Three-Dimensional Ultrastructure of the Normal Rod Photoreceptor Synapse and Degenerative Changes Induced by Retinal Detachment.

The rod photoreceptor synapse is the first synapse of dim-light vision and one of the most complex in the mammalian CNS. The components of its unique structure, a presynaptic ribbon and a single synaptic invagination enclosing several postsynaptic processes, have been identified, but disagreements about their organization remain. Here, we have used EM tomography to generate high-resolution images of 3-D volumes of the rod synapse from the female domestic cat. We have resolved the synaptic ribbon as a single structure, with a single arciform density, indicating the presence of one long site of transmitter release. The organization of the postsynaptic processes, which has been difficult to resolve with past methods, appears as a tetrad arrangement of two horizontal cell and two rod bipolar cell processes. Retinal detachment severely disrupts this organization. After 7 d, EM tomography reveals withdrawal of rod bipolar dendrites from most spherules; fragmentation of synaptic ribbons, which lose their tight association with the presynaptic membrane; and loss of the highly branched telodendria of the horizontal cell axon terminals. After detachment, the hilus, the opening through which postsynaptic processes enter the invagination, enlarges, exposing the normally sequestered environment within the invagination to the extracellular space of the outer plexiform layer. Our use of EM tomography provides the most accurate description to date of the complex rod synapse and details changes it undergoes during outer segment degeneration. These changes would be expected to disrupt the flow of information in the rod pathway.SIGNIFICANCE STATEMENT Ribbon-type synapses transmit the first electrical signals of vision and hearing. Despite their crucial role in sensory physiology, the three-dimensional ultrastructure of these synapses, especially the complex organization of the rod photoreceptor synapse, is not well understood. We used EM tomography to obtain 3-D imaging at nanoscale resolution to help resolve the organization of rod synapses in normal and detached retinas. This approach has enabled us to show that in the normal retina a single ribbon and arciform density oppose a tetrad of postsynaptic processes. In addition, it enabled us to provide a 3-D perspective of the ultrastructural changes that occur in response to retinal detachment.

Three-dimensional organization of nascent rod outer segment disk membranes.

The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

Astrocyte structural reactivity and plasticity in models of retinal detachment.

Although retinal neurodegenerative conditions such as age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, and retinal detachment have different etiologies and pathological characteristics, they also have many responses in common at the cellular level, including neural and glial remodeling. Structural changes in Müller cells, the large radial glia of the retina in retinal disease and injury have been well described, that of the retinal astrocytes remains less so. Using modern imaging technology to describe the structural remodeling of retinal astrocytes after retinal detachment is the focus of this paper. We present both a review of critical literature as well as novel work focusing on the responses of astrocytes following rhegmatogenous and serous retinal detachment. The mouse presents a convenient model system in which to study astrocyte reactivity since the Mϋller cell response is muted in comparison to other species thereby allowing better visualization of the astrocytes. We also show data from rat, cat, squirrel, and human retina demonstrating similarities and differences across species. Our data from immunolabeling and dye-filling experiments demonstrate previously undescribed morphological characteristics of normal astrocytes and changes induced by detachment. Astrocytes not only upregulate GFAP, but structurally remodel, becoming increasingly irregular in appearance, and often penetrating deep into neural retina. Understanding these responses, their consequences, and what drives them may prove to be an important component in improving visual outcome in a variety of therapeutic situations. Our data further supports the concept that astrocytes are important players in the retina's overall response to injury and disease.

The fate of Müller's glia following experimental retinal detachment: nuclear migration, cell division, and subretinal glial scar formation.

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 microg 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 microm. 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.

Retraction and remodeling of rod spherules are early events following experimental retinal detachment: an ultrastructural study using serial sections.

PURPOSE: To describe changes induced by retinal detachment in the ultrastructure and organization of rod terminals and their connections with B-type horizontal cell (HC) axon terminals and rod bipolar cell (RB) dendrites. METHODS: Sections from control, 3 day, 7 day, and 28 day detached feline retinas were prepared for confocal immunofluorescence, light microscopy, and electron microscopy (EM). In addition, 100 mum-thick vibratome sections were immunolabeled with markers for photoreceptor terminals, HCs, and RBs. More than 40 rod spherules were studied in 90 nm-thick serial sections by transmission EM to greater detail changes in their ultrastructure and innervation. RESULTS: Following retinal detachment, many rod terminals retracted varying distances toward their respective cell bodies in the outer nuclear layer (ONL). In retinas detached for 1 to 4 weeks, an altered synaptic vesicle population and associated ribbons were found in all retracting terminals. Many rod somata in the distal ONL seemed to lack synaptic terminal structures altogether. In a retina detached for 1 week, EM showed that less than half of the retracted terminals remain in contact with RB dendrites. In contrast, almost every surviving spherule was contacted by neurite outgrowths from the axon terminals of the B-type HC. Although retracted spherules had several presynaptic structures similar to those in normal retina, numerous changes occurred in their overall synaptic architecture. The spherule's invagination was shallower, contained fewer postsynaptic processes, and often had "opened," allowing swollen HC processes apposing the synaptic ribbon to directly contact other processes of the outer plexiform layer (OPL) neuropil. Whereas in normal cat retina each HC "lobe" comes from a different axon terminal system, after detachment, the opposing lateral elements can stem from the same terminal. The innervating RB dendrites that branched off stout RB dendritic trunks that extended up into the ONL were thinner than normal, unbranched, often electron dense, and lacked organelles. When present, most merely lay adjacent to retracting spherules rather than enter any synaptic invagination that might still occur. CONCLUSIONS: Immunocytochemistry enabled RB and HC neurites to appear postsynaptic to retracted rod terminals. However, at the ultrastructural level, HCs seemed to more consistently retain connection with the retracted spherules than the RBs. The highly conserved architecture of the rod spherule was lost as the invagination opened and postsynaptic contacts became fewer. It would seem that the lack of RB central elements as well as the drastic alterations in the architecture of most retracted terminals would necessarily alter the physiology of this complex synapse.

The effect of alkylphosphocholines on intraretinal proliferation initiated by experimental retinal detachment.

PURPOSE: To determine the effect of alkylphosphocholines (APCs) on intraretinal proliferation induced by experimental retinal detachment in the rabbit. METHODS: Retinal detachments were created in adult pigmented rabbits. APCs, either liposome bound (liposome, L-APC) or unbound (free, F-APC), were injected intravitreally on either day 1 or day 2 after detachment. BrdU was injected on day 3, 4 hours before death. After fixation, retinas were triple labeled with anti-BrdU, anti-vimentin, and the isolectin B4. The number of anti-BrdU-labeled cells was counted per millimeter of retina from sections imaged by laser scanning confocal microscopy. Toxicity was examined using toluidine blue-stained sections imaged by light microscopy and by electron microscopy for ultrastructural evaluation. RESULTS: Retinal detachment initiated proliferation of all non-neuronal cells. After intravitreal injection on day 1 or 2 after experimental induction of retinal detachment, APCs significantly reduced the number of dividing cells at day 3. Liposome-bound drug given on day 2 was more effective on Müller cell proliferation than was unbound drug. Injection of F-APC on day 1 was more effective than when given on day 2. No apparent effect was seen on Müller cell hypertrophy as indicated by vimentin expression. In addition, no evidence of toxicity was observed in the retina at day 3 for any of the conditions. CONCLUSIONS: APCs significantly reduce the number of Müller cells that are stimulated to divide as a result of retinal detachment. The preliminary results indicate no evidence of significant toxicity; however, further studies are needed. APCs have the potential to be used as part of a therapeutic approach if they can be combined with other agents that can suppress the fibrosis that is also a critical event in the pathogenesis of proliferative vitreoretinal diseases such as proliferative vitreoretinopathy (PVR).

Immunocytochemical evidence that rod-connected horizontal cell axon terminals remodel in response to experimental retinal detachment in the cat.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment.

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.

Anatomical and Gene Expression Changes in the Retinal Pigmented Epithelium Atrophy 1 (rpea1) Mouse: A Potential Model of Serous Retinal Detachment.

PURPOSE: The 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). METHODS: Optical 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). RESULTS: By 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. CONCLUSIONS: These 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.

Experimental retinal reattachment: a new perspective.

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 Müller-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.

The ability of rapid retinal reattachment to stop or reverse the cellular and molecular events initiated by detachment.

PURPOSE: To determine the effects of reattachment on the molecular and cellular events initiated by a retinal detachment lasting 1 hour or 1 day. METHODS: Experimental retinal detachments were created in the right eyes of nine cats. Reattachments were performed 1 hour (n = 3) or 1 day (n = 3) after the detachment, and the animals were killed 3 days after detachment. Three-day detached (n = 3) and normal (n = 3) retinas were used for comparisons. Agarose-embedded sections were double labeled with a panel of antibodies. Some sections were also probed with the TUNEL technique to detect apoptotic cells. Wax-embedded sections were labeled with the MIB-1 antibody to the Ki67 protein to detect proliferating cells. RESULTS: The 1-hour and 1-day detachments followed by reattachment showed a very similar and consistent reduction in photoreceptor deconstruction and the Müller cell gliotic response when compared with 3-day retinal detachments without reattachment. Light microscopy and immunolabeling with opsin antibodies showed a significant reduction in both rod and cone outer segment (OS) degeneration, even though OS length was shorter than normal. The reattachments also showed a reduction in opsin redistribution, retraction of rod terminals, TUNEL-labeled photoreceptors, loss of cytochrome oxidase staining in photoreceptors, neurite outgrowth from second-order neurons, the number of proliferating cells, and the increase in intermediate filaments and loss of soluble proteins from Müller cells. The apparent re-ensheathing of the OS by the apical processes of the retinal pigment epithelium had begun but was not completely normal. CONCLUSIONS: These data indicate that, even though the length of the OS is less than normal, retinal reattachment within 1 day of detachment can either greatly retard or reverse many of the molecular and cellular changes initiated by detachment.

Cone photoreceptor recovery after experimental detachment and reattachment: an immunocytochemical, morphological, and electrophysiological study.

PURPOSE: To compare the morphologic and functional recovery of the retina after detachment and reattachment in an animal with a cone-dominant retina, the ground squirrel. METHODS: Ground squirrel (Spermophilus beecheyi) retinas were detached for 1 day and reattached for 7, 35, or 96 days (n = 2, each time point). Flicker ERGs were recorded 1 day after the detachment and at various times after reattachment. Contrast-response functions were measured for isochromatic modulation and for selective modulation of short-wavelength-sensitive (S) and middle-wavelength-sensitive (M) cones. At the end of the experiment, retinas were prepared for light microscopy or immunocytochemical staining with antibodies to rod opsin, S and M cone opsins, cytochrome oxidase, synaptophysin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), interphotoreceptor-binding protein (IRBP), and peanut agglutinin lectin (PNA). Photoreceptor density maps were created from wholemount preparations labeled with biotinylated PNA and anti-S cone opsin. Cell counts of photoreceptor nuclei and cone outer segments (OS) were compared with flicker ERG data. Cell death was examined by the TUNEL method. RESULTS: Reattachment stopped photoreceptor cell death and reversed the disruption of interphotoreceptor matrix as well as the redistribution of Müller cell proteins. It also activated some astrocytes based on anti-GFAP staining. S- and M-cone OS showed a gradual recovery in length after reattachment, and this recovery continued to the longest time points examined. ERG contrast gains also recovered after reattachment, but these reached asymptotic levels by approximately a week after reattachment. There were significant correlations between outer nuclear layer (ONL) cell counts and ERG contrast gains. No differences were noted in the indices of recovery of M and S cones. CONCLUSIONS: The ERG can be used to follow specifically the changes in the retina that occur after retinal detachment and reattachment.

A survey of molecular expression by photoreceptors after experimental retinal detachment.

PURPOSE: To describe changes in the localization patterns and levels of rod and cone photoreceptor proteins after experimental retinal detachment (RD). METHODS: Cat retinas were detached for 1, 3, 7, or 28 days, at which time the eyecups were placed in fixative for immunocytochemical analysis or homogenized for biochemistry. Immunocytochemistry was performed using 19 probes for molecules known to be associated with photoreceptors. Protein concentrations were determined using enzyme-linked immunosorbent assay or Western blot analysis. Cone cell death was analyzed by double labeling with TdT-dUTP terminal nick-end labeling and cone-specific antibodies. RESULTS: Although some cones died, many survived long-term RD. Although their profiles may have changed, rod photoreceptors continued to express most of the molecules studied as long as they were alive. In contrast, the cones failed to label with almost all probes specific to them after 3 to 7 days of detachment. The exception was phosducin, which localized to both rods and cones and, in 28-day detachments, increased to 180% of the amount in normal retina. CONCLUSIONS: Rods and cones respond differently to RD. This difference may account for a faster return of rod vision and for the lingering changes in color vision and acuity that are often reported after successful reattachment surgeries.

Choline acetyltransferase is expressed by non-starburst amacrine cells in the ground squirrel retina.

We have used immunostaining techniques to reveal a new type of amacrine cell that is immunoreactive for choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, in the Ground Squirrel (Spermophilus beecheyi) retina. Cryostat sections and double immunostained wholemount preparations were examined by confocal microscopy. This new ChAT type III cell is distinct in morphology and neurotransmitter content from the well know 'starburst' amacrine cells (types I and II) that are so well represented in the ground squirrel retina [J. Comp. Neurol. 365 (1996) 173-216]. The type III cell colocalizes glycine with the acetylcholine and does not appear to be GABAergic or exhibit calcium-binding proteins like the well-known starburst type. As well, type III cells do not occur as a mirror-symmetric pair with normally placed and displaced varieties. The type III cell is probably a small field amacrine type branching broadly in upper sublamina b of the inner plexiform layer, and is most likely A6 of the Ground Squirrel retina [J. Comp. Neurol. 365 (1996) 173-216]. Type III cells are ideally placed in the architecture of the Ground Squirrel retina to influence ON directionally selective ganglion cell types.

The efficacy of delayed oxygen therapy in the treatment of experimental retinal detachment.

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 Müller 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 Müller 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.

Abnormal reactivity of muller cells after retinal detachment in mice deficient in GFAP and vimentin.

PURPOSE: To determine the roles of glial fibrillary acidic protein (GFAP) and vimentin in Müller cell reactivity. METHODS: Retinal detachments were created in mice deficient for GFAP and vimentin (GFAP(-/-)vim(-/-)) and age-matched wild-type (wt) mice. The reactivity of the retina was studied by immunofluorescence and electron microscopy. RESULTS: Müller cell morphology was different and glutamine synthetase immunoreactivity was reduced in the undisturbed GFAP(-/-)vim(-/-) retinas. After retinal detachment, Müller cells formed subretinal glial scars in the wt mice. In contrast, such scars were not observed in GFAP(-/-)vim(-/-) mice. Müller cells, which normally elongate and thicken in response to detachment, appeared compressed, thin, and "spikey" in the GFAP(-/-)vim(-/-) mice. The end foot region of Müller cells in the GFAP(-/-)vim(-/-) mice often sheared away from the rest of the retina during detachment, corroborating earlier results showing decreased resistance of this region in GFAP(-/-)vim(-/-) retinas to mechanical stress. In regions with end foot shearing, ganglion cells showed intense neurite sprouting, as revealed by anti-neurofilament labeling, a response rarely observed in wt mice. CONCLUSIONS: Müller cells are subtly different in the GFAP(-/-)vim(-/-) mouse retina before detachment. The end foot region of these cells may be structurally reinforced by the presence of the intermediate filament cytoskeleton, and our data suggest a critical role for these proteins in Müller cell reaction to retinal detachment and participation in subretinal gliosis.

Morphological characterization of the retinal degeneration in three strains of mice carrying the rd-3 mutation.

Retinal development in 3 strains of rd-3/rd-3 mutant mice, previously shown to have different rates of degeneration, was studied using light, electron, and immunofluorescence microscopy. The time course and phenotype of the degeneration as well as details on the mechanism of massive photoreceptor cell loss are compared with other known retinal degenerations in mice. Up until postnatal day (P) 10, the retinas of all three strains (RBF, 4Bnr, In-30) develop similarly to those of pigmented and nonpigmented controls. TUNEL-positive cells appear in the outer nuclear layer (ONL) by P14, and reach a maximum in all three mutant strains around P21. Scattered rods and cones form a loose, monolayered ONL by 8 weeks in the albino RBF strain, by 10 weeks in the albino 4Bnr strain, and by 16 weeks in the pigmented In-30 strain. Though the initial degeneration begins in the central retina, there is no preferred gradient of cell death between central and peripheral photoreceptors. Rods and cones are present at all ages examined. During development, stacks of outer segments (OS) form in all three strains though they never achieve full adult lengths, and often have disorganized, atypical OS. Rod opsin is expressed in the developing OS but is redistributed into plasma membrane as OS degeneration proceeds. Retinal pigment epithelial (RPE) cells of all mutant strains contain packets of phagocytosed OS, and their apical processes associate with the distal ends of the OS. At their synaptic sites, photoreceptor terminals contain ribbons apposed to apparently normal postsynaptic triads. As photoreceptors are lost, Müller cells fill in space in the ONL but they do not appear to undergo significant hypertrophy or migration, though during the degeneration, glial fibrillary acidic protein (GFAP) expression is gradually upregulated. Macrophage-like cells are found frequently in the subretinal space after the onset of photoreceptor apoptosis. As OS disappear, the RPE apical processes revert to simple microvilli. Late in the degeneration, some RPE cells die and neighboring cells appear to flatten as if to maintain confluence. In regions of RPE cell loss that happen to lie above retina where the ONL is gone, cells of the inner nuclear layer (INL), wrapped by Müller cell processes, may front directly on Bruch's membrane.

Experimental retinal detachment in the cone-dominant ground squirrel retina: morphology and basic immunocytochemistry.

The cellular responses of the cone-dominant ground squirrel retina to retinal detachment were examined and compared to those in rod-dominant species. Retinal detachments were made in California ground squirrels. The retinas were prepared for light, electron, and confocal microscopy. Tissue sections were labeled with antibodies to cone opsins, rod opsin, glial fibrillary acidic protein (GFAP), vimentin, synaptophysin, cytochrome oxidase, and calbindin D 28K. Wax sections were probed with the MIB-1 antibody to detect proliferating cells. By 10 h postdetachment many photoreceptor cells in the ground squirrel already show structural signs of apoptosis. At 1 day many photoreceptors have collapsed inner segments (IS), yet others still have short stacks of outer segment discs. At 3 days there is a marked increase in the number of dying photoreceptors. Rod and medium-/long-wavelength opsins are redistributed in the cell membrane to their synaptic terminals. At 7 days photoreceptor cell death has slowed. Some regions of the outer nuclear layer (ONL) have few photoreceptor somata. IS remnants are rare on surviving photoreceptors. At 28 days these trends are even more dramatic. Retinal pigmented epithelium (RPE) cells do not expand into the subretinal space. The outer limiting membrane (OLM) appears flat and uninterrupted. Müller cells remain remarkably unreactive; they show essentially no proliferation, only negligible hypertrophy, and there is no increase in their expression of GFAP or vimentin. Horizontal cells show no dendritic sprouting in response to detachment. The speed and extent of photoreceptor degeneration in response to detachment is greater in ground squirrel than in cat retina-only a small number of rods and cones survive at 28 days of detachment. Moreover, the almost total lack of Müller cell and RPE reactivity in the ground squirrel retina is a significant difference from results in other species.