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Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis.

Regenerative medicine possesses the potential to ameliorate damage to tissue that results from a vast range of conditions, including traumatic injury, tumor resection and inherited tissue defects. Adult stem cells, while more limited in their potential than pluripotent stem cells, are still capable of differentiating into numerous lineages and provide feasible allogeneic and autologous treatment options for many conditions. Adipose stem cells are one of the most abundant types of stem cell in the adult human. Here, we review recent advances in the development of synthetic scaffolding systems used in concert with adipose stem cells and assess their potential use for clinical applications.

Tau inclusions are a shared feature of many neurodegenerative conditions and tau mutations lead to frontotemporal dementia. Approaches to treatment of these conditions have focused directly on the tau protein by targeting its post-translational modifications, its levels and its tendency to aggregate. We discovered a novel regulatory pathway for tau degradation that operates through the Rhes protein, a GTPase. Rhes is farnesylated and treatment with the farnesyl transferase inhibitor, lonafarnib, reduced Rhes, attenuated behavioral abnormalities, significantly reduced atrophy, tau inclusions, sumoylation and ubiquitination, as well as microgliosis in the rTg4510 tauopathy mouse. Direct reduction of Rhes levels reproduced the results observed with lonafarnib. The mechanism of lonafarnib action, as mediated by Rhes to reduce tau pathology, operates through the lysosome without involvement of the proteasome. Finally we show that the developmental increase in Rhes levels can be homeostatically regulated in the presence of tau mutations as a protective mechanism through which cells sense abnormal tau before any pathology is present. The extensive human trials of lonafarnib for other conditions, makes this drug ideal for repurposing to treat tauopathies.

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.

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

Classification and detection of biological structures in Electron Micrographs (EM) is a relatively new large scale image analysis problem. The primary challenges are in modeling diverse visual characteristics and development of scalable techniques. In this paper we propose novel methods for synapse detection and localization, an important problem in connectomics. We first propose an attribute based descriptor for characterizing synaptic junctions. These descriptors are task specific, low dimensional and can be scaled across large image sizes. Subsequently, techniques for fast localization of these junctions are proposed. Experimental results on images acquired from a mammalian retinal tissue compare favorably with state of the art descriptors used for object detection.

Segmentation based tracing algorithms detect the extent and borders of an object in a given frame IZ by propagating results from frames I1 ≤ zZ. Although application specific tracers have been forthcoming, techniques that automatically adapt across applications have been less explored. We approach this problem by learning a prior model on topological dynamics that encourages segmentation transitions across frames that are most likely for a given application. Further, we augment a generic tracing technique with a locality sensitive prior derived from dense optic flow fields for deformation guidance. The proposed approach comprises two stages where the generic tracer initially yields multiple segmentation transitions when its parameters are perturbed, and the learnt topology prior subsequently propagates high scoring segmentations. Because the learnt topology model wraps around a generic tracer and adapts it by setting its free parameters, the need for careful parameter tuning is completely obviated. Through extensive experimental validation in surveillance, biological and medical image datasets, we verify the applicability of the proposed model while demonstrating good tracing performance under severe clutter.

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.

Ground squirrels are an increasingly important model for studying visual processing, retinal circuitry, and cone photoreceptor function. Here, we demonstrate that the photoreceptor mosaic can be longitudinally imaged noninvasively in the 13-lined ground squirrel (Ictidomys tridecemlineatus) using confocal and nonconfocal split-detection adaptive optics scanning ophthalmoscopy using 790 nm light. Photoreceptor density, spacing, and Voronoi analysis are consistent with that of the human cone mosaic. The high imaging success rate and consistent image quality in this study reinforce the ground squirrel as a practical model to aid drug discovery and testing through longitudinal imaging on the cellular scale.

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.

Author(s): Sajdak, Benjamin S; Bell, Brent A; Lewis, Tylor R; Luna, Gabriel; Cornwell, Grayson S; Fisher, Steven K; Merriman, Dana K; Carroll, Joseph | Abstract: Purpose:We examined outer retinal remodeling of the euthermic and torpid cone-dominant 13-lined ground squirrel (13-LGS) retina using optical coherence tomography (OCT) imaging and histology. Methods:Retinas and corneas of living 13-LGSs were imaged during euthermic and torpid physiological states using OCT. Retinal layer thickness was measured at the visual streak from registered and averaged vertical B-scans. Following OCT, some retinas were collected immediately for postmortem histologic comparison using light microscopy, immunofluorescence, or transmission electron microscopy. Results:Compared to OCT images from euthermic retinae, OCT images of torpid retinae revealed significantly thicker inner and outer nuclear layers, as well as increases in the distances between outer retinal reflectivity bands 1 and 2, and bands 3 and 4. A significant decrease in the distance between bands 2 and 3 also was seen, alongside significant thinning of the choriocapillaris and choroid. OCT image quality was reduced in torpid eyes, partly due to significant thickening of the corneal stroma during this state. Conclusions:The torpid retina of the hibernating 13-LGS undergoes structural changes that can be detected by OCT imaging. Comparisons between in vivo OCT and ex vivo histomorphometry may offer insight to the origin of hyperreflective OCT bands within the outer retina of the cone-dominant 13-LGS.

Aims: To carry out a histopathological analysis of retinal specimens of patients undergoing translocation surgery for age-related macular degeneration (ARMD). Methods: A histopathological analysis, using confocal microscopy, was performed on six retinal specimens. Results were compared with those from two further retinal specimens, collected during RPE transplantation, to control for the effects of vitrectomy and ARMD. In addition, a third control specimen from a cadaver with no history of ophthalmic disease was also analysed. Results: In the translocation specimens, rods and cones were relatively well preserved but showed reduced density and outer segment length. In four specimens, there were focal areas of rod opsin redistribution to the inner segment, but this was not observed in the controls. Staining with calbindin was decreased in cones compared with controls but normal in horizontal and amacrine cells. Rod bipolar cells were mildly disorganised, and in one there was evidence of neurite sprouting. Glial fibrillar acidic protein was raised in both translocation and transplantation retinae but not in the cadaver control. Conclusions: In this study, there was little evidence of cellular injury following iatrogenic detachment; however, the rate of PVR following translocation surgery infers that cellular events set in motion may continue despite early reattachment.

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.

Motivation: In addition to being involved in retinal vascular growth, astrocytes play an important role in diseases and injuries, such as glaucomatous neuro-degeneration and retinal detachment. Studying astrocytes, their morphological cell characteristics and their spatial relationships to the surrounding vasculature in the retina may elucidate their role in these conditions. Results: Our results show that in normal healthy retinas, the distribution of observed astrocyte cells does not follow a uniform distribution. The cells are significantly more densely packed around the blood vessels than a uniform distribution would predict. We also show that compared with the distribution of all cells, large cells are more dense in the vicinity of veins and toward the optic nerve head whereas smaller cells are often more dense in the vicinity of arteries. We hypothesize that since veinal astrocytes are known to transport toxic metabolic waste away from neurons they may be more critical than arterial astrocytes and therefore require larger cell bodies to process waste more efficiently. Availability and implementation: A 1/8th size down-sampled version of the seven retinal image mosaics described in this article can be found on BISQUE (Kvilekval et al., 2010) at http://bisque.ece.ucsb.edu/client_service/view?resource=http://bisque.ece.ucsb.edu/data_service/dataset/6566968. Contact: arunaj@ece.ucsb.edu or manj@ece.ucsb.edu Supplementary information: Supplementary data are available at Bioinformatics online.

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.

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.

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

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.

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration. J. Comp. Neurol. 387:148–156, 1997. © 1997 Wiley-Liss, Inc.

The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect inLim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis ofLim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.

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.

Cytochalasin D (CD) interferes with the morphogenesis of outer segment disc membrane in photoreceptors. Disruption of either the actin network in the ciliary stalk, where membrane evagination is initiated, or the actin core of the calycal processes, whose position could define the disc perimeter, could be responsible. We have attempted to determine which of these local F-actin populations is involved in membrane morphogenesis and what step in the process is actin-dependent. Biocytin accumulation in nascent discs, detected by fluorescent avidin and laser scanning confocal microscopy (LSCM), provided a means of labeling abnormal discs and a measure of disc membrane addition. F-actin content and distribution were assessed using fluorescent phalloidin and LSCM. First, we examined the effects of a range of CD dosages (0.1, 1.0, or 10.0 pM) on rod photoreceptors inXenopus laevis eyecup cultures. Ectopic outgrowth of discs, evaluated by LSCM and transmission electron microscopy (TEM), occurred at each concentration. Phalloidin labeling intensified in the ciliary stalk with increasing CD concentration, indicating F-actin aggregation. In contrast, it diminished in the calycal processes, indicating dispersal; TEM showed that calycal process collapse ensued. Disruption was evident at a lower concentration in the ciliary stalk (0.1 pM) than in the calycal processes (1.0 pM). TEM confirmed that the calycal processes remained intact at 0.1 pM. Thus, CD’s action on the ciliary stalk network is sufficient to disrupt disc morphogenesis. Second, we examined the effect of CD on temperature-induced acceleration of the rate of disc formation. In the absence of CD, a 10°C temperature shift increased the disc formation rate nearly three-fold. CD (5 pM) caused a 94% inhibition ( P < 0.025) of this response; yet, the rate of membrane addition to ectopically growing discs exhibited the expected three-fold increase. Thus, CD’s action interferes with the generation of new discs. 0 1996 Wiley-Liss, Inc. Indexing terms: cytoskeleton, cytochalasin D, morphogenesis, morphology, confocal

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.

Although the optic nerve fibers of the cone-dominant ground squirrel retina have been well studied physiologically, the morphological details of the retinal neurons have not. To that end, retinal neurons of the California ground squirrel have been studied in Golgi-impregnated wholemounts. Two types of horizontal cell have been identified: H1 has an axon and axon terminal, whereas H2 is axonless. The dendritic field of H1 cells enlarges in a nonuniform manner with increasing displacement from the central retina. The smallest examples lie centrally in the visual streak, and the largest occur in the superior periphery. Eight types of bipolar cell are distinguished by morphological differences in dendritic branching pattern and field size in the outer plexiform layer, cell body size, and layering within the inner nuclear layer and by the morphology and stratification of axon terminals in the inner plexiform layer. A large bistratified bipolar cell (B8) is introduced here; the other 7 types closely resemble those in the retinas of other sciurid species described by R.W. West (1976, J. Comp. Neurol. 168:355–378; 1978, Vision Res. 18:129–136). The B1 type is proposed as a blue cone bipolar cell. Amacrine cells are classified into 27 cell types. Six of these occur as mirror-image pairs across the inner plexiform layer, the soma of one of each pair being “displaced” to the ganglion cell layer. The best described of these pairs is the very elaborate starburst amacrine cell, A5, which stains regularly in these wholemounted retinas. Changes in dendritic field size of both A5 subtypes with retinal location are quantified. The morphology of three amacrine cell types identified in Spermophilus beecheyi suggests that their possible counterparts in S. mexicanus (West, 1976) were, as displaced amacrine cells, misidentified as ganglion cells. Amacrine cell types that may play roles in the rod pathway, the blue cone pathway, and ganglion cell directional selectivity are discussed. No type of interplexiform cell was observed. Ganglion cells are classified into 19 cell types, 9 of which probably correspond to the ganglion cells described by West (1976) in the Mexican ground squirrel. The bistratified G11 cell is proposed as an ON-OFF directionally selective type. © 1996 Wiley-Liss, Inc.

One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg–Gly–Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration.

Markov Random Fields (MRF) have proven to be extremely useful models for efficient and accurate image segmentation.Recent literature points to an increased effort towards incorporating useful priors (shape, geometry, context) in a MRF framework. However, topological priors, considered extremely crucial in biological and natural image sequences have been less explored. This work proposes a strategy wherein free parameters of the MRF are used to make it topology aware using a semantic graphical model working in conjunction with the MRF. Estimation of free parameters is constrained by prior knowledge of an object's topological dynamics encoded by the graphical model. Maximizing a regional conformance measure yields parameters for the frame under consideration. The application motivating this work is the tracing of neuronal structures across 3D serial section Transmission Electron Micrograph (ssTEM) stacks. Applicability of the proposed method is demonstrated by tracing 3D structures in ssTEM stacks.

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.

Golgi-impregnated horizontal cells (HCs) as viewed in whole mount human retinas have been studied by light microscopic (LM) techniques. Impregnated HCs have been drawn by camera lucida and by the Eutectics neuron tracing method to provide quantitative data on dendritic tree sizes, dendritic tree shapes, and dendritic terminals for statistical treatment and cluster analysis. In addition, fractal analyses of HC dendritic branching patterns have been performed. Three significantly different HCs can be classified on both subjective and objective morphological criteria in central and peripheral human retina. In the fovea all HCs are so small that it is difficult to achieve a clear separation of the subtypes, although they can be distinguished by the experienced observer. HI types are the classic HCs of Polyak (The Retina, Chicago: University of Chicago Press, 1941) with distinct dendritic terminal clusters going to cones and a fan-shaped axon terminal consisting of large numbers of rod-destined terminals. HII cells have profusely branched, overlapping dendrites, with poorly defined terminals going to cones and a short curled axon bearing small terminals also going to cones. The HIII types exhibit larger diameter, more asymmetrically shaped dendritic trees and 30% more dendritic terminal clusters than HI cells at any location on the retina. Many HIII cells appear to emit a process from the cell body in the inner nuclear layer (INL) that descends into the outer strata of the inner plexiform layer (IPL). The axon of the HIII cell may end in a loosely organized, sprawling arborization. Fractal dimensions of the horizontal cells also show significant differences between the three groups. HII cells exhibit the highest fractal dimension followed by HI and HIII cells with lower and lowest fractal dimensions, respectively. The fractal dimension of HII cells of rhesus monkey, as determined from drawings by other authors in other publications, are the same as HII cells of human retina.

Analyzing high-resolution images of astrocytes is important in understanding the diseases, such as glaucoma and retinal detachment, to which astrocytes are known to become reactive. This is challenging because the cells are small, homogeneous, and closely packed. We propose an interactive visualization system designed for such images. Our system employs a probabilistic segmentation algorithm to help distinguish between cells. Design decisions on visualization and interactions were made based on the needs of the scientists, resulting in a visualization that shows the details of individual cells in the context of a large image mosaic. Our interactive system brings patterns of information to the surface, conveys uncertainty, and serves as a tool for astrocyte research.

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.

Injury to the mammalian retina produces a number of cellular wound-healing types of responses, with similarities and differences to injury responses in the brain and spinal cord. Retinal detachment is one such serious, sight-threatening injury that produces responses from almost all cell types including cellular degeneration and death, neuronal remodeling, some poorly understood immune responses, and glial cell reactivity. Glial cell reactivity is particularly important in glial scar formation which may, in turn, have future downstream effects on visual recovery. The glial cell response is prominent in all injuries to the central nervous system and, yet, its exact purpose and regulatory mechanisms are poorly understood.

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.