted retinas from Sprague–Dawley (SD), Brown–Norway (BN) and RCS rat. During dark adaptation, immunoreactivities toward phosphorylated 338Ser and 334Ser sites were diminished within several hours (0.2–2 h) in SD and BN rat retinas. However, those toward phosphorylated 338Ser and 334Ser sites were diminished within 4 to 7 days in RCS rat retinas. In vitro studies demonstrated decreased levels of both Rho phosphorylation and dephosphorylation reactions in RCS retinas. However, the dephosphorylation reaction was much more greatly affected than the phosphorylation reaction. Extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.

Mapping photoreceptor and postreceptoral labelling patterns using a channel permeable probe (agmatine) during development in the normal and RCS rat retina.

The aim of this study was to determine whether agmatine, a channel permeable probe, can identify photoreceptor dysfunction in the Royal College of Surgeons (RCS) retina at an earlier stage to that shown by apoptosis or anatomical markers, and also characterize the neurochemical development of the inner retina in the normal and degenerating rat. We used isolated retinas at different ages incubated in physiological media containing agmatine. Subsequently, postembedding immunocytochemistry was used to determine the number of labelled photoreceptors and the labelling pattern within postreceptoral neurons. Agmatine labelling patterns revealed a sequential development of retinal neurons beginning at postnatal day (PND) 11/12 with most horizontal cells, a few ganglion and amacrine cells, showing a strong signal. The neurochemical development progressed rapidly, and reflects to a large part the known distribution of glutamate receptors, with inner nuclear labelling being evident by PND14, continuing with the same pattern of labelling in adulthood for the control retina. The RCS retina showed markedly reduced agmatine labelling in the inner retina at PND20. A rapid increase in photoreceptor AGB labelling was evident during the degeneration phase. Multiple samples at PND14 and PND16 confirmed a significant increase of labelled photoreceptors in the RCS retina.

Graft Location Affects Functional Rescue Following RPE Cell Transplantation in the RCS Rat

Photoreceptor (PRC) rescue in the dystrophic Royal College of Surgeons (RCS) rat has been well documented following a variety of interventions. Although the dystrophic process is asymmetric with respect to the horizontal meridian, little attention has been paid to the effect of topographic position on treatment outcome. In this study, RPE cells from adult congenic nondystrophic animals were injected into the subretinal space of 1-month-old dystrophic RCS rats in either the dorsal or the ventral equatorial region. Animals were followed longitudinally during the degenerative process using the pupillary light reflex (PLR). The parameter of the PLR most sensitive to PRC rescue is latency at low light levels. At 3 months of age this parameter showed statistically better performance (ANOVA, P = 0.016) for eyes with grafts placed dorsally compared to those placed ventrally or untreated controls. There was no treatment effect on amplitude. By 6 months of age the dorsal/ventral disparity in latency was less apparent and amplitude remained equivalent across groups. Late analysis of retinal whole-mounts using RT-97 fluorescent labeling showed extensive irregularities in ventral axonal morphology in all treatment groups. These results indicate that functional rescue of the RCS retina is significantly influenced by the local degenerative timetable. The role of initial local conditions on treatment outcome is worthy of consideration in other models of neuroprotection.

Neurochemical architecture of the normal and degenerating rat retina

We used post-embedding immunocytochemistry to determine the cellular localization of glutamate, γ-amino butyric acid (GABA), glycine, aspartate, glutamine, arginine, and taurine in the normal and degenerating rat retina. Müller's cell function was also evaluated by determining the uptake and degradation characteristics for glutamate. Immunocytochemical localization of amino acids in adult Royal College of Surgeons (RCS) and control rat retinas were similar with respect to cell classes. Differences in the intensity of labelling for glutamate, aspartate, glutamine, and glycine were observed in several classes of neurons, but the most prominent differences were shown by bipolar cells of the adult RCS rat retina. In addition, glutamine labelling within Müller's cells was higher in the RCS rat than the control. These changes may have occurred because of alterations in the glutamate production or degradation pathways. We tested this hypothesis by determining Müller's cells glutamate uptake and degradation characteristics in adult and postnatal day 16 RCS retinas. High affinity uptake of 3[H]-glutamate revealed an accumulation of grains over Müller's cell bodies in the adult RCS retina implying glutamate degradation anomalies. We confirmed anomalies in glutamate metabolism in RCS Müller's cells by showing that exogenously applied glutamate was degraded over a longer time course in postnatal day 16 RCS retinas, compared to control retinas. Differences in arginine immunoreactivity in adult and immature RCS retinas conform to the presumed dysfunction of Müller's cells in these degenerating retinas. The anomalies of amino acid localization, uptake and degradation lead us to conclude that Müller's cells in the RCS retina show abnormal function by postnatal day 16; an earlier time to previously reported anatomical and functional changes in this animal model of retinal degeneration. © 1996 Wiley-Liss, Inc.

Neurochemical architecture of the normal and degenerating rat retina.

We used post-embedding immunocytochemistry to determine the cellular localization of glutamate, gamma-amino butyric acid (GABA), glycine, aspartate, glutamine, arginine, and taurine in the normal and degenerating rat retina. Müller's cell function was also evaluated by determining the uptake and degradation characteristics for glutamate. Immunocytochemical localization of amino acids in adult Royal College of Surgeons (RCS) and control rat retinas were similar with respect to cell classes. Differences in the intensity of labelling for glutamate, aspartate, glutamine, and glycine were observed in several classes of neurons, but the most prominent differences were shown by bipolar cells of the adult RCS rat retina. In addition, glutamine labelling within Müller's cells was higher in the RCS rat than the control. These changes may have occurred because of alterations in the glutamate production or degradation pathways. We tested this hypothesis by determining Müller's cells glutamate uptake and degradation characteristics in adult and postnatal day 16 RCS retinas. High affinity uptake of 3[H]-glutamate revealed an accumulation of grains over Müller's cell bodies in the adult RCS retina implying glutamate degradation anomalies. We confirmed anomalies in glutamate metabolism in RCS Müller's cells by showing that exogenously applied glutamate was degraded over a longer time course in postnatal day 16 RCS retinas, compared to control retinas. Differences in arginine immunoreactivity in adult and immature RCS retinas conform to the presumed dysfunction of Müller's cells in these degenerating retinas. The anomalies of amino acid localization, uptake and degradation lead us to conclude that Müller's cells in the RCS retina show abnormal function by postnatal day 16; an earlier time to previously reported anatomical and functional changes in this animal model of retinal degeneration.

Photoreceptor repair in response to RPE transplants in RCS rats: outer segment regeneration.

We have previously shown that transplants of normal rat neonatal RPE cells rescued photoreceptor cells in retinas of Royal College of Surgeons (RCS) dystrophic rats for up to one year. In this study, we investigated the photoreceptor rescue effects in RCS rats within the first three weeks following transplantation in an attempt to determine if RPE transplants initiate repair mechanisms, specifically, outer segment (OS) regeneration. Freshly isolated RPE cells from neonatal pigmented Long Evans rats were transplanted into the subretinal space of 22-23 day-old RCS rats using a transscleral approach. For controls, vehicle was similarly injected. When analyzed at 10 days post-transplantation, long inner segments were observed with short buds of outer segment growth in the area of the RPE-cell transplants. The outer segments were of insufficient length to be measured at 10 days, but by 14 and 21 days, OS were 2.02 +/- 0.32 microns and 18.80 +/- 2.78 microns, respectively. In vehicle-injected retinas from 10 to 21 days postsurgery, outer segments were not observed and the inner segments were three-fold shorter than in RPE-transplanted retinas. At 10 days post-transplantation, most RPE cells were seen in the subretinal space, but a few had attached to Bruch's membrane; however, by 21 days, many of the transplanted RPE cells had attached to Bruch's membrane, although a few were found free in the subretinal space. This study has shown that transplants of normal rat neonatal RPE cells have the capacity to support not only photoreceptor cell survival but also initiate early repair mechanisms as exhibited by outer segment regeneration in RCS retinas. These results also conclusively show the important role that the RPE plays in outer segment growth and maturation.

Early abnormalities of retinal dopamine pathways in rats with hereditary retinal dystrophy.

The dopaminergic pathway that affects rod-driven horizontal cells has been studied in the Royal College of Surgeons (RCS) rat during the period preceding photoreceptor degeneration (postnatal day 17-24). The experiments were performed by intracellular recording from single horizontal cells in vitro. Horizontal cells from the recessive control animals (postnatal day 17-24) were depolarized by dopamine (10 microM) and hyperpolarized by the D1 antagonist SCH 23,390 (10 microM). In contrast, cells from age-matched dystrophic retinas, though depolarized by dopamine, were unaffected by SCH 23390 (10-100 microM), suggesting a significant reduction in the level of endogenous dopamine release. Histologic examination for catecholaminergic neurons revealed no differences in either the cell number or anatomy between the retinas of the control and dystrophic animals. Furthermore, perfusion of the control retinas with melatonin (500 nM-1 microM) yielded response characteristic of the dystrophic type. In the period preceding degeneration, the RCS retina thus displays a discrete abnormality in dopaminergic pathways, such that there is a gross reduction in endogenous dopamine release below that required to activate D1 receptors. Since melatonin levels have been shown to be high in these retinas, we propose that abnormalities in the dopamine-melatonin systems give rise to an electrophysiologic deficit in the postphotoreceptoral retina of the RCS rat.

Muller cell expression of glial fibrillary acidic protein (GFAP) in RPE-cell transplanted retinas of RCS dystrophic rats.

In Royal College of Surgeons (RCS) rats with inherited retinal dystrophy, photoreceptor cell degeneration is accompanied by Muller cell changes, in particular, increased expression of the intermediate filament protein, glial fibrillary acidic protein (GFAP). In this study, we examined the expression of GFAP in retinas of 4 month-old RCS dystrophic rats either transplanted with normal retinal pigment epithelial (RPE) cells or injected with vehicle (sham control). The sham-injected and non-treated retinas showed increased expression of GFAP in Muller cells with advancing age. GFAP-immunostained Muller processes were observed in the region of the subretinal space of these RCS retinas beginning by 4 months. However, in the retinas of RCS rats transplanted with normal RPE cells, GFAP expression by Muller cells was significantly reduced. Specifically, under the transplanted RPE cells, GFAP-immunolabeled Muller radial processes were not observed and GFAP immunostaining was not present in the subretinal space at any time period examined. Immunoblot analysis of the superior hemisphere of RPE-cell transplanted retinas of 4 month-old RCS dystrophic rats showed less GFAP immunostaining than sham-injected and the inferior hemisphere of retinas of age-matched RCS rats. This study showed that in normal RPE-cell transplanted retinas of RCS rats, in addition to rescued photoreceptor cells, there was an accompanying stabilization of Muller cells as demonstrated by a reduction in the expression of GFAP.

Immunohistochemical localization of basic fibroblast growth factor in mature and developing retinas of normal and RCS rats.

Basic fibroblast growth factor (bFGF) delays photoreceptor degeneration when injected intraocularly in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy. In the present study, we have determined the localization of endogenous bFGF in retinas of normal and RCS rats during the normal developmental period (postnatal days 0-20) and the period of photoreceptor degeneration in RCS rats (days 20-90). bFGF was localized immunohistochemically by indirect immunoperoxidase using two different polyclonal antibodies and one monoclonal antibody against bFGF. bFGF was present in retinas as early as birth, and remained through adult age. Controls using either PBS, non-immune IgG or antibody preabsorbed with bFGF peptide were devoid of label. In normal rats between the ages of birth and postnatal day (P) 4, bFGF was found in developing ganglion cells, superficial blood vessels, some of the innermost cells in the neuroblastic layer, developing horizontal cells, and retinal pigment epithelial (RPE) cells. Between P0 and P4, the intensity of staining increased significantly in horizontal cells. From P6-P10, some cells in the inner nuclear layer remained positive, but horizontal cell staining became less intense in the central retina. The superficial vessels, ganglion cells and RPE cells also remained positive for bFGF. At P20-25, when the retina was essentially mature, bFGF was found in RPE cells, most cells of the ganglion cell layer, and many cells of the inner nuclear layer, but horizontal cells and blood vessels showed a lower concentration of bFGF than they did at younger ages. At P45 and older, blood vessels no longer showed bFGF immunoreactivity. The staining pattern in RCS rats was indistinguishable from that for normal rats at all ages examined. These results show that bFGF is present in the developing and adult rat retina in some neural cells, in addition to vessels and RPE cells. The transient elevated expression of bFGF immunoreactivity in developing horizontal cells and blood vessels suggests a possible role for this growth factor in retinal development. In addition, if RCS retinas possess any difference in bFGF localization or concentration compared to normal retinas, it must be too small to detect by immunohistochemical means, or at least with the reagents used.

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas

Stress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.

Examination of sialic acid binding on dystrophic and normal retinal pigment epithelium

In order to study differences in cell-surface sugars which may be involved in the phagocytic defect in Royal College of Surgeons (RCS) retinas, we have examined the presence or absence of lectin binding to carbohydrates on retinal pigment epithelial (RPE) plasma membranes of dystrophic (RCS-p+) and normal (Long-Evans) rats. A lectin which binds to both sialic acid and N-acetylglucosamine sugar residues, wheat germ agglutinin-ferritin (WGA-fe), was used. The specificity of WGA-fe binding to each sugar was studied by either pre-treating the tissue with neuraminidase enzyme which removes sialic acid residues, or by incubating the WGA-fe lectin with one of the haptens, N-acetylglucosamine. In non-enzyme-treated tissue, RPE cell-surface membranes from RCS retinas were densely labeled with WGA-fe as compared with the labeling on normal RPE, which appeared less dense and patchy in distribution. Wheat germ agglutinin-ferritin labeling in the presence of N-acetylglucosamine was blocked on both RCS and normal RPE surface membranes. After pre-treatment with neuraminidase, WGA-fe labeling on dystrophic RPE membranes was similar to non-enzyme-treated tissue but was enhanced on normal RPE. Labeling was blocked when N-acetylglucosamine was present with the lectin after enzyme pre-treatment. Other lectins which specifically bind to sialic acid, Limulus polyphemus agglutinin-ferritin (LPA-fe) and Limax flavus agglutinin (LFA) demonstrated sparse or no labeling on both RCS and normal RPE membranes. Our data suggests that N-acetylglucosamine residues predominate on RCS and normal RPE cell-surface membranes and that sialic acid binding sites are either not accessible to the lectins or may not be present.