Epiretinal membrane in a subject after transvitreal delivery of palucorcel (CNTO 2476).

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.

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.

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.

Proteomic Analysis of the Vitreous following Experimental Retinal Detachment in Rabbits.

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, and α-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.

Remodelling of retinal on- and off-bipolar cells following experimental retinal detachment.

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.

Quantifying spatial relationships from whole retinal images.

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. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optomotor and immunohistochemical changes in the juvenile S334ter rat.

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.

Protein changes in the retina following experimental retinal detachment in rabbits.

PURPOSE: Retinal detachment leads to the widespread cellular remodeling of the retina. The purpose of this study was to identify protein changes that accompany these cellular alterations by comparing the proteomic profiles of sham and experimentally detached rabbit retina. Elucidation of the proteins most dramatically affected by retinal detachment would add further understanding to the pathophysiology of this condition, and potentially identify therapeutic targets useful in preventing the deleterious effects of detachment, including photoreceptor cell death and the activation of non-neuronal microglial and Müller cells. METHODS: Retinal detachments were induced in the right eyes of six New Zealand Red pigmented rabbits. Sham surgery was performed in the right eyes of six other rabbits that were used as controls. At seven days, the eyes were enucleated and the retinal tissue was harvested. The individual retinal samples were subjected to high resolution two-dimensional polyacrylamide gel electrophoresis. Differentially expressed protein spots were processed for identification by liquid chromatography-tandem mass spectrometry. Further investigation was undertaken with western blotting, and immunocytochemical studies on a further set of four sham and four detached retinas. RESULTS: Eighteen protein spots were found to be at least twofold differentially expressed between the sham and detached retinas. These protein spots were identified as: vimentin; tubulin ß-2C; fragments of α-enolase; fructose-bisphosphate aldolase A; ATP synthase subunit ß; mitochondrial creatine kinase; N-terminal fragments of albumin; prohibitin; and transducin-ß(1). CONCLUSIONS: The differentially expressed proteins determined in this study may play an important role in the cellular responses of the retina after its detachment, subsequent ability to recover following surgical reattachment, as well as in serious complications such as subretinal fibrosis and proliferative vitreoretinopathy.

Cell proliferation in human epiretinal membranes: characterization of cell types and correlation with disease condition and duration.

PURPOSE: To quantify the extent of cellular proliferation and immunohistochemically characterize the proliferating cell types in epiretinal membranes (ERMS) from four different conditions: proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy, post-retinal detachment, and idiopathic ERM. METHODS: Forty-six ERMs were removed from patients undergoing vitrectomy and immediately fixed in paraformaldehyde. The membranes were processed whole and immunolabeled with either anti-MIB-1 or anti-SP6 to detect the K(i)-67 protein in proliferating cells, in combination with anti-glial fibrillary acidic protein or anti-vimentin to identify glia, anti-ezrin to identify retinal pigment epithelial cells, Ricinus communis to identify immune cells, and Hoechst to label nuclei. Digital images were collected using a laser scanning confocal microscope. The cell types were identified, their combined proliferative indices were tabulated as the average number of anti-K(i)-67-positive cells/mm(2) of tissue, and the number of dividing cells was related to the specific ocular condition and estimated disease duration. RESULTS: ERMs of all four types were shown to be highly cellular and contained proliferating cells identified as glia, retinal pigment epithelium, and of immune origin. In general, membranes identified as PVR had many more K(i)-67-positive cells in comparison to those in the other three categories, with the average number of K(i)-67-positive cells identified per mm(2) of tissue being 20.9 for proliferative diabetic retinopathy, 138.3 for PVR, 12.2 for post-retinal detachment, and 19.3 for idiopathic ERM. While all membrane types had dividing cells, their number was a relatively small fraction of the total number of cells present. CONCLUSIONS: The four ERM types studied demonstrated different cell types actively dividing at the time of removal, confirming that proliferation is a common event and does continue over many months. The low number of dividing cells at the time of removal in comparison to the total number of cells present, however, is an indicator that proliferation alone may not be responsible for the problems observed with the ERMs. Treatment strategies may need to take into consideration the timing of drug administration, as well as the contractile and possibly the inflammatory characteristics of the membranes to prevent the ensuing effects on the retina.

Evidence that neurites in human epiretinal membranes express melanopsin, calretinin, rod opsin and neurofilament protein.

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.

Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin.

PURPOSE: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. METHODS: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days' duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti-glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT-PCR) techniques. RESULTS: Immunocytochemistry showed increased Müller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Müller cells. Nestin was consistently observed in the foremost processes of those Müller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Müller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Müller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. CONCLUSIONS: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Müller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Müller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries.

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.

Assessment of the integrin alpha5beta1 antagonist JSM6427 in proliferative vitreoretinopathy using in vitro assays and a rabbit model of retinal detachment.

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 (Müller 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.

Alkylphosphocholines: a new approach to inhibit cell proliferation in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy represents the major complication in retinal detachment surgery and occurs in about 5-15% of cases resulting in a significant loss of vision despite multiple surgical procedures. Although successful anatomical reattachment is usually achieved, the reduction in central vision often remains permanent due to the intraretinal changes induced by retinal detachment and the subsequent proliferative response within the retina. Retinal Muller glial cells play a pivotal role in this process together with retinal pigment epithelial cells which are dispersed in the vitreous and stimulated by growth factors and serum in the vitreous after the breakdown of the blood-retinal barrier.

The effects of transient retinal detachment on cavity size and glial and neural remodeling in a mouse model of X-linked retinoschisis.

PURPOSE: To determine the cellular consequences of retinal detachment in retinoschisin knockout (Rs1-KO) mice, a model for retinoschisin in humans. METHODS: Experimental retinal detachments (RDs) were induced in the right eyes of both Rs1-KO and wild-type (wt) control mice. Immunocytochemistry was performed on retinal tissue at 1, 7, or 28 days after RD with antibodies to anti-GFAP, -neurofilament, and -rod opsin to examine cellular changes after detachment. Images of the immunostained tissue were captured by laser scanning confocal microscopy. Quantitative analysis was performed to measure the number of Hoechst-stained photoreceptor nuclei and their density, number, and size of inner retinal cavities, as well as the number of subretinal glial scars. RESULTS: Since detachments were created with balanced salt solution, by examination, all retinas had spontaneously reattached by 1 day. Cellular responses common to many photoreceptor degenerations occurred in the nondetached retinas of Rs1-KO mice, and, of importance, RD did not appear to significantly accentuate these responses. The number of schisis cavities was not changed after detachment, but their size was reduced. CONCLUSIONS: These data indicate that large short-term RD in Rs1-KO mice, followed by a period of reattachment may cause a slight increase in photoreceptor cell death, but detachments do not accentuate the gliosis and neurite sprouting already present and may in fact reduce the size of existing retinal cavities. This finding suggests that performing subretinal injections to deliver therapeutic agents may be a viable option in the treatment of patients with retinoschisis without causing significant cellular damage to the retina.

Muller cell reactivity and photoreceptor cell death are reduced after experimental retinal detachment using an inhibitor of the Akt/mTOR pathway.

PURPOSE: To test the effect of Palomid 529, an inhibitor of the Akt/mTOR pathway, on Müller cell proliferation, subretinal glial scar formation, and photoreceptor survival after experimental retinal detachment (RD). METHODS: Palomid 529 (600 microg) in balanced salt solution or balanced salt solution alone was injected intravitreally immediately after RD into the right eyes of 12 rabbits. Ten micrograms of BrdU was injected intravitreally on day 3. Animals were killed on day 3 or 7, at which time retinal sections were labeled with anti-BrdU to detect dividing cells, with anti-vimentin to identify Müller cells, and with the isolectin B4 to identify microglia and macrophages. Outer nuclear layer (ONL) thickness was measured from fluorescence-labeled nuclear-stained sections. Labeling was imaged using confocal microscopy. Six additional animals received either drug or balanced salt solution injections into normal eyes, and paraffin sections were stained with hematoxylin and eosin. RESULTS: In the drug-treated eyes there was a significant decrease in the number of anti-BrdU-labeled Müller cells, the number and size of subretinal scars, and the number of isolectin B4-labeled cells. The ONL was also significantly thicker, and there was no evidence of toxic effects. CONCLUSIONS: Palomid 529 is an effective suppressor of Müller cell proliferation, glial scar formation, and photoreceptor cell death in a rabbit model of RD. This suggests that inhibiting the Akt/mTOR signal transduction pathway may be an effective strategy to decrease proliferation and photoreceptor cell death induced by detachment and perhaps represents a novel therapy for related human diseases such as proliferative vitreoretinopathy.

Clinicopathological case series of four patients with inherited macular disease.

PURPOSE: To correlate the phenotype of four patients with inherited macular disease with the immunohistopathology of retinal tissue collected at the time of retinal pigment epithelium (RPE)-choroidal transplantation. METHODS: A clinicopathologic case series describing the phenotype of four patients, including confocal immunohistochemistry and electron microscopy (EM), and the results of genetic testing. RESULTS: In Case 1, electrophysiology showed only macular dysfunction. Confocal microscopy revealed minor abnormalities. EM showed abnormal cone inner segments with swollen mitochondria. In case 2 (R172W mutation in RDS), electrophysiology demonstrated generalized cone system dysfunction with severe macular involvement. Peripherin labeling of outer segments was nonuniform, and EM showed discs arranged in whorllike structures. Case 3 showed severe central macular dysfunction on multifocal electroretinogram (ERG). Peripherin staining was irregular and disorganized. EM revealed abnormal inner segment morphology, particularly in rods, and disorganized irregular outer segments. Case 4 had localized central macular dysfunction on multifocal ERG. Confocal microscopy was grossly normal, with evidence of early redistribution of cone opsin to the inner segment. EM showed variable rod morphology and normal cones. CONCLUSIONS: RPE transplantation provides a unique opportunity to gain insight into retinal disorders by enabling phenotypic correlation with the immunohistopathology of retinal tissue collected during surgery.

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.

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.