Search

Rand Spencer,1 Steven Fisher,2 Geoffrey P Lewis,3 Terri Malone4 1Texas Retina Associates, Dallas, TX, USA; 2Molecular, Cellular, and Developmental Biology, 3Center for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, CA, 4Cell Therapy, Janssen Research & Development, LLC, Spring House, PA, USA Background: A 70-year-old woman with retinitis pigmentosa experienced an epiretinal membrane (ERM) formation and a tractional retinal detachment (RD) following subretinal administration of palucorcel (CNTO 2476), a novel human umbilical tissue-derived cell-based therapy, as part of a Phase I study. The clinical course and results of a histologic examination of the ERM, which was peeled during surgery to repair the RD, are described here.Methods: In this open-label, first-in-human, Phase I study (NCT00458575), two of seven subjects developed RD, with an ERM formation reported in a woman receiving a targeted dose of 3.0×105 palucorcel administered via a transvitreal route. A sample of the ERM was retained for analysis following the ERM peeling procedure. Clinical outcomes and ERM histology, based on immunocytochemistry analyses and fluorescence in situ hybridization (FISH) staining, were evaluated.Results: We first noted the RD and formation of the ERM at 26 days after palucorcel administration. The ERM was cellular and contained multiple cell types, including Müller glial cells, immune cells, neurites, retinal pigment epithelial cells, and palucorcel. The majority of cells were not actively dividing. FISH staining showed a subset of Y chromosome-positive cells in the ERM from this woman, supporting the presence of palucorcel (derived from umbilical cord tissue of male neonate). Palucorcel did not differentiate into Müller glia, immune cells, neurites, or retinal pigment epithelial cells.Discussion: The development of an ERM containing both subject (self) cells and palucorcel suggests that palucorcel egress in the vitreal cavity after retinotomy may contribute to ERM formation and RD and that an alternative delivery method will be required before further studies are conducted. Subsequent clinical research using alternative subretinal delivery methods for palucorcel in other indications suggests that membrane development does not occur when palucorcel is delivered without retinal perforation. Keywords: cell therapy, epiretinal membrane, retinitis pigmentosa

Background To study the response of ON and OFF bipolar cells in experimental retinal detachment. Methods Domestic cat retinas were detached for 7 days. The retinas were prepared for immunocytochemical staining with antibodies to Go alpha (α), glutamate transporter GLT-1, protein kinase C and rod opsin, which serve as markers for ON bipolar cells, OFF bipolar cells, rod bipolar cells and rod photoreceptors, respectively. Both sections and whole-mounts were labelled with antibodies to Goα and GLT-1. Results Following 7 days of detachment, ON bipolar cell processes extended into the outer nuclear layer and had neurites extending beyond their target layer into the inner plexiform layer. In contrast, OFF bipolar cell processes were reduced in the outer plexiform layer following detachment. Conclusion ON and OFF bipolar cells undergo significant remodelling of their processes in response to retinal detachment, and the ON and OFF pathways may be differentially affected. The remodelling may be due to morphological changes that have previously been shown to occur in photoreceptor synaptic terminals or as a result of loss of synaptic connections due to photoreceptor cell death.

Motivation: Microscopy advances have enabled the acquisition of large-scale biological images that capture whole tissues in situ. This in turn has fostered the study of spatial relationships between cells and various biological structures, which has proved enormously beneficial toward understanding organ and organism function. However, the unique nature of biological images and tissues precludes the application of many existing spatial mining and quantification methods necessary to make inferences about the data. Especially difficult is attempting to quantify the spatial correlation between heterogeneous structures and point objects, which often occurs in many biological tissues. Results: We develop a method to quantify the spatial correlation between a continuous structure and point data in large (17 500 × 17 500 pixel) biological images. We use this method to study the spatial relationship between the vasculature and a type of cell in the retina called astrocytes. We use a geodesic feature space based on vascular structures and embed astrocytes into the space by spatial sampling. We then propose a quantification method in this feature space that enables us to empirically demonstrate that the spatial distribution of astrocytes is often correlated with vascular structure. Additionally, these patterns are conserved in the retina after injury. These results prove the long-assumed patterns of astrocyte spatial distribution and provide a novel methodology for conducting other spatial studies of similar tissue and structures. Availability: The Matlab code for the method described in this article can be found at http://www.cs.ucsb.edu/∼dbl/software.php. Contact: bruttenberg@cra.com or ambuj@cs.ucsb.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Author(s): Luna, Gabriel; Lewis, Geoffrey P; Linberg, Kenneth A; Chang, Bo; Hu, Quiri; Munson, Peter J; Maminishkis, Arvydas; Miller, Sheldon S; Fisher, Steven K | Abstract: PurposeThe purpose of this study was to examine the rpea1 mouse whose retina spontaneously detaches from the underlying RPE as a potential model for studying the cellular effects of serous retinal detachment (SRD).MethodsOptical coherence tomography (OCT) was performed immediately prior to euthanasia; retinal tissue was subsequently prepared for Western blotting, microarray analysis, immunocytochemistry, and light and electron microscopy (LM, EM).ResultsBy postnatal day (P) 30, OCT, LM, and EM revealed the presence of small shallow detachments that increased in number and size over time. By P60 in regions of detachment, there was a dramatic loss of PNA binding around cones in the interphotoreceptor matrix and a concomitant increase in labeling of the outer nuclear layer and rod synaptic terminals. Retinal pigment epithelium wholemounts revealed a patchy loss in immunolabeling for both ezrin and aquaporin 1. Anti-ezrin labeling was lost from small regions of the RPE apical surface underlying detachments at P30. Labeling for tight-junction proteins provided a regular array of profiles outlining the periphery of RPE cells in wild-type tissue, however, this pattern was disrupted in the mutant as early as P30. Microarray analysis revealed a broad range of changes in genes involved in metabolism, signaling, cell polarity, and tight-junction organization.ConclusionsThese data indicate changes in this mutant mouse that may provide clues to the underlying mechanisms of SRD in humans. Importantly, these changes include the production of multiple spontaneous detachments without the presence of a retinal tear or significant degeneration of outer segments, changes in the expression of proteins involved in adhesion and fluid transport, and a disrupted organization of RPE tight junctions that may contribute to the formation of focal detachments.

The aim of this study was to examine the temporal relationship between behaviorally measured visual thresholds, photoreceptor degeneration and dysfunction, synaptic and neuronal morphology changes in the retina in the S334ter line 4 rat. Specifically, we examined the optokinetic tracking (OKT) behavior in S334ter rats daily and found that OKT thresholds reflected normal values at eye opening but quickly reduced to a non-response level by postnatal day (P) 22. By contrast, the scotopic electroretinogram (ERG) showed a much slower degeneration, with substantial scotopic function remaining after P90 as previously demonstrated for this line of rats. Photopic b-wave amplitudes revealed functional levels between 70 and 100% of normal between P30 and P90. Histological evidence demonstrated that photoreceptor degeneration occurred over many months, with an outer nuclear layer (ONL) roughly half the thickness of a normal age-matched control at P90. Immunohistochemical analysis revealed a number of changes in retinal morphology in the Tg S334ter line 4 rat that occur at or before P40 including: elevated levels of rod opsin expression in the ONL, cone photoreceptor morphology changes, glial cell activation, inner retinal neuron sprouting, and microglial cell activation. Many of these changes were evident at P30 and in some cases as early as eye opening (P15). Thus, the morphological changes occurred in concert with or before the very rapid loss of the behavioral (OKT) responses, and significantly before the loss of photoreceptors and photoreceptor function.

Die proliferative Vitreoretinopathie (PVR) gilt als eine der schwersten Komplikationen nach Ablatio-Chirurgie. Bisher konnte sich keine Pharmakotherapie zur Kontrolle der zellbiologischen Komponente dieser Erkrankung klinisch etablieren. Ziel unserer Studie war es, den Einfluss von Alkylphosphocholinen (APCs; hier: Erucylphosphocholin; ErPC), neuen pharmakologischen Substanzen mit antiproliferativen Eigenschaften, auf die intraretinale Proliferation nach experimentell induzierter Netzhautablosung an einem etablierten in vivo Modell zu untersuchen. Am Kaninchenauge wurde eine Netzhautablosung durch subretinale Injektion mit einer Mikropipette induziert. An Tag 1 oder an Tag 2 erfolgte die intravitreale Injektion von Erucylphosphocholin (ErPC). Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) wurde an Tag 3 injiziert. Nach Fixierung des Gewebes erfolgte eine Dreifachmarkierung der Netzhaut mit Anti-BrdU (Proliferationsmarker), Isolectin B4 (Marker fur retinale Mikrogliazellen) und Anti-Vimentin (Marker fur retinale Muller-Gliazellen). Die Anzahl proliferierender, also Anti-BrdU-markierter Zellen pro mm Netzhaut wurde am konfokalen Laser-Scanning-Mikroskop bestimmt. Die Toxizitat wurde anhand von gefarbten Gewebeschnitten am Lichtmikroskop sowie ultrastrukturell am Elektronenmikroskop beurteilt. Nach einmaliger intravitrealer Gabe von Erucylphosphocholin (ErPC) zeigte sich eine signifikante Abnahme der Proliferation nichtneuraler retinaler Zellen an Tag 3 nach Induktion einer Netzhautablosung am Kaninchenauge. Eine Injektion an Tag 1 war der Injektion an Tag 2 nach Netzhautablosung uberlegen. Fur keine der applizierten ErPC-Konzentrationen war morphologisch eine retinale Toxizitat nachweisbar. Alkylphosphocholine sind neue Substanzen, die eine Inhibition der intraretinalen Proliferation nach Netzhautablosung im Tiermodell bewirken. Moglicherweise konnten APCs zukunftig zur Prophylaxe einer PVR bei der Ablatio-Chirurgie in noch zu definierenden Situationen intravitreal gegeben werden, um die zellbiologische Komponente der Erkrankung zu kontrollieren. Unabdingbare Voraussetzung dafur sind jedoch weitere, langfristige Untersuchungen zur Toxizitat dieser neuen Substanzgruppe.

Purpose. The pathogenesis of rhegmatogenous retinal detachment (RRD) remains incompletely understood, with no clinically effective treatment for potentially severe complications such as photoreceptor cell death and proliferative vitreoretinopathy. Here we investigate the protein profile of the vitreous following experimental retinal detachment using a comparative proteomic based approach.Materials and Methods. Retinal detachment was created in the right eyes of six New Zealand red pigmented rabbits. Sham surgery was undertaken in five other rabbits that were used as controls. After seven days the eyes were enucleated and the vitreous was removed. The vitreous samples were evaluated with two-dimensional polyacrylamide gel electrophoresis and the differentially expressed proteins were identified with tandem mass spectrometry.Results. Ten protein spots were found to be at least twofold differentially expressed when comparing the vitreous samples of the sham and retinal detachment surgery groups. Protein spots that were upregulated in the vitreous following retinal detachment were identified as albumin fragments, and those downregulated were found to be peroxiredoxin 2, collagen-Iα1 fragment, andα-1-antiproteinase F.Conclusions. Proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications.

Purpose To evaluate the ability of delayed hyperoxia to slow or prevent degenerative and gliotic changes initiated by retinal detachment. Design An experimental study. Methods Rhegmatogenous detachments were produced in the right eyes of eight cats. After 1 day in room air (21% O 2 ), four cats were placed in chambers with the O 2 concentration regulated at 70%; the other four were left in room air. At 7 days the retinas were harvested and examined by light and confocal microscopy. Cell specific antibodies, TUNEL and proliferation assays, outer segment length, and photoreceptor counts, were used to assess the condition of the retina. The contralateral unoperated eyes were used as controls. Results Animals maintained in elevated O 2 showed a dramatic preservation of rod and cone outer segments as well as in the organization of the outer plexiform layer. The number of surviving photoreceptors was increased in the hyperoxia-treated animals. Neurite sprouting, a characteristic of detached retina, was rarely observed in the experimental eyes. Proliferation of non-neuronal cells was reduced, but not halted, by hyperoxia. GFAP and vimentin expression was not effected by hyperoxia; these intermediate filament proteins increased in Muller cells similar to that observed in control detachments. Conclusions Exposure to hyperoxia, delayed by 1 day after the onset of retinal detachment, was highly effective in preserving photoreceptor cells and in reducing proliferation within the retina. It did not, however, reduce the hypertrophy of Muller glia. There were no apparent detrimental effects of exposure to 70% O 2 for 6 days. These results suggest that human patients may benefit from breathing elevated oxygen levels while awaiting reattachment surgery, even if the hyperoxia is delayed relative to the time of detachment.

Recent evidence suggests that the adult mammalian retina is far more plastic than was previously thought. Retinal detachment induces changes beyond the degeneration of outer segments (OS). Changes in photoreceptor synapses, second- and even third-order neurons may all contribute to imperfect visual recovery that can occur after successful reattachment. Changes that occur in Müller cells have obvious effects through subretinal fibrosis and proliferative vitreoretinopathy, but other unidentified effects seem likely as well. Reattachment of the retina induces its own set of responses aside from OS re-growth. Reattachment halts the growth of Müller cell processes into the subretinal space, but induces their growth on the vitreal surface. It also induces the outgrowth of rod axons into the inner retina.

In people, retinal detachment often leads to a significant loss in cone-based vision. Most of the animal models commonly used for studying the consequences of retinal detachment have rod-dominated retinas. The purpose of this investigation was to evaluate the possibility that the ground squirrel, a rodent with a heavily cone-dominated retina, might provide a useful model for studying cone function in retinal detachment. Corneal ERGs were recorded from ground squirrels for large-field temporal modulations presented on a computer-controlled color monitor. Modulations were chosen to selectively stimulate either of the two classes of cone found in the ground squirrel retina. Under these test conditions, large and reliable cone ERGs could be readily recorded. In animals in which the retina had been surgically detached, the loss of cone signal was directly related to the number of cones in the detachment zone relative to the total cone population and that relationship did not differ for short-wavelength sensitive (S) and middle-wavelength sensitive (M) cones. Surgical reattachment produced a progressive recovery of cone-based signals. The ground squirrel seems likely to provide a useful animal model for studying the dynamics of cone function in retinal detachment and subsequent events.

Supported by NIH grants EY03323 and Research to Prevent Blindness to H.K., EY 00888 to S.K.F. and Generalitat Valenciana CT1DIB/2002/146 to N.C.

The lectin peanut agglutinin (PNA) and antibodies to short (S)- and medium to long wavelength (M/L)-sensitive cones were utilized in order to define the relative distributions of the two spectral types of cone across the domestic cat’s retina. These values, in turn, were compared to those from retinas that had been experimentally detached from the retinal pigment epithelium. The pattern of cone distribution in the normal cat’s retina is established by the preponderance of M-cones that constitute between 80% and 90% of all cones. Their peak density of over 26,000 cells/mm 2 resides at the area centralis. Though M-cone density decreases smoothly to the ora serrata where they have densities as low as 2,200 cells/mm 2 , the density decrease along the nasotemporal axis is slower,creating a horizontal region of higher cone density. S-cones constitute between 10% and 20% of all cones, the number being quite variable even between individual animals of similar age. The highest S-cone densities are found in three distinct locations: at the superior far periphery near the ora serrata, immediately at the area centralis itself, and in a broad zone comprising the central and lower half of the inferior hemiretina. S-cones in the cat retina do not form a regular geometrical array at any eccentricity. As for the detached cat retina, the density of labeled S-cone outer segments (OS) decreases rapidly as early as 1 day postdetachment and continues decreasing to day 28 when the density of cones labeling with anti-S opsin has dropped to less than 10% of normal. This response points to a profound difference between rods and cones; essentially all rods, including those without OS, continue to express their opsin even in long-term detachments. The implications of these results for visual recovery after retinal reattachment are discussed. J. Comp. Neurol. 430:343‐356, 2001. © 2001 Wiley-Liss, Inc. Indexing terms: photoreceptors; blue cones; lectins; antibodies; retinal detachment

This review will summarize the properties of five variant forms of human growth hormone: a disulfide dimer, a glycosylated form, 20 kD-hGH, and two peptides made up of portions of 22 kD-hGH. The two pituitary peptides (hGH(1-43) and hGH(44-191)) have, respectively, insulin-potentiating and anti-insulin properties. Both have been detected in serum. The shorter peptide may prove to be useful in decreasing the amount of exogenous insulin required by diabetics. The larger, strongly anti-insulin peptide, may be involved in diabetic retinopathy. We believe that this peptide is the long sought after diabetogenic substance of the pituitary gland.

PURPOSE: To assess the role of hypoxia in inducing the proliferation, hypertrophy, and dysfunction of Muller cells in detached retina and the effectiveness of supplemental oxygen in limiting these reactions. METHODS: Retinal detachments were produced in the right eye of each of 13 cats; the cats survived surgery for 3 days, during which six were kept in normoxia (room air, 21%) and seven in hyperoxia (70% oxygen). Retinas were labeled for proliferation with an antibody (MIB-1) to a cell cycle protein (Ki-67), for evidence of hypertrophy employing antibodies to the intermediate filament protein glial fibrillary acidic protein (GFAP) and to β-tubulin and for disturbance of glutamate neurochemistry employing antibodies to glutamate to a glutamate receptor (GluR-2) and to glutamine synthetase. RESULTS: Results from the two animals kept in normoxia after retinal detachment confirmed previous reports that detachment caused the proliferation of Muller cells, the hypertrophy of Muller cell processes, and the disruption of glutamate recycling by Muller cells. Oxygen supplementation during detachment reduced Muller cell proliferation and hypertrophy and reduced the abnormalities in the distributions of glutamate, GluR-2, and glutamine synthetase. CONCLUSIONS: Oxygen supplementation reduced the reaction of retinal Muller cells to retinal detachment, limiting their proliferation and helping to maintain their normal structure and function. In the clinical setting, oxygen supplementation between diagnosis and reattachment surgery may reduce the incidence and severity of glial-based complications, such as proliferative vitreoretinopathy.

Cellular retinaldehyde binding protein (CRALBP) is present in Müller glia and in cells of the retinal pigment epithelium, but we have recently observed CRALBP-like immunoreactivity near the inner limiting membrane in the newborn mouse retina. The present study has examined whether this protein is present in developing retinal astrocytes. Retinal tissue was collected at various embryonic and postnatal ages and in adulthood. Tissue for immunohistochemistry was fixed by immersion in 4% paraformaldehyde and immunostained using rabbit polyclonal antisera to CRALBP or glial fibrillary acidic protein (GFAP), while fresh tissue was homogenized for Western analysis. Specificity of the antiserum for the 33 kDa protein was shown in retinal homogenates by immunoblotting, with expression of the protein increasing steadily from E15.5 through adulthood. Immunostaining of sections from fetal eye-cups revealed faint labeling of cells in the optic nerve, with progressive migration of CRALBP-immunoreactive cells into the retina at the inner limiting membrane during the perinatal period. By the day of birth, these cells were intensely immunoreactive, showing a morphology characteristic of migrating astrocytes. These CRALBP-immunoreactive cells mimicked the progressive infiltration of GFAP-positive astrocytes which are known to migrate into the retina from the optic nerve head, many of which were double-labeled with GFAP. Their distribution across the retina is distinct from that of the lighter-staining Müller glial somata during these stages, and they are not misidentified Müller glial endfeet. Astrocytes are only transiently CRALBP-immunoreactive, no longer containing the protein after the second post-natal week. Preincubation of the antiserum with purified CRALBP abolished all staining of astrocytes. Coupled with the fact that only a single (approximately 33 kDa) molecular weight protein is labeled by the antiserum, it was concluded that retinal astrocytes contain CRALBP during a limited period of development.

To study the response of ON and OFF bipolar cells in experimental retinal detachment.Domestic cat retinas were detached for 7 days. The retinas were prepared for immunocytochemical staining with antibodies to Go alpha (α), glutamate transporter GLT-1, protein kinase C and rod opsin, which serve as markers for ON bipolar cells, OFF bipolar cells, rod bipolar cells and rod photoreceptors, respectively. Both sections and whole-mounts were labelled with antibodies to Goα and GLT-1.Following 7 days of detachment, ON bipolar cell processes extended into the outer nuclear layer and had neurites extending beyond their target layer into the inner plexiform layer. In contrast, OFF bipolar cell processes were reduced in the outer plexiform layer following detachment.ON and OFF bipolar cells undergo significant remodelling of their processes in response to retinal detachment, and the ON and OFF pathways may be differentially affected. The remodelling may be due to morphological changes that have previously been shown to occur in photoreceptor synaptic terminals or as a result of loss of synaptic connections due to photoreceptor cell death.

Exogenous basic fibroblast growth factor (bFGF) stimulates proliferation of non-neuronal retinal cells in vivo. To help understand how this proliferative effect is mediated, we followed the fate of biotinylated bFGF after injection into the vitreous of normal rabbit eyes. The retinal distributions, binding, and processing of biotinylated bFGF (bFGF-biotin) was examined from 2hr to 7 days after intravitreal injection using laser scanning confocal microscopy, electron microscopy and Western blot analysis. At 2hr, bFGF-biotin was detected throughout the extracellular space and on retinal basement membranes. At 6hr, discrete punctate material first appeared within the cytoplasm of Muller cells, astrocytes, endothelial cells, retinal pigment epithelial (RPE) cells, and ganglion cells. Labeling was also present in the invaginations of the photoreceptor synaptic terminals at this time. This general pattern persisted up to 4 days after injection but was greatly attenuated by post-injection day 7. Labeling in the inner retina decreased progressively over the seven days; whereas labeling in the outer retina, primarily within the RPE, increased at 4 days post-injection and then gradually decreased to nearly undetectable levels by 7 days. Western analysis of retinal protein homogenates following injection showed that an 18kDa component representing intact bFGF, can be identified up to 1 week following injection. This component, as well as a 15 and 9kDa biotinylated fragment, showed a progressive reduction during the one week post-injection period. Cross-linking experiments demonstrated that bFGF-biotin binds to three putative receptors with approximate molecular weights of 54, 62, and 110kDa. These data are consistent with binding of exogenous bFGF to: (a) low affinity bFGF receptors associated with retinal basement membranes; (b) invaginations at the base of photoreceptor synapses; and (c) putative high affinity bFGF receptors on the plasma membranes of glial cells, endothelial cells, RPE cells and ganglion cells. bFGF-biotin apparently binds to, and is then internalized by, the same non-neuronal cell types that are stimulated to proliferate following retinal injuries such as detachment.

We examined the expression of several proteins normally present in Müller's glia after the production of experimental retinal detachment in adult cats. Retinas were detached for one-half to seven days, after which the tissue was processed for correlative immunocytochemistry and biochemistry. Previous studies demonstrated that the intermediate filament proteins glial fibrillary acidic protein and vimentin, increase after long-term retinal detachment (30 to 60 days), whereas glutamine synthetase, carbonic anhydrase C, and cellular retinaldehyde-binding protein all decrease to barely detectable levels. Alterations in Müller cell protein expression are rapid and specific events that can be detected as early as two days after retinal detachment. By seven days, levels of protein expression are similar to those in the long-term retinal detachments. Within the first week after injury the Müller cell processes hypertrophy and begin forming glial scars, which indicates that early intervention may be required to halt or reverse the effects of detachment.

Aims We have previously identified neurofilament-protein-containing neurites in human epiretinal membranes (ERMs). The aim of this study was to further characterise these neurites by examining the expression of additional specific proteins in human ERMs and to correlate this expression with various retinal disease conditions. Methods Epiretinal membranes originating from 43 patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) or with no known pathology (idiopathic epiretinal membrane; iERM) were removed during vitrectomy at varying durations after diagnosis and immediately placed in fixative. The membranes were labelled immunohistochemically with different combinations of antibodies to the proteins melanopsin, calretinin and neurofilament (to identify subclasses of ganglion cells), rod opsin (to identify rod photoreceptors), synaptophysin and synaptic vesicle glycoprotein 2A (SV2) (identifies synaptic vesicles) and vimentin (identifies glial cells). Results Anti-melanopsin-, anti-calretinin-, anti-neurofilament- and anti-rod-opsin-labelled neurites were routinely observed in the epiretinal membranes. Their presence did not appear to correlate with a specific disease condition or duration of the membrane. Generally neurites were observed in regions of glial cells. Conclusions Based on the expression of selected markers for neurites, we show neurite processes in human ERMs of various aetiologies originating from rod photoreceptors and different populations of retinal ganglion cells, although there was no obvious correlation with specific disease condition. In addition, synaptophysin and SV2 labelling was observed associated with all types of neurites, indicating the presence of at least one component necessary for synaptic transmission. Our data suggest that the adult human retina retains a significant capacity for neuronal remodelling under various disease conditions.

Purpose To study the fate of Müller’s glia following experimental retinal detachment, using a “pulse/chase” paradigm of bromodeoxyuridine (BrdU) labeling for the purpose of understanding the role of Müller cell division in subretinal scar formation. Methods Experimental retinal detachments were created in pigmented rabbit eyes, and 3 days later 10 µg of BrdU was injected intravitreally. The retinas were harvested 4 h after the BrdU was administered (i.e., day 3) or on days 4, 7, and 21 post detachment. The tissue was fixed, embedded in agarose, and sectioned at 100 µm. The sections were labeled with various combinations of probes, including anti-vimentin and anti-S100 (as markers for Müller cells), anti-BrdU, anti-phosphohistone H3 (to identify mitotic cells), and the isolectin B4 (to identify macrophages and microglia). Images were captured using an Olympus Fluoview 500 confocal microscope. To aid in our understanding of how Müller cell nuclei undergo cell division, two additional procedures were used: 1) electron microscopy of normal cat and rabbit retinas and 2) a new method using 5-fluorouracil and subsequent anti-BrdU labeling to detect all Müller cell nuclei, using confocal imaging. Results Three days after detachment, anti-vimentin labeled all Müller cells, some of which were also labeled with anti-BrdU. On day 4, many of the anti-BrdU-labeled Müller cell nuclei appeared in columns with one labeled nucleus in the inner nuclear layer and another directly sclerad to it in the outer nuclear layer. By day 7, most anti-BrdU-labeled nuclei were observed in subretinal scars. At 3 weeks, some anti-BrdU-labeled nuclei that remained within the retina did not express vimentin or S100. Anti-phosphohistone H3-labeled (i.e., mitotic) cells, some of which were also labeled with anti-BrdU, were only observed in the outer nuclear layer on day 4, and these nuclei were surrounded by an accumulation of vimentin filaments. Isolectin B4-labeled microglia and macrophages also incorporated BrdU and were observed throughout the retina and in subretinal scars during all times of detachment. Electron microscopy and immunofluorescence labeling of the 5-fluorouracil-injected eyes revealed the presence of a unique structural relationship between Müller cell nuclei and intermediate filament proteins. Conclusions Following retinal detachment, many Müller cell nuclei initially migrate to the outer retina, undergo mitosis, and eventually reside in subretinal glial scars, suggesting a possible link between the early division of Müller cells and the process of subretinal gliosis. In addition, a subpopulation of anti-BrdU-labeled cells, presumably once Müller cells, appears to stop expressing well accepted Müller cell marker proteins, suggesting a potential dedifferentiation of some of these cells over time. Additionally, Müller cell nuclei may use intermediate filaments as a “track” for migration into the outer retina and later as an important component of cell division by the accumulation of vimentin filaments around the mitotic nuclei.

We have previously demonstrated that glial fibrillary acidic protein (GFAP) containing intermediate filaments in retinal Müller cells undergo both quantitative induction and subcellular reorganization as a response to long-term retinal detachment (an induced CNS degeneration wherein the Müller cells form a multicellular scar). This study demonstrates by RNA blotting analysis that normal retina expresses a low basal level of GFAP mRNA, which is induced approximately 500% within 3 days of retinal detachment. At the cellular level, electron microscopic in situ hybridization analysis readily detects GFAP mRNA in Müller cells of detached retinas, but not in normal retinas. On the other hand, GFAP mRNA was readily detected in retinal astrocytes (which appear to express GFAP mRNA at high, constitutive levels). In both cell types, the ultrastructural localization of GFAP mRNA was the same. In the nuclei, the GFAP mRNA was associated with amorphous, electron-dense regions within the euchromatin. In the cytoplasm, the GFAP mRNA was associated with intermediate filaments near the nuclear pores, along the filaments when no other structures were apparent, and when the filaments appeared to be associated with ribosomes and polysomes. The ultrastructural location of the GFAP mRNA (especially along the intermediate filaments) may be unique to this mRNA or may represent a more generalized mRNA phenomenon.

Purpose To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model. Methods Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections. Results Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Muller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment. Conclusions JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells.

Proliferative vitreoretinopathy represents the major complication in retinal detachment surgery andoccurs in about 5–15% of cases resulting in a significant loss of vision despite multiple surgical pr

The distribution of opsin was examined immunocytochemically after experimental retinal detachment in adult cats. Retinal detachments were produced by injecting fluid between the retinal pigment epithelium and neural retina. One to 60 days later the animals were killed. Tissue areas from detached and attached retinal regions from the eye with the detached retina, as well as normal (control) retinas, were processed for post-embedding light and electron microscopic immunocytochemistry. In normal and attached retinal regions, anti-opsin labeled the outer segments and Golgi apparatus most heavily, although the entire photoreceptor plasma membrane was labeled at a low level. Beginning at 2 days after retinal detachment, immunolabeling increased in the photoreceptor inner segment, cell body and synaptic terminal plasma membranes. This pattern of anti-opsin labeling continued at all intervals up through the 60-day detachment time-point. Injection of radiolabeled amino acid in detachments from 1 to 30 days show that radiolabeled protein is still transported to the truncated outer segments of the photoreceptor cells. In addition, these outer segment disks label with anti-opsin. These data imply that opsin continues to be transported and incorporated into the outer segments of photoreceptors showing severe degeneration as a result of long-term detachment from the RPE.

The interphotoreceptor matrix (IPM) occupies the extracellular space between the photoreceptors of the retina and the apical surface of the retinal pigmented epithelium. A large proportion of the IPM is composed of aqueous-insoluble glycoconjugates, including chondroitin sulfate-containing proteoglycans, the distribution of which exhibits both apical-basal and photoreceptor cell type-specific heterogeneities. The precise function of most insoluble IPM constituents is unknown, although the available evidence suggests some may contribute to retinal adhesion or photoreceptor survival. We have now identified basic fibroblast growth factor (bFGF), or an immunologically related protein from the FGF family, within the IPM. The IPM is labeled on sections of primate retinas by a battery of polyclonal antibodies (Abs) directed against various peptide sequences of bFGF and by an Ab to bovine brain bFGF. bFGF Abs also bind to purified preparations of aqueous-insoluble IPM. All bFGF Abs utilized cross-react with equivalent low molecular mass components of 16.5-17.5 kDa on Western blots of insoluble IPM proteins, purified bFGF, and recombinant bFGF. The Abs do not bind any aqueous-soluble IPM components, suggesting that the bFGF is normally bound to an insoluble IPM constituent(s) in situ. The fact that bFGF is sequestered in the IPM and is located in such close proximity to the photoreceptors, the retinal pigmented epithelium, and Mueller's glia raises the strong possibility that it is synthesized by and regulates the activities of one or more of these three cell types in vivo.

Cats have two types of horizontal cell (HC); one is axon-bearing (B-type), the other is axonless (A-type). We have previously described neurite sprouting from HCs in response to experimental retinal detachment. Here we sought to determine whether one or both types elaborate these outgrowths.Sections as well as wholemounts of retinas detached for 3, 7 and 28 days together with control retinas were double or triple labeled with antibodies to the calcium binding proteins calretinin and calbindin, to the synaptic vesicle-associated membrane protein 2 (VAMP2), and to the 70 and 200 kDa subunits of the neurofilament protein. Digital immunofluorescence images were collected by both confocal and two-photon microscopy.In control retina, both HC types label with antibodies to calretinin and calbindin D, but only the A-type also intensely labels with the neurofilament protein antibody. After 3, 7 and 28 days of detachment, these staining patterns persist, but there is a moderate upregulation of neurofilament protein in the B-type cell. In the detached retina, HC processes sprout neurites that appear most commonly as a loose array of fine beaded processes rising from the outer plexiform layer (OPL) into the outer nuclear layer (ONL), or, especially at 28 days, as stout unbranching processes that often cross the ONL en route to the subretinal space where some expand and arborize. Both types are strongly calretinin-positive while being somewhat less positive for antibodies to calbindin D and neurofilament protein. Moreover, they all arise from similarly labeled processes in the distal-most domain of the OPL where the narrowly stratified field of axon terminal boutons of the B-type HC normally innervates rod spherules, two to three thousand per cell. Our data indicate that the HC sprouts apparently arise specifically from the axon terminal of the B-type cell since outgrowths were never seen arising from either type of HC perikaryon or from processes identifiable as A-type dendrites.The data described here point to the specific remodeling of the rod-connected axon terminals of the B-type cell through neurite outgrowth. Rods respond to detachment by withdrawing synaptic terminals from the OPL while cones do not. Those HC outgrowths that terminate within the ONL appear to retain their connection with the retracted terminals. Others apparently have lost their presynaptic targets and cross the ONL in association with hypertrophied Müller cell processes.

Aim: To determine whether neural elements are present in subretinal and epiretinal proliferative vitreoretinopathy (PVR) membranes as well as in diabetic, fibrovascular membranes removed from patients during vitrectomy surgery. Methods: Human subretinal and epiretinal membranes of varying durations were immunolabelled with different combinations of antibodies to glial fibrillary acidic protein, vimentin, neurofilament protein and laminin. Results: Anti-neurofilament-labelled neurites from presumptive ganglion cells were frequently found in epiretinal membranes and occasionally found in subretinal membranes. In addition, the neurites were only observed in regions that also contained glial processes. Conclusions: These data demonstrate that neuronal processes are commonly found in human peri-retinal cellular membranes similar to that demonstrated in animal models. These data also suggest that glial cells growing out of the neural retina form a permissive substrate for neurite growth and thus may hold clues to factors that support this growth.

To develop an automated tool that provides reliable, consistent, and accurate results for counting cell nuclei in tissue sections.We propose a novel method based on an image processing algorithm to analyze large sets of digital micrographs. The nucleus detector design is based on a Laplacian of Gaussian filter. We use the leave-one-out cross validation method for estimating the generalization error, which is then used to choose the model and parameters of the proposed nucleus detector with both fluorescent and dye stained images. We also evaluate the performance of a nucleus detector by comparing the results with manual counts.When our nucleus detector is applied to previously unanalyzed images of feline retina, it correctly counts nuclei within the outer nuclear layer (ONL) with an average error of 3.67% ranging from 0 to 6.07%, and nuclei within the inner nuclear layer (INL) with an average error of 8.55% ranging from 0 to 13.76%. Our approach accurately identifies the location of cell bodies. Even though we have a relatively large error in the INL due to the large intra-observer variation, both manual counting and nucleus detector result in the same conclusion. This is the first time that cell death in the INL in response to retinal detachment is analyzed quantitatively. We also test the proposed tool with various images and show that it is applicable to a wide range of image types with nuclei varying in size and staining intensity.The proposed method is simple and reliable. It also has widespread applicability to a variety of sample preparation and imaging methods. Our approach will be immediately useful in quantifying cell number in large sets of digital micrographs and from high-throughput imaging. The tool is available as a plug-in for Image J.

Intravitreal bevacizumab (Avastin; Genentech Inc., San Francisco, CA) is a new treatment for age-related macular degeneration. The aim of this study was to evaluate retinal penetration and toxicity of bevacizumab.Ten albino rabbits were injected intravitreally with 0.1 mL (2.5 mg) of Avastin into one eye and 0.1 mL saline into the fellow eye. The electroretinogram (ERG) was recorded after 3 hours, 3 days, and 1, 2, and 4 weeks. The visual evoked potential (VEP) was recorded after 4 weeks. Confocal immunohistochemistry was used to assess retinal penetration.The ERG responses of the control and experimental eyes were similar in amplitude and pattern throughout the follow-up period. The flash VEP responses of the experimental eyes were of normal pattern and amplitude and did not differ from those recorded by stimulation of the control eye alone. Full thickness retinal penetration was present at 24 hours and was essentially absent at 4 weeks.Bevacizumab was found to be nontoxic to the retina of rabbits based on electrophysiologic studies. Full thickness retinal penetration may explain observed clinical effects of intravitreal bevacizumab. Although it is difficult to directly extrapolate to humans, our study supports the safe use of intravitreal bevacizumab injection.

To compare the activation of microglia in response to retinal detachment in four species.Experimental detachments were created in cats, rabbits, and ground squirrels and the retinas harvested 1, 3, 7, or 28 days later. Retinal reattachments of 28 days in duration were also performed in cats following a 3-day detachment. Human tissue was obtained during reattachment surgery. Microglia and macrophages were labeled with the lectins Griffonia simplicifolia and Ricinus communis and the antibody CD11b. Müller cell and photoreceptor responses were followed immunocytochemically on the same tissue sections labeled with microglial markers. Images were collected by laser scanning confocal microscopy.Lightly labeled microglia were observed primarily in the inner retina of control tissue. In the cat and rabbit, a progressive increase in the number of labeled cells occurred in the outer retina beginning at 1 day of detachment. In both long term human and cat detachments numbers of microglia were elevated throughout the retina. This is in contrast to the rabbit and ground squirrel retinas where microglial activation was dramatically diminished in longer term detachments. Presumptive macrophages (anti-CD11b labeled cells) occurred only in the subretinal space. Retinal reattachment in cats significantly attenuated the response except in areas of poor outer segment regeneration.The robust microglial response to retinal detachment is an indicator of the importance of this cell type in the overall response of the retina. Our data suggest that the feline retina is a particularly appropriate model system for understanding this response in humans. Inhibiting the microglial response in that species should help us understand more precisely its potential role in photoreceptor survival in human pathology.

Retinal detachment, the separation of the neural retina from the retinal pigmented epithelium, starts a cascade of events that results in cellular changes throughout the retina. While the degeneration of the light sensitive photoreceptor outer segments is clearly an important event, there are many other cellular changes that have the potential to significantly effect the return of vision after successful reattachment. Using animal models of detachment and reattachment we have identified many cellular changes that result in significant remodeling of the retinal tissue. These changes range from the retraction of axons by rod photoreceptors to the growth of neurites into the subretinal space and vitreous by horizontal and ganglion cells. Some neurite outgrowths, as in the case of rod bipolar cells, appear to be directed towards their normal presynaptic target. Horizontal cells may produce some directed neurites as well as extensive outgrowths that have no apparent target. A subset of reactive ganglion cells all fall into the latter category. Muller cells, the radial glia of the retina, undergo numerous changes ranging from proliferation to a wholesale structural reorganization as they grow into the subretinal space (after detachment) or vitreous after reattachment. In a few cases have we been able to identify molecular changes that correlate with the structural remodeling. Similar changes to those observed in the animal models have now been observed in human tissue samples, leading us to conclude that this research may help us understand the imperfect return of vision occurring after successful reattachment surgery. The mammalian retina clearly has a vast repertoire of cellular responses to injury, understanding these may help us improve upon current therapies or devise new therapies for blinding conditions.

Intermediate filament proteins are a heterogeneous group of proteins that form 10-nm-diameter filaments, a highly stable cytoskeletal component occurring in various cell types. The up-regulation of one of these intermediate filament proteins, glial fibrillary acidic protein (GFAP), historically has been an indicator of "stress" in central nervous system (CNS) astrocytes. The retina also responds similarly to "stress" but the up-regulation of intermediate filaments occurs primarily in the Müller cells, the radial glia of the retina. This is a remarkably ubiquitous response in that a similar up-regulation can be observed in numerous forms of retinal degeneration. As a consequence of retinal detachment, a "mechanical" injury to the retina, GFAP, and another intermediate filament protein, vimentin, dramatically increase in Müller cells. Concomitant with this up-regulation is the hypertrophy of these cells both within the retina and onto the photoreceptor and vitreal surfaces of the retina. The function of this distinctive intermediate filament up-regulation in glial cells is unknown, but in the retina their expression is differentially regulated in a polarized manner as the Müller cells hypertrophy, suggesting that they play some role in this process. Moreover the response of intermediate filaments and the Müller cells differs depending on whether the retina has been detached or reattached to the retinal pigment epithelium. The differential expression of these proteins may give insight into their role in the formation of glial scars in the retina and elsewhere in the CNS.

PURPOSE Drusen are variably sized extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. They are commonly found in aged eyes, however, numerous and/or confluent drusen are a significant risk factor for age-related macular degeneration. The purpose of this study was to investigate the impact of drusen on overlying cells of the retina. METHODS Tissue containing retina and RPE/choroid was dissected from human donor eyes, embedded in agarose, and sectioned at 100 micro m using a vibratome. Sections were immunostained with a panel of antibodies that labeled glial cells, first-, second-, and third-order retinal neurons and processed for confocal microscopy. RESULTS Retinal cells that overlie both soft and hard drusen exhibited numerous structural and molecular abnormalities. Normally detectable only in the outer segments of rod photoreceptors, rod opsin immunolabeling was also observed in the inner segment, cell body, axon, and axon terminal of photoreceptors that overlie drusen. Labeling with this antibody also revealed the deflection and shortening of rod inner and outer segments. Cone photoreceptors displayed similar structural abnormalities, as well as a decrease in cone opsin immunoreactivity. Drusen-associated abnormalities in the synaptic terminals of photoreceptor cells were also observed. In addition, an increase in intermediate filament protein immunoreactivity (vimentin and glial fibrillary acidic protein) was observed within Muller glial cells in areas of retina overlying drusen. Both soft and hard drusen were associated with a similar spectrum of effects in both macular and extramacular regions. Second- and third-order neurons, including bipolar, horizontal, amacrine, and ganglion cells all appeared unaffected. The structural and molecular abnormalities observed in photoreceptors and Muller glial cells were confined to retinal regions directly overlying and immediately adjacent to drusen; more distant retinal regions appeared unperturbed. Remarkably, significant abnormalities were observed over small subclinical drusen. CONCLUSIONS Retinal cells overlying both soft and hard drusen exhibit structural and molecular abnormalities indicative of photoreceptor degeneration and Muller glial activation. These abnormalities resemble the degenerative effects common to many forms of retinal degeneration, but are confined to areas directly overlying drusen. This suggests that photoreceptor cell function is compromised as a consequence of drusen formation.

Ground squirrel retinas were immunostained with antibodies against calcium binding proteins (CBPs) and classical neurotransmitters in order to describe neuronal phenotypes in a diurnal mammalian retina and to then compare these neurons with those of more commonly studied nocturnal retinas like cats' and rabbits'. Double immunostained tissue was examined by confocal microscopy using antibodies against the following: rhodopsin and the CBPs, calbindin, calretinin, parvalbumin, calmodulin and recoverin (CB, CR, PV, CM, RV), glycine, GABA, choline acetyltransferase (CHAT) and tyrosine hydroxylase (TOH). In ground squirrel retina, the traditional cholinergic mirror symmetric amacrine cells colocalize CHAT with PV and GABA and faintly with glycine. A second cholinergic amacrine cell type colocalizes glycine alone. CR is found in at least 3 different amacrine cell types. The CR-immunoreactive (IR) cell population is a mixture of glycinergic and GABAergic types. The dopamine cell type IR to tyrosine hydroxylase has the typical morphology of a wide field cell with dendrites in S1 but the "rings" seen in cat or rabbit retina are not as numerous. TOH-IR amacrine cells send large club-shaped processes to the outer plexiform layer. CB and CR are in bipolar cells, A- and B-type horizontal cells and several amacrine cell types. Anti-rhodopsin labels the low density rod photoreceptor population in this species. Anti-recoverin labels cones and some bipolar cells while PKC is found in several different bipolar cell types. One ganglion cell with dendritic branching in S3 is strongly CR-IR. We find no evidence for an AII amacrine cell in the ground squirrel, with either anti-CR or anti-PV. An amacrine cell with similarity to the DAP1-3 cell of rabbit is CR-IR and glycine-IR. We discuss this labeling pattern in relationship to other mammalian species. The differences in staining patterns and phenotypes revealed suggest a functional diversity in the populations of amacrine cells according to whether the retinas are rod or cone dominated.

In the feline model, retinal detachment initiates a cascade of changes that include photoreceptor- cell "deconstruction," apoptotic death of some photoreceptors, neurite outgrowth from second- and third-order neurons, remodeling of photoreceptor synaptic terminals, and Muller-cell gliosis. We have previously shown that reattachment within 24 h halts or reverses many of these presumed detrimental changes. However, in patients with retinal detachments, reattachment cannot always be performed within this 24-h window. Moreover, recovery of vision following successful reattachment surgery in the macula is often imperfect. Here, we examine the ability of relatively long-term reattachment (28 d) to stop or reverse several cellular events that occur at 3 d of detachment. In contrast to earlier studies of reattachment, which focused on the regeneration of outer segments, we focus our attention here on other cellular events such as neuronal remodeling and gliosis. Some of these changes are reversed by reattachment, but reattachment itself appears to stimulate other changes that are not associated with detachment. The implications of these events for the return of vision are unknown, but they do indicate that simply reattaching the retina does not return the retina to its pre-detachment state within 28 d.

This chapter shows that retinal detachment in the ground squirrel model does not have a larger impact on S-cone than on M-cone function as indexed by signals originating in the outer retina. To the contrary, to the extent that there is any differential effect of detachment on function subserved by the two cone types, the results suggest that the loss may be slightly in the opposite direction, that is, a proportionally greater effect of detachment on M-cone than on the S-cone function.