Biochemical interruption of membrane phospholipid renewal in retinal photoreceptor cells

The rabbit retina's synthesis of new phosphatidylcholine from extracellular choline was interrupted by an intravitreal injection of the choline analogue hemicholinium-3. This disrupted the process by which new membrane is added to the rod photoreceptor outer segments and eventually caused outer segment degeneration. During the first 2 days after hemicholinium-3 was injected, rows of vesicles replaced the newly formed membrane discs at the outer segment's base. The region of vesicles then expanded, and the distal outer segment detached and was quickly phagocytosed by the pigment epithelium. Two weeks after hemicholinium-3 injection, all of the retina's outer segments had been lost and the inner segments were reduced in length. The threshold concentration of hemicholinium-3 was approximately 20 microM intraocularly. At this dose, the cell bodies, intracellular organelles, and synapses of the rod cells survived. A small group of amacrine cells, possibly those that synthesize acetylcholine, became pyknotic; but the other retinal neurons remained normal to both light and electron microscopy even upon exposure to intraocular concentrations as high as 1 mM. Biochemical experiments indicated that at 20 microM hemicholinium-3, the perturbation of choline metabolism is partial and transient. That it has major selective consequences for the outer segments probably reflects the large amount of new phospholipid required for renewal of their membranes. The selectivity of the lesion was also evidenced by electrophysiological activity recorded from hemicholinium-3-treated retinas. Hemicholinium-3 was injected in vivo, and at various times retinas were isolated and incubated in vitro. The normal components of the electroretinogram were observed, but its amplitude rapidly declined; 12 days after injection, no response to light could be detected. Spontaneous firing of single ganglion cells was observed at all times following hemicholinium-3 injection. As the outer segments degenerated and the threshold of the electroretinogram rose, the thresholds of the ganglion cells also rose, but normal ganglion cell receptive fields could sometimes be plotted. Fourteen days after injection, when all of the outer segments were gone, ganglion cell responses to bright light could still be recorded; their thresholds were about 3.5 log units above normal. This finding is consistent with a previous report of light-evoked responses in mice after outer segment degeneration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydroxyindole-O-methyltransferase in rat retinal bipolar cells: persistence following photoreceptor destruction

The presence of hydroxyindole-O-methyltransferase (HIOMT) activity and localization of HIOMT immunoreactivity was examined in albino rat retinas following photoreceptor destruction. Male Sprague-Dawley rats were exposed to high intensity fluorescent light for 4 consecutive days, then placed on a 14:10 h light:dark cycle for two weeks to allow for phagocytic removal of damaged cells from the retina. Histologic examination revealed almost complete destruction and removal of all photoreceptors. The damaged retinas exhibited an increase in HIOMT activity relative to controls, when expressed as activity per mg of protein. HIOMT activity in the pineal glands was not affected. When control and light damaged retinas were examined for HIOMT localization by immunocytochemistry, the control retinas displayed intense HIOMT immunoreactivity in all photoreceptors, and a somewhat lighter labeling in a population of bipolar cells, whereas the light damaged retinas (lacking photoreceptors) showed intense HIOMT immunoreactivity in bipolar cells. These results suggest that the increase in HIOMT activity following photoreceptor destruction is due to increased synthesis of this enzyme in a population of bipolar cells. These HIOMT-immunoreactive bipolar cells may perhaps respond in a compensatory manner to changing levels of melatonin in the retina.     

小鼠视网膜前体细胞的视网膜下腔移植

The development of cell replacement techniques is promising as a potential treatment for photoreceptor loss. However, the limited integration ability of donor and recipient cells presents a challenge following transplantation. In the present study, retinal progenitor cells (RPCs) were harvested from the neural retinas of enhanced green fluorescent protein mice on postnatal day 1, and expanded in a neurobasal medium supplemented with fetal bovine serum without endothelial growth factor. Using a confocal microscope, immunohistochemistry demonstrated that expanded RPCs in vitro maintain retinal stem cell properties and can be differentiated into photoreceptor cells. Three weeks after transplantation, subretinal transplanted RPCs were found to have migrated and integrated into the outer nuclear layer of recipient retinas with laser injury, some of the integrated cells had differentiated into photoreceptors, and a subpopulation of these cells expressed photoreceptor specific synaptic protein, appearing to form synaptic connections with bipolar cells. These results suggest that subretinal transplantation of RPCs may provide a feasible therapeutic strategy for the loss of retinal photoreceptor cells.     

Caspase-3 mRNA expression in rats with apoptosis of retinal photoreceptor cells

BACKGROUND: Apoptosis of retinal photoreceptor cells is a commonly pathological characteristic of various eye diseases, while caspase-3 is an important regulating gene and plays a key role in apoptosis. OBJECTIVE: To measure the expression of caspase-3 mRNA in rats with apoptosis of retinal photoreceptor cells by using real-time fluorescent quantitative polymerase chain reaction and compare with those of the normal rats. DESIGN: Randomized controlled animal study. SETTING: Zhongshan Ophthalmological Center of Sun Yat-sen University. MATERIALS: A total of 36 female SD rats of 50 days old and clean grade and weighing (150±10) g were provided by Experimental Animal Center of Northern Area of Sun Yat-sen University. All rats were randomly divided into normal control group (n =6) and N-methyl-N-nitrosourea (MNU) group (n =30), and they were observed at 12 hours, 1, 2, 3 and 5 days after model establishment, with 6 rats at each time point. METHODS: The experiment was carried out at Zhongshan Ophthalmological Center, Key Laboratory of Ophthalmology by State Ministry of Education from March to December 2004. Rats in the normal control group were intraperitoneally injected with saline and rats in the MNU group were intraperitoneally injected with 40 mg/kg MNU. And then, retinal photoreceptor injured models were established. At 12 hours, 1, 2, 3 and 5 days after model establishment, the rats were sacrificed for enucleating right eyeballs, isolating retina immediately and extracting total RNA. The expression of caspase-3 mRNA in retina was measured with real-time fluorescent quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: Expression of caspase-3 mRNA in retina of rats in the two groups. RESULTS: A total of 36 SD rats were involved in the final analysis. The expressions of caspase-3 mRNA in the rat retina of both groups at the five time points (12 hours, 1, 2, 3 and 5 days) after model establishment were 1.52×105, 18.35×105, 25.14×105, 29.25×105, 13.72×105 and 12.24×105, respectively. The expression of caspase-3 mRNA in the MNU group increased after 12 hours of intraperitoneal injection, and rose to the top on the 2nd day, which was 19 times as many as that of the normal control group. Then, it decreased gradually and was still 8 times as many as that of the normal control group on the 5th day. CONCLUSION: The expression of caspase-3 mRNA is related to apoptosis of retinal photoreceptor cells, while caspase-3 plays an important role in occurrence and development of apoptosis of retinal photoreceptor cells.     

Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.     

bFGF-mediated redox activation of the PI3K/Akt pathway in retinal photoreceptor cells

In many retinal diseases, it is the death of photoreceptors that leads to blindness. In previous in vitro and in vivo studies, basic fibroblast growth factor (bFGF) has been shown to increase retinal cell survival. More recently, reactive oxygen species (ROS) have also been shown to promote cell survival, contrary to the traditional view that they are solely destructive molecules. Due to this possible link, we hypothesised that bFGF could stimulate the production of ROS, which in turn stimulates the protein kinase B (Akt) survival pathway. Flow cytometry was used to measure the fluorescence of oxidised dihydrorhodamine, a ROS indicator, in the murine 661W photoreceptor cell line under several different conditions. Expression of cyclooxygenase (Cox) enzymes was evaluated by immunohistochemistry, and the response of photoreceptor cells to exogenous bFGF in the explanted mouse retina was studied by confocal microscopy. Exogenous addition of bFGF to 661W cells resulted in an increase in ROS production that lasted for 24 h. When this ROS production was inhibited, bFGF-induced phosphorylation of Akt was prevented. Through the use of inhibitors and small interfering RNA in the cell line, the source of this production was shown to be Cox and to involve the activation of phospholipases A(2) + C. This pathway may also occur in the mouse retina, as we showed that the retina expressed Cox1&2, and that photoreceptors in explanted retina respond to bFGF by increasing their ROS levels. These results demonstrate that exogenous bFGF can stimulate ROS production through the activation of Cox, and activate the Akt pathway.     

神经干细胞移植对视网膜节细胞再生的影响

目的探讨神经干细胞（NSCs）在视神经损伤后对视网膜节细胞轴突再生的作用及其在视神经内迁移和分化。方法实验动物分对照组（PBS组），实验组（NSCs组）；成年SD大鼠在眼球后1min处切断视神经。移植NSCs或PBS至视神经断端；4周后以霍乱毒素B亚基顺行标记观察轴突再生情况，并观察NSCs在视神经内的迁移及免疫组织化学法检测移植后的细胞表达神经丝蛋白（NF）、2，3-环核苷酸磷酸二酯酶（CNP）、胶质纤维酸性蛋白（GFAP）的情况。结果4周后视网膜节细胞再生轴突穿过视神经断端到达远端，移植的NSCs分化为星形胶质细胞并在视神经内迁移0．5～1min。免疫组织化学法检测NSCs部分呈GFAP阳性，未见NF、CNP表达。结论NSCs移植可促进视网膜节细胞轴突再生，能在视神经内迁移并在视神经周围分化为星形胶质细胞。     

Absence of phosphoglucose isomerase-1 in retinal photoreceptor, pigment epithelium and Muller cells

Macroarray analysis was used to compare equal amounts of cDNA from wild-type and rd/rd (retinal degeneration) mice, collected at P90 when photoreceptor degeneration is virtually complete. A stronger signal for the glycolytic enzyme phosphoglucose isomerase (Gpi1) was observed in the rd/rd sample. Extracellularly, Gpi1 may act as a cytokine, independently described as neuroleukin and autocrine motility factor. Retinal Gpi1 expression was investigated by Northern and Western blot analysis and immunohistochemistry. Double-labelling was performed with antibodies against Gpi1 and calbindin-D, glutamine synthetase, RPE65, calretinin and ultraviolet opsin in order to provide positive cell type identification. Northern and Western blots showed double expression levels per microgram of RNA and protein, respectively, in the rd/rd retina compared with wild-type. However, the total amount of Gpi1 protein per retina was indistinguishable. Gpi1 immunoreactivity was found in ganglion, amacrine, horizontal and bipolar cells, but not in rods, cones, pigment epithelium and Muller cells. This distribution explains why the absolute amounts of Gpi1 protein were not appreciably different between wild-type and the rd/rd phenotype, where rods and cones are absent, whilst the relative contribution of Gpi1 to the total protein and RNA pools differed. Some extracellular immunoreactivity was observed in the photoreceptor matrix around cones in freshly fixed tissue only, which could possibly reflect a role as a cytokine. We propose that glycolysis in Gpi1-negative cells proceeds entirely through the pentose phosphate pathway, creating NADPH at the cost of organic carbon. We hypothesize that the unique metabolic needs of photoreceptors justify this trade-off.     

Localization of specific autoantibodies in the retinal photoreceptor cell layer in experimental retinal autoimmunity

The presence and localization of autoantibodies was determined in strain 13 guinea pigs with experimental retinal autoimmunity (ERA) induced by immunization with rhodopsin and rod outer segments (ROS). Sera were obtained from rhodopsin-immunized and from ROS-immunized guinea pigs before, during, and after onset of clinical uveitis. Autoantibodies were detected by indirect immuno-fluorescent staining of autogenic retinas as well as normal guinea pig retinas. Sera from animals with clinical disease showed specific labeling of the photoreceptor cell layer of the retina. The rhodopsin autoantibody showed a more defined specificity than the ROS autoantibody staining, only the retinal photoreceptors and retinal pigment epithelium. Specific fluorescence was localized only in the retina, and not in any other ocular or nonocular tissues. Neither the rhodopsin nor the ROS antibodies stained the uvea. Sera from animals taken before the onset of clinical disease did not demonstrate the presence of retinal-binding autoantibodies. These findings suggest that photoreceptor-binding autoantibodies appear in the sera of animals immunized with rhodopsin and with ROS, but only in animals with clinical disease. However, these antibodies probably are not the primary cause of pathology, since previous passive transfer experiments (data not shown here) could not be achieved with anti-ROS or with anti-rhodopsin antibodies. These autoantibodies could occur secondarily as a response to the bovine antigens which cross-reacted with the autologous guinea pig antigens. Subsequently these antibodies could be of primary importance in further tissue alteration and destruction.     

Impacts of the retinal environment and photoreceptor type on functional regeneration

Retinal regeneration is a promising central nervous system (CNS) target amongst the various stem cell ther-apy pursuits, due to its accessibility for manipulation and its disposition towards longitudinal monitoring of treatment safety and efficacy. We offer our perspective on current hurdles towards functional regeneration of cone photoreceptors. Cones are key: For patients suffering vision loss, cone photoreceptors are a required cellular component to restoring daytime vision, colour vision, and high acuity vision. The challenges of regenerating cones contrast with logistic challenges of regenerating rod photoreceptors, which underlines the importance of evaluating context in degeneration and regeneration studies. Foundational research is required to delineate the factors required to generate a diversity of cones in the human macula, and to coax both remaining and newly regenerating cones to rewire towards restoring daytime colour vision. A complex interplay between cell-intrinsic factors and the retinal environment determine both the specifica-tion of cone fates and the synaptic plasticity enabling their functional integration. Recent revelations that cellular materials are transferred amongst photoreceptor progenitors further emphasize the critical role of neighbouring cells in directing stem cell fates. From our vantage point, translation of stem cell therapies to restore the cone-rich human macula must be borne upon foundational research in cone-rich retinas. Re-search frameworks centered on patient outcomes should prioritize animal models and functional outputs that enable and report functional restoration of cone-mediated vision.     

From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells

Cells from dissociated embryonic avian retinae have the capacity to re-aggregate in rotation culture and form cellular spheres reconstituting a complete arrangement of all retinal layers. This exquisite phenomenon is based upon in vitro proliferation of multipotent precursor stem cells and spatial organization of their differentiating descendants. The addition of soluble factors from cultured retinal pigmented epithelial (RPE) or radial glial cells is essential to revert inside-out spheres (rosetted retinal spheres) into correctly laminated outside-out spheres (stratified spheres). Such complete restoration of a laminated brain tissue by cell re-aggregation has been achieved only for the embryonic avian retina, but not the mammalian retina, nor for other brain parts. This review summarises the history of the re-aggregation approach, presents avian retinal re-aggregate models, and analyses roles of the RPE and Müller cells for successful retinal tissue regeneration. It is predicted that these results will become biomedically relevant, as stem cell biology will soon open ways to produce large amounts of human retinal precursors.     

Reciprocal retinal transplantation: A tool for the study of an inherited retinal degeneration

The pathogenesis of retinal degeneration in rd mutant mice has been extensively studied, the gene responsible for the defect has been cloned, and the neural retina has been identified as the primary site for the degeneration. However, the possible contributory role of the ocular environment in this form of retinal degeneration remains undetermined. Retinal transplantation, which provides the opportunity to implant the neural retinal into a genetically defined intraocular environment, was used to examine this possibility. A reciprocal retinal transplantation paradigm was designed based on three experimental groups: (1) normal immature retina transplanted into rd/rd mutant eyes, (2) rd/rd immature retina transplanted into normal eye, and (3) normal immature retina transplanted into normal eyes. The rates of survival and histological characteristics of the grafts were compared between the three groups. At post-transplantation Day 3 (PTD 3), there were no differences between the three groups. Between PTD 10 and 15, the retinal grafts in group 1 showed degeneration. In contrast, the retinal grafts in groups 2 and 3 survived and developed well. At PTD 30, the retinal grafts in both groups 1 and 2 showed degeneration, but the retinal grafts in group 3 survived and remained differentiated well. These results suggest that the retinal degeneration of rd mice may be caused by both a deficit of the neural retina and intraocular environmental changes which are elicited either as a result of mutation or as a sequel to retinal degeneration.     

The lateral ganglionic eminence is the origin of cells committed to striatal phenotypes: neural transplantation and developmental evidence

In order to determine whether the lateral ganglionic eminence (LGE) of the fetal telencephalon is the primary source of striatal precursors in striatal transplants and tissue cultures, cells derived exclusively from the LGE of fetal rat brains were transplanted into the quinolinic-acid-lesioned striatum of adult rats. After 2–3 months they produced grafts that were almost entirely AChE-positive as well as DARPP-32-, TH-, and calbindin-immunoreactive. The grafts were integrated into the host striatum so that host corticofugal fiber tracts interdigitated with graft tissues similar to the way they penetrate the gray matter of the normal striatum. Fast Blue dye injected into the ipsilateral globus pallidus of LGE grafted produced retrogradely labeled neurons within the grafts, but Fluorogold dye injected into the ipsilateral substantia nigra did not. In a separate experiment using DARPP-32-immunohistochemistry as a striatal marker, fetal (E16) and neonatal (P2) rat brains showed DARPP-32 immunoreactivity in the LGE but not in the adjacent medial ganglionic eminence (MGE). In summary, both fetal LGE cells and LGE grafts express specific striatal markers, and LGE grafts integrate into the host striatum and innervate the major striatal efferent target within the host brain. These data suggest that the LGE is the origin of cells committed to striatal phenotypes in the developing brain.     

Generation of retinal ganglion cells is modulated by caspase-dependent programmed cell death

Programmed cell death occurs during both early and late neural development. The mechanisms for the regulation and execution of the early cell death as well as its developmental role are still not fully understood. In this work we have studied the early programmed cell death in the retinal neuroepithelium. Apoptotic cells were selectively located around the optic nerve head in the retinal neuroepithelium of 2- to 6-day-old chick embryos. TUNEL-positive cells and cells which were immunostained for activated caspase-3 showed overlapping distributions suggesting that caspase-3 is involved in the early retinal cell death. Caspase-3 involvement in early retinal cell death was also demonstrated by in vivo treatment with caspase inhibitors z-DEVD-fmk and Boc-D-fmk. After 6 h of treatment, the number of TUNEL-positive cells was reduced by 50%. Sustained treatments (20 h) resulted in a slight widening in the central part of the neural retina but the retinal ganglion cell axons maintained their organization and navigation towards the optic fissure. The most prominent result after inhibition of cell death was an increase in the number of retinal ganglion cells which also produced an enlargement of the ganglion cell layer and an increased number of ganglion cell axons. In conclusion, our results show that caspase-dependent programmed cell death occurs in the embryonic chick retina and that it plays a role to modulate the generation of retinal ganglion cells.     

Interleukin-4 blocks proliferation of retinal progenitor cells and increases rod photoreceptor differentiation through distinct signaling pathways

Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been related to the differentiation of the rodent retina in vitro, but constitutive presence of either IL-4 or of IL-4 receptor in the retina has not been reported. In this work we examined the expression of IL-4 and its specific receptor alpha subunit (IL-4Ralpha). IL-4Ralpha is expressed both in neural retina and non-neural ocular tissue, while IL-4 was found mainly in non-neural tissue. We characterized a novel trophic effect of IL-4 upon the retina. We showed that IL-4 can inhibit the proliferation of retinal cells (approximately 40%) through the cAMP-PKA pathway and associated with a reduction of cyclin D1 and increase of p27(kip1). IL-4 also promotes the differentiation of rod photoreceptors. Activation of tyrosine kinases, protein kinase C, and mitogen-activated kinases of the Erk family were required for IL-4-induced rod photoreceptor differentiation, independent of the release of other trophic factors in culture. Taken together, our results show, for the first time, that IL-4 directly modulates proliferation of retinal cells and rod photoreceptor differentiation, through distinct signaling pathways.     

Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy

To investigate the role(s) of protein‐tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1^?/?/Tpst2 ^?/?) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein‐tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.     

Undersized dendritic arborizations in retinal ganglion cells of the rd1 mutant mouse: a paradigm of early onset photoreceptor degeneration

Retinitis pigmentosa (RP) is a family of inherited diseases causing progressive photoreceptor death. Retinal ganglion cells (RGCs) form the biological substrate for various therapeutic approaches designed to restore vision in RP individuals. Assessment of survival and preservation of RGCs in animal paradigms mimicking the human disease is of key importance for appropriate implementation of vision repair strategies. Here we studied the survival of RGCs in the rd1 mutant mouse, a known model of early onset, autosomic recessive RP, at various stages of photoreceptor degeneration. Furthermore, we analyzed the morphology of various types of RGCs using the newly generated transgenic mouse rd1/Thy1-GFP, in which the rd1 mutation is associated with green fluorescent protein (GFP) expression in a small population of different RGCs. We found excellent survival of cells at up to 1 year of age, a time at which the inner retina is known to have severely reorganized and partially degenerated. However, 50% of the cells analyzed within all RGC types exhibit an undersized dendritic tree, spanning about half of the normal area. Undersized cells are found both in adult and in very young (1-month-old) mice. This suggests that their aberrant phenotype is due to incomplete dendritic development, possibly as a consequence of altered visual input at the time of dendritic arbor refinement. These data show the importance of the timing of photoreceptor death in RGC dendritic development.