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Retinitis pigmentosa initially presents as night blindness owing to defects in rods, and the secondary degeneration of cones ultimately leads to blindness. Previous studies have identified active roles of microglia in the pathogenesis of photoreceptor degeneration in RP. However, the contribution of microglia to photoreceptor degeneration remains controversial, partly due to limited knowledge of microglial phenotypes during RP.

Background Human umbilical cord mesenchymal stem cells (UCMSCs) transplantation is a promising therapy for the treatment of retinitis pigmentosa (RP). However, intravenously infused cells may be blocked in the lung, increasing the risk of vascular obstruction, which needs to be optimized to further improve safety and efficacy. Methods We derived small UCMSCs (S-UCMSCs) from filtering UCMSCs with a 10-μm filter, and compared with UCMSCs by flow cytometry, directional differentiation culture and transcriptome sequencing. Then the S-UCMSCs and UCMSCs were intravenously infused in the Royal College Surgeons (RCS) rats to evaluate the safety and the efficacy. Results The diameter of S-UCMSCs ranged from 5.568 to 17.231 μm, with an average diameter of 8.636 ± 2.256 μm, which was significantly smaller than that of UCMSCs. Flow cytometry, immunofluorescence and transcriptome sequencing demonstrated that the S-UCMSCs and UCMSCs were the same kind of MSCs, and the S-UCMSCs were more proliferative. After the S-UCMSCs and UCMSCs were intravenously infused into the Royal College of Surgeons (RCS) rats at a dose of 1 × 106 cells/rat, the S-UCMSCs blocked in the lungs were significantly fewer and disappeared more quickly than UCMSCs. The b wave of the flash electroretinogram was improved at 7 d, and the retinal outer nuclear layer thickness was thicker at 7 d and 14 d. The expression level of inflammation was inhibited, and the expression level of neurotrophic factors was upregulated in the retina and serum after transplantation. Conclusions S-UCMSCs intravenous infusion was safer than UCMSCs and could delay retinal degeneration and protect visual function in RCS rats, which may be a preferable therapeutic approach for RP.

To study whether ectopic human melanopsin (hMel) in retinal ganglion cells (RGCs) could restore the visual function in end-stage retinal degeneration, AAV2/8-CMV-hMel/FYP was injected into the intravitreal space of Royal College of Surgeons (RCS) rats. It was observed that ectopic hMel/yellow fluorescent protein (YFP) was dominantly expressed in the RGCs of the RCS rat retinae. At 30-45 days after administration of AAV2/8-CMV-hMel/FYP in RCS rats, the flash visual evoked potentials and behavioral results demonstrated that visual function was significantly improved compared to that in the control group, while no improvement in flash electroretinography was observed at this time point. To translate this potential therapeutic approach to the clinic, the safety of viral vectors in the retinae of normal macaques was then studied, and the expression profile of exogenous hMel with/without internal limiting membrane peeling was compared before viral vector administration. The data revealed that there was no significant difference in the number of RGCs containing exogenous hMel/YFP between the two groups. Whole-cell patch-clamp recordings demonstrated that the hMel/YFP-positive RGCs of the macaque retinae reacted to the intense light stimulation, generating inward currents and action potentials. This result confirms that the ectopic hMel expressed in RGCs is functional. Moreover, the introduction of AAV2/8-CMV-hMel/FYP does not cause detectable pathological effects. Thus, this study suggests that AAV2/8-CMV-hMel/FYP administration without internal limiting membrane peeling is safe and feasible for efficient transduction and provides therapeutic benefits to restore the visual function of patients suffering photoreceptor loss.

Olfactory ensheathing cells (OECs) have been shown to be a leading candidate in cell therapies for central nervous system (CNS) injuries and neurodegenerative diseases. Rapid clearance of neuron debris can promote neuronal survival and axonal regeneration in CNS injuries and neurodegenerative diseases. The phagocytic removal of neuron debris by OECs has been shown to contribute to neuronal outgrowth. However, the precise molecular and cellular mechanisms of phagocytic removal of neuron debris by OECs have not been explored. In this study, we found that OECs secreted anti-inflammatory cytokine transforming growth factor β1 (TGF-β1) during the phagocytic removal of neuron debris. TGF-β1 enhanced phagocytic activity of OECs through regulating integrin/MFG-E8 signaling pathway. In addition, TGF-β1 shifted OECs towards a flattened shape with increased cellular area, which might also be involved in the enhancement of phagocytic activity of OECs. Furthermore, the removal of neuron debris by OECs affected neuronal survival and outgrowth. TGF-β1 enhanced the clearance of neuron debris by OECs and increased neuronal survival. These results reveal the role and mechanism of TGF-β1 in enhancing the phagocytic activity of OECs, which will update the understanding of phagocytosis of OECs and improve the therapeutic use of OECs in CNS injuries and neurodegenerative diseases.

Stem cell therapy may replace lost photoreceptors and preserve residual photoreceptors during retinal degeneration (RD). Unfortunately, the degenerative microenvironment compromises the fate of grafted cells, demanding supplementary strategies for microenvironment regulation. Donor cells with both proper regeneration capability and intrinsic ability to improve microenvironment are highly desired. Here, we use cell surface markers (C-Kit+/SSEA4−) to effectively eliminate tumorigenic embryonic cells and enrich retinal progenitor cells (RPCs) from human embryonic stem cell (hESC)-derived retinal organoids, which, following subretinal transplantation into RD models of rats and mice, significantly improve vision and preserve the retinal structure. We characterize the pattern of integration and materials transfer following transplantation, which likely contribute to the rescued photoreceptors. Moreover, C-Kit+/SSEA4− cells suppress microglial activation, gliosis and the production of inflammatory mediators, thereby providing a healthier host microenvironment for the grafted cells and delaying RD. Therefore, C-Kit+/SSEA4− cells from hESC-derived retinal organoids are a promising therapeutic cell source., Stem cell transplantation to treat retinal degeneration could be limited by the degenerative microenvironment. Here, the authors show that C-Kit+/SSEA4– progenitor cells enriched from human embryonic stem cell derived retinal organoids protect retinal structure, suppress microglial activation, gliosis and inflammation.

Graphene and its derivatives are the new carbon nanomaterials with the prospect for great applications in electronics, energy storage, biosensors and medicine. However, little is known about the toxicity of graphene or its derivatives in the case of occasional or repeated ocular exposure. We performed in vitro and in vivo studies to evaluate the toxicity of graphene oxide (GO) exposure to the eye. Primary human corneal epithelium cells (hCorECs) and human conjunctiva epithelium cells (hConECs) were exposed to GO (12.5-100 μg/mL). Acute GO exposure (2 h) did not induce cytotoxicity to hCorECs. However, short-term GO exposure (24 h) exerted significant cytotoxicity to hCorECs and hConECs with increased intracellular reactive oxygen species (ROS). Glutathione (GSH) reduced the GO-induced cytotoxicity. We further performed acute eye irritation tests in albino rabbits according to the Organization for Economic Cooperation and Development (OECD) guidelines, and the rabbits did not exhibit corneal opacity, conjunctival redness, abnormality of the iris, or chemosis at any time point after the instillation of 100 μg/mL of GO. However, 5-day repeated GO exposure (50 and 100 μg/mL) caused reversible mild corneal opacity, conjunctival redness and corneal epithelium damage to Sprague-Dawley rats, which was also alleviated by GSH. Therefore, our study suggests that GO-induced time- and dose-dependent cytotoxicity to hCorECs and hConECs via oxidative stress. Occasional GO exposure did not cause acute eye irritation; short-term repeated GO exposure generally resulted in reversible damage to the eye via oxidative stress, which may be alleviated by the antioxidant GSH.

Retinal regeneration and repair are severely impeded in higher mammalian animals. Although Muller cells can be activated and show some characteristics of progenitor cells when injured or under pathological conditions, they quickly form gliosis scars. Unfortunately, the basic mechanisms that impede retinal regeneration remain unknown. We studied retinas from Royal College of Surgeon (RCS) rats and found that let-7 family molecules, let-7e and let-7i, were significantly overexpressed in Muller cells of degenerative retinas. It demonstrated that down-regulation of the RNA binding protein Lin28B was one of the key factors leading to the overexpression of let-7e and let-7i. Lin28B ectopic expression in the Muller cells suppressed overexpression of let-7e and let-7i, stimulated and mobilized Muller glia de-differentiation, proliferation, promoted neuronal commitment, and inhibited glial fate acquisition of de-differentiated Muller cells. ERG recordings revealed that the amplitudes of a-wave and b-wave were improved significantly after Lin28B was delivered into the subretinal space of RCS rats. In summary, down-regulation of Lin28B as well as up-regulation of let-7e and let-7i may be the main factors that impede Muller cell de-differentiation and proliferation in the retina of RCS rats.

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Resident microglia are the main immune cells in the retina and play a key role in the pathogenesis of retinitis pigmentosa (RP). Many previous studies on the roles of microglia mainly focused on the neurotoxicity or neuroprotection of photoreceptors, while their contributions to synaptic remodeling of neuronal circuits in the retina of early RP remained unclarified. In the present study, we used Royal College of Surgeons (RCS) rats, a classic RP model characterized by progressive microglia activation and synapse loss, to investigate the constitutive effects of microglia on the synaptic lesions and ectopic neuritogenesis. Rod degeneration resulted in synapse disruption and loss in the outer plexiform layer (OPL) at the early stage of RP. Coincidentally, the resident microglia in the OPL increased phagocytosis and mainly engaged in phagocytic engulfment of postsynaptic mGluR6 of rod bipolar cells (RBCs). Complement pathway might be involved in clearance of postsynaptic elements of RBCs by microglia. We pharmacologically deleted microglia using a CSF1 receptor (CSF1R) inhibitor to confirm this finding, and found that it caused the accumulation of postsynaptic mGluR6 levels and increased the number and length of ectopic dendrites in the RBCs. Interestingly, the numbers of presynaptic sites expressing CtBP2 and colocalized puncta in the OPL of RCS rats were not affected by microglia elimination. However, sustained microglial depletion led to progressive functional deterioration in the retinal responses to light in RCS rats. Based on our results, microglia mediated the remodeling of RBCs by phagocytosing postsynaptic materials and inhibiting ectopic neuritogenesis, contributing to delay the deterioration of vision at the early stage of RP.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Choroidal neovascularization (CNV) is the abnormal angiogenesis that causes severe visual loss in AMD. Fibulin-5 (Fbln5), which functions as an angiogenesis inhibitor, plays an important role in the pathogenesis of AMD. Here, we investigated whether subretinal transplantation of Fbln5-overexpressing retinal pigment epithelial (RPE) cells can inhibit CNV in vivo. Adult Long-Evans rats were used in this study. CNV was induced by laser photocoagulation. One week after laser-induced CNV, RPE cells expressing pZlen-Fbln5-IRES-GFP or the control pZlen-IRES-GFP vectors were transplanted into the subretinal space of the right and left eyes, respectively. CNV was evaluated using fundus photography, fundus fluorescein angiography (FFA), and hematoxylin and eosin staining. We found that CNV occurred at 1 week after photocoagulation, reaching peak activity at 3 weeks and remaining at a high level at 4–5 weeks after photocoagulation. Transplanted RPE cells survived for at least 4 weeks and migrated toward the retina. Subretinal transplantation of Fbln5-overexpressing RPE cells resulted in a significant reduction in the total area of leakage and the number of leakage spots compared with transplantation of RPE cells expressing only green fluorescent protein. Our findings suggest that subretinal transplantation of Fbln5-overexpressing RPE cells inhibits laser-induced CNV in rats and thus represents a promising therapy for the treatment of AMD.

Our aim is to define related molecular events on how dormant Müller glia cells re-enter the cell cycle, proliferate and produce new retinal neurons from initial injury to glial scar formation. Sodium iodate (NaIO3) was used to induce acute retinal injury. Long-Evans rats were administered with NaIO3 or phosphate-buffered saline by intraperitoneal injection. The proliferation, dedifferentiation and neurogenesis of Müller cells were analyzed by double-labeled fluorescence immunohistochemistry with primary antibodies – against Müller cells and specific cell markers. Possible molecules that limit the regenerative potential of Müller cells were also determined by immunofluorescence staining, quantitative RT-PCR, protein array, ELISA and Western blot. In the first 3–7days after NaIO3 administration, Müller cells were activated and underwent a fate switch, including transient proliferation, dedifferentiation and neurogenesis. Nerve growth factor (NGF) signaling concomitantly increased with the downregulation of p27Kip1 in Müller cells, which may promote Müller cells to re-enter the cell cycle. The transient increase of NGF signaling and the transient decrease of Notch signaling inhibited Hes1, which might enhance the neuronal differentiation of dedifferentiated Müller cells and suppress gliosis. Upregulated Notch and decreased NGF expressions limit dedifferentiation and neurogenesis, but induces retinal Müller cell gliosis at a later stage. We conclude that transient NGF upregulation and Notch1 downregulation may activate the transient proliferation, dedifferentiation and neurogenesis of Müller cells during NaIO3-induced acute retinal injury; which could be a therapeutic target for overcoming Müller cell gliosis. Such therapy could be potentially used for treating retinal-related diseases.

Müller cells can completely repair retinal injury by acting as endogenous stem/progenitor cells in lower-order vertebrates. However, a safe and effective approach to activate progenitor potential of retinal Müller cells in higher-order vertebrates, which rarely re-enter the cell cycle, is a bottleneck problem. In the present study, Royal College of Surgeon's (RCS) rats were subjected to rat bone marrow mesenchymal stem cells (rBMSCs) subretinal space transplantation. Electroretinography (ERG) recordings showed that the b-wave amplitudes and ONL thicknesses statistically increased after transplantation. The number of Müller cells expressing proliferative, stem/progenitor and neuronal markers significantly increased after rBMSCs transplantation in vivo or after co-culturing with rBMSCs in vitro. The cultured rBMSCs could secrete nerve growth factor (NGF). In addition, we confirmed that NGF or NGF-neutralizing antibody could activate or depress Müller cells dedifferentiation, both in vivo and in vitro. Furthermore, Müller cells expressing high levels of the NGF receptor neurotrophic tyrosine kinase receptor type 1 (TrkA) were observed in the retinas of rats transplanted with rBMSCs. Moreover, the protein expression of downstream elements of NGF/TrkA signaling, such as p-PI3K, p-Akt and p-CREB, increased in Müller cells in the retinas of rBMSCs-treated rats in vivo or in Müller cells co-cultured with rBMSCs in vitro. Blocking TrkA with K-252a reduced the number of dedifferentiated Müller cells and the expression of NGF/TrkA signaling in vitro. Thus, rBMSCs might initiate endogenous regenerative mechanisms, which may constitute a new therapeutic strategy for retinal dystrophic diseases.

Müller cells maintain retinal synaptic homeostasis by taking up glutamate from the synaptic cleft and transporting glutamine back to the neurons. To study the interaction between Müller cells and photoreceptors, we injected either DL-α-aminoadipate or L-methionine sulfoximine–both inhibitors of glutamine synthetase–subretinally in rats. Following injection, the a-wave of the electroretinogram (ERG) was attenuated, and metabotropic glutamate receptor 5 (mGluR5) was activated. Selective antagonism of mGluR5 by 2-methyl-6-(phenylethynyl)-pyridine increased the ERG a-wave amplitude and also increased rhodopsin expression. Conversely, activation of mGluR5 by the agonist, (R,S)-2-chloro-5-hydroxyphenylglycine, decreased both the a-wave amplitude and rhodopsin expression, but upregulated expression of Gq alpha subunit and phospholipase C βIII. Overexpression of mGluR5 reduced the inward-rectifying potassium ion channel (Kir) current and decreased the expression of Kir4.1 and aquaporin-4 (AQP4). Further experiments indicated that mGluR5 formed a macromolecular complex with these two membrane channels. Lastly, increased expression of mGluR5 was found in Royal College of Surgeons rats–a model of retinitis pigmentosa (RP). Inhibition of mGluR5 in this model restored the amplitude of ERG features, and reduced the expression of glial fibrillary acidic protein. These results suggest that mGluR5 may be worth considering as a potential therapeutic target in RP.

Following retinal degeneration, retinal remodeling can cause neuronal microcircuits to undergo structural alterations, which particularly affect the dendrites of bipolar cells. However, the mechanisms and functional consequences of such changes remain unclear. Here, we used Royal College of Surgeon (RCS) rats as a model of retinal degeneration, to study structural changes in rod bipolar cells (RBCs) and the underlying mechanisms of these changes. We found that, with retinal degeneration, RBC dendrites extended into the outer nuclear layer (ONL) of the retina, and the ectopic dendrites formed synapses with the remaining photoreceptors. This ectopic neuritogenesis was associated with brain-derived neurotrophic factor (BDNF) – expression of which was negatively regulated by miR-125b-5p. Overexpression of miR-125b-5p in the retinae of RCS rats diminished RBC ectopic dendrites, and compromised the b-wave of the flash electroretinogram (ERG). In contrast, down-regulation of miR-125b-5p (or exogenous BDNF treatment) increased RBC ectopic dendrites, and improved b-wave. Furthermore, we showed that the regulation of ectopic neuritogenesis by BDNF occurred via the downstream modulation of the TrkB-CREB signaling pathway. Based on these findings, we conclude that ectopic dendrites are likely to be providing functional benefits and that, in RCS rats, miR-125b-5p regulates ectopic neuritogenesis by RBCs through modulation of the BDNF-TrkB-CREB pathway. This suggests that therapies that reduce miR-125b-5p expression could be beneficial in human retinal degenerative disease.

Retinitis pigmentosa (RP) is one of hereditary retinal diseases characterized by the loss of photoreceptors. Cell transplantation has been clinically applied to treat RP patients. Human retinal progenitor cells (HRPCs) and human bone marrow-derived mesenchymal stem cells (HBMSCs) are the two commonly and practically used stem cells for transplantation. Since combined transplantation could be a promising way to integrate the advantages of both stem cell types, we transplanted HRPCs and HBMSCs into the subretinal space (SRS) of Royal College of Surgeons (RCS) rats. We report that HRPCs/HBMSCs combined transplantation maintains the electroretinogram results much better than HRPCs or HBMSCs single transplantations. The thickness of outer nuclear layer also presented a better outcome in the combined transplantation. Importantly, grafted cells in the combination migrated better, both longitudinally and latitudinally, than single transplantation. The photoreceptor differentiation of grafted cells in the retina of RCS rats receiving combined transplantation also showed a higher ratio than single transplantation. Finally, activation of microglia and the gliosis of Müller cells were more effectively suppressed in combined transplantation, indicating better immunomodulatory and anti-gliosis effects. Taken together, combining the transplantation of HRPCs and HBMSCs is a more effective strategy in stem cell-based therapy for retinal degenerative diseases.

Background Retinitis pigmentosa is a common genetic disease that causes retinal degeneration and blindness for which there is currently no curable treatment available. Vision preservation was observed in retinitis pigmentosa animal models after retinal stem cell transplantation. However, long-term safety studies and visual assessment have not been thoroughly tested in retinitis pigmentosa patients. Methods In our pre-clinical study, purified human fetal-derived retinal progenitor cells (RPCs) were transplanted into the diseased retina of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. Based on these results, we conducted a phase I clinical trial to establish the safety and tolerability of transplantation of RPCs in eight patients with advanced retinitis pigmentosa. Patients were studied for 24 months. Results After RPC transplantation in RCS rats, we observed moderate recovery of vision and maintenance of the outer nuclear layer thickness. Most importantly, we did not find tumor formation or immune rejection. In the retinis pigmentosa patients given RPC injections, we also did not observe immunological rejection or tumorigenesis when immunosuppressive agents were not administered. We observed a significant improvement in visual acuity (P

Retinal regeneration and self-repair, whether in response to injury or degenerative disease, are severely impeded by glial scar formation by Müller cells (specialized retinal macroglia). We have previously demonstrated that the activation of Müller cells and gliosis in the degenerative retina are significantly suppressed by the subretinal transplantation of a mixture of olfactory ensheathing cells (OECs) and olfactory nerve fibroblasts. However, the underlying molecular mechanism has remained elusive. Here we transplanted purified rat OECs into the subretinal space of pigmented Royal College of Surgeons (RCS) rats, a classic rodent model of retinal degeneration. Using behavioral testing and electroretinography, we confirmed that the grafted OECs preserved the visual function of rats for 8 weeks, relative to vehicle controls (phosphate-buffered saline). Histological evaluation of outer nuclear layer thickness and composition demonstrated that more photoreceptors and ON-bipolar cells were preserved in the retinas of OEC-treated RCS rats than in controls. The grafted OECs migrated into the outer plexiform layer, inner nuclear layer, and inner plexiform layer. They interacted directly with Müller cells in the retina of RCS rats, in three distinct patterns, and secreted matrix metalloproteinases 2 and 3. Previous studies have demonstrated that rat OECs express delta-like ligand (DLL), while Müller cells express Notch3, the receptor for DLL. Here we found that the grafted OECs significantly decreased the expression, by retinal cells, of Notch signaling pathway components (including Notch3, Notch4, DLL1, DLL4, Jagged1, Hes1, and Hes5) 2 weeks after the cell transplantation and that this effect persisted for a further 2 weeks. Based on these findings, we suggest that transplanted OECs inhibit the activation of Müller cells and the associated gliosis, at least partly through suppression of the Notch pathway.

Purpose of the Study: Protein tyrosine phosphatase σ (PTPσ) acts as a neuronal receptor for chondroitin sulfate proteoglycans (CSPGs). CSPGs have inhibitory effects on experience-dependent plasticity and usually form lattice-like cell coatings that surround the parvalbumin (PV) interneurons in the visual cortex (VC). We investigated developmental changes and the effect of binocular form deprivation (BFD) on PTPσ, perineuronal nets (PNNs) and their tempo-spatial relationships with PV neurons in the VC.Double-immunostaining was used to observe the coexpression pattern of PNNs staining by biotinylated wisteria floribunda lectin (WFA) with PV neurons. The expression of PTPσ in the VC of Long Evans rats was detected by real-time quantitative PCR, immunohistochemistry and western blots. The changes in the number of PV/WFA/PTPσ labeled cells in layer IV of the VC and its proportion of PV neurons were examined during development and after BFD.The expression of PV neurons wrapped by PNNs was increased, particularly in the first half of the critical period, and the ratio for PV neurons reached the highest level (over 75%) at adulthood, indicating that PNNs may play an important role in the maturation of PV neurons during the critical period. BFD decreased the density of PNNs and the percentage of PV neurons with PNNs. This result suggests that the number of PNNs surrounding PV neurons may be experience-dependent. Meanwhile, the CSPG receptor PTPσ was maintained at its lowest level during the critical period and could be modulated by BFD after the critical period. The percentage of PV/WFA/PTPσ-positive cells in PV population increased during development and reached its highest ratio at adulthood, which could also be reversed by BFD.The changes in the coexpression of PNNs, PV and PTPσ provide valuable insights into the connection between CSPGs and PV neurons.

Hydrogen sulphide (H2S) is one of three gaseous signaling molecules after nitric oxide and carbon monoxide. Various H2S donor compounds have been synthesized to study its physiological function. Among these compounds sodium hydrosulphide (NaHS), a donor of releasing H2S rapidly have shown to be protective in certain neuronal cell line but several in vivo studies have generated conflicting data. Furthermore several slow releasing H2S donors have been shown to have positive effects on cells in culture. The intracellular concentration of H2S and hence its rate of production may be a factor in keeping the balance between its neuroprotective and toxic effects. The present study was undertaken to deduce how a rapid releasing H2S donor (NaHS) as opposed to a slow releasing donor (ADTOH), affect oxidative stress related intracellular components and survival of RGC-5 cells. It was concluded that when RGC-5 cells are exposed to the toxic effects of glutamate in combination with buthionine sulfoxime (Glu/BSO), ADTOH was more efficacious in inhibiting apoptosis, scavenging reactive oxygen species (ROS), stimulation of glutathione (GSH) and gluthathione-S-transferase (GST). Western blot and qPCR analysis showed ADTOH increased the levels of Nrf2, HO-1, PKCα, p-Akt, Bcl-2 and XIAP but caused a decrease of Nfκβ and xCT greater than NaHS. This study is first to compare the efficacy of two H2S donor drugs as potential neuroprotectants and demonstrate that slow regulated release of H2S to cell culture can be more beneficial in inhibiting oxidative stress induced cell death.

The aim of the present study was to investigate glutamate homeostasis in retinal degeneration-induced changes and the potential mechanisms of glutamate-mediated excitotoxicity in a rat model. The expression of vesicular glutamate transporter-1 (VGLUT-1) and protein kinase Cα (PKCα) in wild-type and Royal College of Surgeons (RCS) rat retinas, at postnatal Day 15 (P15), P30, P60 and P90, were detected using quantitative real-time polymerase chain reaction and immunohistochemistry. The levels of glutamine synthetase (GS) and L-glutamate/L-aspartate transporter (GLAST) were evaluated by western blotting. Compared with wild-type rats, outer nuclear layer thickness was significantly thinner and VGLUT-1 expression was upregulated in a time-dependent pattern in RCS rats. The ratio of VGLUT-1 to PKCα in RCS rats peaked at P60 (p

The aim of this study was to determine whether α-lipoic acid (LA) is effective in blunting the detrimental effect of light to transformed retinal ganglion cells (RGC-5 cells) in culture. In this study, RGC-5 cells were exposed to light (400-760 nm; 1000 lx) for 48 h with or without LA. For cell assessment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4-[3-(-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetzolio]-1,3-benzene disulfonate (WST-1) reduction assays were used to assess cell and mitochondrial viability respectively. Furthermore, cells were stained for reactive oxygen species (ROS), Apoptosis DNA breakdown and Apoptosis membrane alteration. Antioxidant-capacity, glutathione (GSH) and gluthathione-S-transferase (GST) were determined as well. Light reduced cell viability, affected mitochondrial function, increased the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells and enhanced labelling for ROS. These effects were all attenuated by the presence of LA. LA also stimulated GSH and GST. These findings support the view that light can affect mitochondria which could lead to retinal ganglion cell apoptosis and LA can blunt by decreasing ROS generation and stimulating GSH and GST.

In lower-order vertebrates, Muller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Muller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Muller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Muller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Muller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Muller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Muller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Muller cells toward characteristics of retinal progenitors.

Increased intraocular pressure (IOP) damages the retinal ganglion cell axons as they pass through the optic nerve head (ONH). The massive connective tissue structure of the human lamina cribrosa is generally assumed to be the pressure transducer responsible for the damage. The rat, however, with no lamina cribrosa, suffers the same glaucomatous response to raised IOP. Here, we show that the astrocytes of the rat ONH are "fortified" by extraordinarily dense cytoskeletal filaments that would make them ideal transducers of distorting mechanical forces. The ONH astrocytes are arranged as a fan-like radial array, firmly attached ventrally to the sheath of the ONH by thick basal processes, but dividing dorsally into progressively more slender processes with only delicate attachments to the sheath. At 1 week after raising the IOP by an injection of magnetic microspheres into the anterior eye chamber, the fine dorsal processes of the ONH astrocytes are torn away from the surrounding sheath. There is no indication of distortion or compression of the axons. Subsequently, despite return of the IOP toward normal levels, the damage to the ONH progresses ventrally through the astrocytic cell bodies, resulting in complete loss of the fortified astrocytes and of the majority of the axons by around 4 weeks. We propose that the dorsal attachments of the astrocytes are the site of initial damage in glaucoma, and that the damage to the axons is not mechanical, but is a consequence oflocalized loss of metabolic support from the astrocytes (Tsacopoulos and Magistretti (1996) J Neurosci 16:877-885).

Intravitreal injection of α-crystallin can promote axons from optic nerve regeneration after crushing in rats. We have previously demonstrated that α-crystallin can counteract the effect of myelin inhibitory factors and stimulate neurite growth. And a common crucial signaling event for myelin inhibitory factors is the activation of RhoA. To investigate whether α-crystallin counteracts the inhibitory effect of myelin inhibitory factors through regulation of RhoA/Rock signaling pathway, α-crystallin (10(-4) g/L) was injected into rat vitreous at the time the optic nerve crushed. The RhoA protein activity and the expression of RhoA and Rock were evaluated after 3 days of optic nerve axotomy. Rock downstream effectors, phosphorylated cofilin, and phosphorylated myosin light chain were detected when retinal neurons were cultured for 3 days. Axonal regeneration and neurites growth of cultured cells were observed also. Our results showed that α-crystallin decreased the RhoA protein activity and the phosphorylation of both cofilin and myosin light chain, and promoted the axonal growth. However, the expression of RhoA and Rock was not affected by α-crystallin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors through the regulation of RhoA/Rock signaling pathway.

The temporal and spatial characteristics of cone degeneration in the Royal College of Surgeons (RCS) rat were studied to provide information for treatment strategies of retinitis pigmentosa.Nonpigmented dystrophic RCS rats (RCS) and pigmented nondystrophic RCS rats (controls) were used. Cone processes were visualized with peanut agglutinin (PNA).Cone development appears to have been completed by postnatal day 21 (P21) in both the RCS and control rats. Signs of cone degeneration were obvious by P30, with shorter outer segments (OSs) and enlarged inner segments (ISs). At that time, 81.7% of the cones retained stained ISs. The rate of IS density decline was slower in the peripheral, nasal, and superior retina, and only 43.6% of the cones with ISs were present at P45. By P60, PNA-labeled cone ISs were distorted and restricted to the peripheral retina, and by P90, few cone pedicles were detected.Our findings indicate that therapeutic strategies aimed at rescuing cones in the degenerating retina should be applied before P21 and no later than P45 while substantial numbers of cones retain their ISs. Either the middle or peripheral regions of the nasal and superior retina are the best locations for transplantation strategies.

Transplants of olfactory ensheathing cells (OECs) cultured from the olfactory bulb are able to induce structural regeneration of severed central axons and return of function in rat models. For clinical purposes it would be preferable to obtain the cells from the more accessible olfactory mucosa in the nasal lining. However, in our laboratory preparations cultures from mucosal samples yielded around 5% of OECs compared with the 50% obtained from samples cultured from the bulb, and when transplanted these mucosal cell preparations were less effective at repair. There are a number of manipulations which may increase the OEC content and the effectiveness of mucosal preparations, but in vivo transplantation would be a highly labour intensive method for evaluating them. As a candidate for a high throughput assay to screen for beneficial effects of modifications to mucosal cells we here report the effects of co-culture of the cells with retinal explants. Both bulbar and mucosal cell preparations prolong the survival of the explants. Counts of the surviving retinal ganglion cells, identified by beta-III-tubulin immunohistochemistry and by their axon trajectory, show that the bulbar cell preparations have around twice the potency of those from the mucosa. This in vitro system, therefore, provides a bioassay that discriminates bulbar and mucosal cell preparations, and a useful tool for evaluating the functional effects of manipulations of cultured mucosal preparations.

Background: Optic nerve regeneration has previously been achieved by injuring the lens, which results in the release of lentogenic factors. However, these lentogenic factors are still unknown. Objectives: To investigate what were the lentogenic factors by examining the effects of lens extract and macrophage-conditioned medium (MCM) on the survival and the neurite outgrowth of rat retinal neurons in vitro. Methods: Retinal neurons were cultured in 4 groups: (1) Dulbecco’s modified Eagle’s medium (DMEM), (2) DMEMcontaining lens extract, (3) DMEM containing macrophage-conditioned medium (MCM-D), (4) DMEM and medium from macrophages grown with lens extract (MCM-L). Neurite outgrowth and neuron survival time were observed. The density of retinal neurons with neurites and the longest neurites of the cells were measured on days 1, 3 and 5. Results: Retinal neurons survive for 12–14 days in DMEM containing lens extract. However, the cells only survive for 6 days in DMEM and only 7 days in DMEM containing MCM-L or MCM-D. The present results indicate that lens extract may directly promote survival of rat retinal neurons and neurite outgrowth in vitro. The MCM also promoted cell survival and neurite outgrowth but its effects were weaker than that of the lens extract. We postulate that lens extract exerts its effect by direct neurotrophic effects and/or indirectly by activating macrophagesin vitro.

Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system treatment. Retinal stem cells (RSC) are a substrate for cell-replacement therapy, and previous studies have shown that RSCs from different developmental stages have distinct properties in proliferative capacity and differentiation potential. The tailbud stage is of special interest in retinogenesis, because RSCs commence differentiation after this period. However, no information about the characteristics of RSCs from the tailbud stage is available. In this study, the characteristics of cell cultures from the rat optic cup (referred to as optic-cup-derived RSCs; OC-RSCs) at embryonic day 12.5 (tailbud stage) were analyzed. OC-RSCs grew either as monolayers or as neurospheres in the presence of basic fibroblast growth factor and could be dissociated into a single cell suspension. Using the MTT assay, immunochemistry, cytogenetic analysis, and flow cytometry, we found that OC-RSCs were easily enriched to 92% by three passages, had a normal diploid karyotype, and exhibited no obvious differences in proliferative rate during eight passages (doubling time: 36 h). OC-RSCs produced retinal specific cells after the addition of serum to the medium, but the differentiation potential was affected by serum concentration. Preliminary results showed that transplanted OC-RSCs were incorporated into the degenerated retina of RCS rats and differentiated into rhodopsin-positive cells. Thus, OC-RSCs, after suitable enrichment, provide a population of stem cells with distinct growth and differentiation properties that make them suitable for research into RSC differentiation and transplantation.

We investigated the regulation of primary neurite growth and expression of specific growth-associated genes by lens epithelium-derived growth factor (LEDGF) in rat retinal ganglion cells (RGCs). A pAd-LEDGFp52 adenovirus vector and a siRNA-LEDGFp52 eucaryotic expression vector were transfected into cultured RGCs. Transfection with pAd-LEDGFp52 significantly increased the number of neurites and their lengths compared with untransfected control RGCs. The expression of growth associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and low-molecular-weight neurofilament (NF-L) genes and proteins were also significantly up-regulated. In contrast, the introduction of siRNA-LEDGFp52 significantly decreased the number and length of neurites, and significantly down-regulated the expression GAP43, NF-L and MAP2 genes and proteins compared with controls. Our findings suggest that LEDGFp52 might act as a dendritic arborization gene as well as an axonal elongation gene in RGCs and that it might be beneficial to the functional recovery of regenerating RGCs.

Lens epithelium-derived growth factor (LEDGF) can be alternatively spliced to produce two isoforms-LEDGFp52 and LEDGFp75, however, LEDGFp52 has rarely been investigated. The LEDGFp52 protein or its monoclonal antibody was added to primary rat retinal ganglion cell cultures and their impact on neurite number and length, and the mRNA and protein expression levels of GAP-43, NF-L and MAP-2 quantified. LEDGFp52 was compared to the addition of ciliary neurotrophic factor (CNTF). LEDGFp52 protein significantly increased primary neurite growth compared to control conditions. In addition, the expression of GAP-43, NF-L and MAP2 genes and proteins were also significantly up-regulated. The positive action of the LEDGFp52 protein was similar to or more efficacious than CNTF. LEDGFp52 appears to be an important regulatory protein for the growth of cell processes.

To investigate the molecular mechanism involved in the termination of the critical period of visual cortex plasticity in the rat.The rats were divided into two groups, one group was dark-reared for 67 days after birth, while the other group was dark-reared for 60 days and then put under a normal light/dark cycle for 7 days. A subtracted cDNA library was constructed from the visual cortex, and differentially expressed genes were screened by nested PCR, reverse Northern hybridization, sequencing, and homology analysis.Fourteen genes were found to be up-regulated in the visual cortex. These included 13 known genes and a novel fragment (Genbank submission EB174193). Of the known genes, three genes encoding beta -tubulin, myelin basic protein, and cyclophilin were previously reported to be associated with visual cortex plasticity.A set of candidate genes related to the termination of the critical period was identified using the subtracted cDNA library. This work provides an important basis for understanding the molecular mechanisms involved in the termination of plasticity in the visual cortex.

Retinitis pigmentosa (RP) is a pathological condition leading to progressive visual decline resulting from continual loss of photoreceptor cells and outer nuclear layers of the retina. The aim of the present study was to explore whether melanopsin was able to restore retinal function and inhibit its degeneration by acting in a similar manner to channel rhodopsins. Royal College of Surgeons rats, which were used as an animal model of inherited retinal degeneration, were subjected to sub-retinal injection with melanopsin overexpression vector (AV‑OPN4‑GFP). Immunohistochemical and western blot analyses were used to detect the distribution and protein expression of melanopsin in the retina, revealing that melanopsin was gradually reduced with increasing age of the rats, which was due to loss of dendritic axons of intrinsically photosensitive retinal ganglion cells. Animals injected into both eyes were subjected to a behavioral open-field test, revealing that melanopsin overexpression reduced the loss of light sensitivity of the rats. In a flash electroretinography experiment, the b‑wave and response to light flash stimuli at three and five weeks following injection with AV‑OPN4‑GFP were higher compared to those in eyes injected with AV‑GFP (P

To explore the mechanism of optic nerve damage in glaucoma by study on structure of glial lamina cribrosa(LC) in rats.Experimental study. Albino Swiss(AS) rats were divided into 3 groups. Bilateral eyes of 10 normal rats were employed to be group I (right eye ) and group II (left eye) . Group III was from the left eyes of 13 rats underwent artificially intraocular hypertension in the right eyes. All rats were perfused and fixed with electronic microscopy fixative (2% paraformaldehyde +2% glutaraldehyde). Trimmed optic nerves were embedded with resin. Serial 1.5 µm thick 'semithin' sections were cut, either (2 eyes from group III) longitudinally, through the optic nerve head (ONH) from the retinal end to the commencement of the optic nerve, or (31 eyes) transversely (cross-sections). Ultrathin sections were cut in the middle of glial LC. The morphological observation of glial LC was obtained by light microscopy and transmission electron microscopy. Bonferroni correction was used to counteract the multiple comparison of each group.Fortified astrocytes formed the main supportive structure of glial LC in all rats, including group I, group II and group III. Astrocytes were ranked as a fan-like radial array, firmly attached ventrally to the sheath of the LC by thick basal processes, but dividing dorsally into progressively more slender processes with only delicate attachments to the sheath. These fortified astrocytes form ventral stout basal end feet, radial array, axon free-'preterminal' layer before terminating in a complex layer of fine interdigitating delicate branches at the dorsal. LC astrocytes were highly and uniformly electron dense throughout all the cell processes. An equally striking feature of the astrocytic processes was their massive cytoskeletal 'strengthening' of longitudinal massed filaments and tubules. Especially, massive filaments accumulated as cytoskeletal cores to form 'scaffold' of fortified astrocytes. There was vulnerable area in the dorsal of glial LC. This vulnerable area was isomerisation in bilateral eyes and different rats. There was different space in the vulnerable area. These space could be divided into 3 grades, (-), (+) and (++) . The number of (-), (+) and (++)were 1, 6, 3 eyes in group I, 1, 5, 4 eyes in group II, 1, 7, 3 eyes in group III. The Kruskal-Wallis test was used for statistical evaluations. There was no statistical differences of the ratio of (-), (+) and (++) in group I, group II and group III(χ(2) = 3.35, P = 0.1870.05;group I vs group II, Z = -1.048, P = 0.294;group I vs group III Z = -1.691, P = 0.091;group II vs group III,Z = -1.343, P = 0.179). The ratio of space (-)was significantly less than space (+) and space (++) in group I, group II and group III(χ(2) = 23.88, P0.05; (-) vs (+) , Z = -2.821, P = 0.005; (-) vs (++) , Z = -2.726, P = 0.006). The ratio of space (+)was much more than space (++) in group I, group II and group III(Z = -4.410, P0.05).Glial isomerisation in LC may play a key role in glaucomatous optic nerve damage.

This study aimed to show if two different sulphur containing drugs sulbutiamine and acetylcysteine (NAC) could attenuate the effects of two different insults being serum deprivation and glutamate/buthionine sulfoximine (GB)-induced death to transformed retinal ganglion cell line (RGC-5) in culture. Cells were exposed to either 5 mM of GB for 24 h or serum deprivation for 48 h with inclusion of either NAC or sulbutiamine. Cell viability, microscopic evidence for apoptosis, caspase 3 activity, reactive oxygen species (ROS), glutathione (GSH), catalase and gluthathione-S-transferase (GST) were determined. The effects of NAC and sulbutiamine on the oxidative stress related transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) levels and its dependent phase II enzyme haemeoxygenase-1 (HO-1) were carried out using Western blot and quantitative-polymerase chain reaction (PCR). NAC and sulbutiamine dose-dependently attenuated serum deprivation-induced cell death. However NAC but not sulbutiamine attenuated GB-induced cell death. NAC and sulbutiamine both independently stimulated the GSH and GST production but scavenged different types of ROS with different efficacy. Moreover only sulbutiamine stimulated catalase and significantly increased Nrf-2 and HO-1 levels. In addition, the pan caspase inhibitor, benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) attenuated the negative effect of serum deprivation while the necroptosis inhibitor (necrostatin-1) counteracted solely an insult of GB. The neuroprotective actions of NAC and sulbutiamine in GB or serum-deprivation insult are therefore different.

All know that retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive dysfunction of photoreceptors and associated with progressive cells loss; nevertheless, little is known about how rods and cones loss affects the surviving inner retinal neurons and networks. Retinal ganglion cells (RGCs) process and convey visual information from retina to visual centers in the brain. The healthy various ion channels determine the normal reception and projection of visual signals from RGCs. Previous work on the Royal College of Surgeons (RCS) rat, as a kind of classical RP animal model, indicated that, at late stages of retinal degeneration in RCS rat, RGCs were also morphologically and functionally affected. Here, retrograde labeling for RGCs with Fluorogold was performed to investigate the distribution, density, and morphological changes of RGCs during retinal degeneration. Then, patch clamp recording, western blot, and immunofluorescence staining were performed to study the channels of sodium and potassium properties of RGCs, so as to explore the molecular and proteinic basis for understanding the alterations of RGCs membrane properties and firing functions. We found that the resting membrane potential, input resistance, and capacitance of RGCs changed significantly at the late stage of retinal degeneration. Action potential could not be evoked in a part of RGCs. Inward sodium current and outward potassium current recording showed that sodium current was impaired severely but only slightly in potassium current. Expressions of sodium channel protein were impaired dramatically at the late stage of retinal degeneration. The results suggested that the density of RGCs decreased, process ramification impaired, and sodium ion channel proteins destructed, which led to the impairment of electrophysiological functions of RGCs and eventually resulted in the loss of visual function.

Purpose: To study the electrophysiological properties of the light-evoked currents in ganglion cells in situations of retinal degeneration. Methods: We investigated light-evoked currents in ganglion cells by performing whole-cell patch-clamp recordings from ganglion cells using a retina-stretched preparation from Royal College of Surgeons (RCS) rats, a model of retinal degeneration and congenic controls at different ages. Pharmacological inhibitors of the AMPA receptor (NBQX), GABA receptor (BMI), and sodium channels (TTX) were used to identify the components of the light-evoked currents in ON, OFF and ON-OFF retinal ganglion cells. Results: We found that the light-evoked currents in ganglion cells from control rats were inhibited by NBQX, BMI and TTX, suggesting that AMPA receptors, GABA receptors and sodium channels contribute to these currents in ganglion cells. However, only AMPA receptor-mediated currents were recorded in RCS rats. Light-evoked inward currents were absent in the majority of ganglion cells from RCS rats, particularly at the later stages of retinal degeneration. At earlier stages of retinal degeneration, we found that both the timing and amplitude of light-evoked currents are significantly different in ganglion cells from RCS and control rats. Conclusions: Our study furthers the understanding of the electrophysiological characteristics of retinal ganglion cells during retinal degeneration, and provides insight into the optimal timing for the treatment of retinal degeneration.

Sodium iodate (NaIO(3))-induced retina injury is one of models that is commonly used to study various retinal diseases caused by retinal pigment epithelium (RPE) injury such as AMD. Previous researches have revealed that RPE and photoreceptors are main impaired objects in this model. By comparison, intra-retinal layer has not been studied in detail after NaIO(3) administration. In this study, we present evidences that intra-retinal neurons can be directly injured by NaIO(3) at early stage and that the morphology had taken obvious changes, the decreased areas of dendritic fields of dopaminergic amacrine cells (DA-ACs), horizontal cells, and melanopsin-expressing retinal ganglion cells (mRGCs). Moreover, we found that miRNA 133b that was considered specifically to express in midbrain dopaminergic neurons was markedly upregulated in retinal DA-ACs after NaIO(3) administration. The overexpression of mir-133b negatively regulated the expression of pitx3, an important transcription factor, and led to a series of deficits of DA-ACs such as TH and D2 receptor expression and DA producing, which may play a causative role in pathological events of horizontal cells and mRGCs. After mir-133b was interfered with mir-133b/RNAi, not only those deficits were rescued, but also the amplitude of b-wave and summed OPs of ERG were improved significantly. In conclusion, our data demonstrate, for the first time, that intra-retinal neurons can be directly injured by NaIO(3) at early stage, and that mir-133b level effectively controls synaptic contacts or neural interactions among DA-ACs, horizontal cells, and mRGCs. Delivering mir-133b/RNAi intravitreally can rescue NaIO(3)-induced failure and improve visual function by restoring synaptic contacts.

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss. Fibulin-5 (FBLN5) plays a pleiotropic role in the pathogenesis of AMD. We examined whether the in vitro overexpression of FBLN5 in retinal pigment epithelial (RPE) cells alters the proliferation and migration of cocultured choroidal endothelial cells (CECs) and explored the possible mechanisms involved.A recombinant lentiviral vector carrying the Fbln5 gene was generated to transduce rat RPE cells. The expression of FBLN5 in transduced RPE cells was detected by quantitative real-time PCR and Western blot. The transduced RPE cells were then cocultured with rhesus macaque CECs in a Transwell coculture system. The impact of overexpression of FBLN5 in RPE cells on CEC proliferation and migration was assessed, as well as the impact on the mRNA expressions of vascular endothelial growth factor (VEGF), C-X-C chemokine receptor type 4 (CXCR4), and transforming growth factor β1 (TGFB1).Our results showed that a recombinant lentivirus carrying the Fbln5 gene, which could induce overexpression of FBLN5 in RPE cells, was successfully generated. Overexpression of FBLN5 in RPE cells inhibited cell proliferation and migration and downregulated the mRNA expressions of VEGF, CXCR4, and TGFB1 in cocultured CECs.These findings suggest that FBLN5 may interfere with choroidal neovascularization by downregulating VEGF, CXCR4, and TGFB1 expression and inhibiting CEC proliferation and invasion, intensifying interest in FBLN5 as a target for therapeutic intervention in neovascular AMD.

Proteoglycans have a number of important functions in the central nervous system. Aggrecan (hyaluronan-binding proteoglycan, CSPG-cs56) is found in the extracellular matrix of cartilage as well as in the developing brain. We compared the postnatal distribution of CSPG-cs56 in Long Evans (LE) and Royal College of Surgeons (RCS) rat retinas to determine if this proteoglycan played a role in the development of dystrophic retinas. CSPG-cs56 expression was examined in rat retinas aged between birth (postnatal day 0, P0) and P150 using immunofluorescence and Western-blots. Immunofluorescence was quantified using ImageJ. GFAP staining was used to compare Muller cell labeling and the distribution of CSPG-cs56. Both rat strains showed a significant rise in total retinal CSPG-cs56 between P0 and P21; values peaked on P21 in LE rats and P14 in RCS rats. CSPG-cs56 then significantly decreased to lower levels (P35) in both strains before reaching significantly higher levels by P90–P150. CSPG-cs56 positive staining was present in the ganglion cell layer at birth and clear layering of the inner plexiform layer was seen between P7 and P21 due to dendritic staining of retinal ganglion cells. Staining was less intense and diffuse within the outer plexiform over a similar time-course. Light CSPG-cs56 labeling in the region of the outer segments was present at (P14) and became more intense as the retina approached maturity. CSPG-cs56 in the outer segments was the main contributor to the higher expression in older animals. Substantial differences in CSPG-cs56 labeling were not seen between LE and RCS rats. There was no evidence to suggest that Muller cells were the source of CSPG-cs56 in either rat strain, although their staining distributions had a degree of overlap. The lack of significant differences between LE and RCS rats indicates that CSPG-cs56 may not be involved in the degenerative process or the reorganization of the RCS rat retina. We suggest that the main role of CPSG-cs56 is to maintain retinal ganglion cell dendritic structure in the inner plexiform layer and is closely related to providing adequate support and flexibility for the photoreceptor outer segments, which is necessary to maintain their function.

In several vertebrate classes, the Müller glia are capable of de-differentiating, proliferating, and acquiring a progenitor-like state in response to acute retinal injury or in response to exogenous growth factors. Our previous study has shown that Müller cells can be activated and de-differentiated into retinal progenitors during Royal College of Surgeons (RCS) rats' degeneration, although the limited proliferation cannot maintain retinal function. We now report that rat retinal stem cells (rSCs) transplanted into RCS rats slowed the progression of retinal morphological degeneration and prevented the functional disruption. Further, we found that retinal progenitor cells labeled with Chx10 were increased significantly after rSCs transplantation, and most of them are mainly from activated Müller cells. rSCs transplantation also enhances neurogenic potential by producing more recoverin-positive photoreceptors, which was proved coming from Müller glia-derived cells. These results provide evidence that stem cell-based therapy may offer a novel therapeutic approach for the treatment of retinal degeneration, and that Müller glia in mammalian retina can be activated and de-differentiated by rSC transplantation and may have therapeutic effects.

To study the influence of the ectopic synapse for electrophysiological characteristics modification in ON retinal bipolar cells (ON-RBCs) of RCS rat.Immunofluorescence of the retinal frozen section was taken in P60 d, P90 d of RCS rat (RCS) and control rat (CTR) with the anti-mGluR6 and anti-Synaptophysin, Lucifer Yellow staining solo ON-RBCs was taken in all the group. The whole cell recording was performed in the retinal slice of P60 d, P90 d in RCS and CTR. The modification of the passive membrane properties and the outward currents properties in RCS-ON-RBCs, CTR-ON-RBCs and CTR-OFF-RBCs were observed.RCS-ON-RBCs stretched out the ectopic neurite in different direction and the activity of synapse could be detected around the ectopic neurite. From Pn60d, passive membrane properties of RCS-ON-RBCs kept immature, The RMP in RCS-ON-RBCs and CTR-ON-RBCs were (-61.8 ± 3.07), (-50.44 ± 1.36) mV and (-63.1 ± 2.59), (-48.37 ± 3.69) mV when P60 d and P90 d, there ware significantly higher than CTR group (t = 2.191, 2.435, 5.817, 6.912;P0.05). The IR in RCS-ON-RBCs and CTR-ON-RBCs were (323.3 ± 42.6), (337.6 ± 71.3) MΩ and (321.2 ± 58.6), (340.3 ± 62.8) MΩ when P60 d and P90 d, there ware significantly higher than CTR group (t = 3.561, 1.987, 5.211, 4.034; P0.05). Outward currents were recorded when giving hyper- and depolarized voltage steps. In retinal degeneration, the amplitude of outward currents in RCS-ON-RBCs is significantly different with CTR-ON-RBCs (t = 5.561, 6.341; P0.05) or CTR-OFF-RBCs (t = 5.357, 6.997; P0.05).The ectopic neurite from RCS-ON-RBCs has the possibility for translating the signal. In retinitis pigmentosa, the modification of electrophysiology characteristics in RCS-ON-RBCs was significantly different with CTR-ON-RBCs and CTR-OFF-RBCs. Influence with the ectopic neurite is the possible cause.

In retinitis pigmentosa (RP), the slow and progressive death of inner retinal neurons is thought to be inevitable after the death of photoreceptors. However, even in the advanced stage of RP, all inner retinal neurons are not completely lost. The morphological and electrophysiological modifications in ON-retinal bipolar cells (ON-RBCs) of Royal College of Surgeons (RCS) rats (RCS-ON-RBCs) were investigated to elucidate the mechanisms of survival of RCS-ON-RBCs in RP.Control (CTR) and RCS rats were divided into age groups according to postnatal stage: postnatal day 21 (Pn21d), postnatal day 30 (Pn30d), postnatal day 60 (Pn60d), and postnatal day 90 (Pn90d). Lucifer yellow staining of single ON-RBCs and double-immunofluorescence of the retinal frozen sections were used to detect the morphological modifications and loss of RCS-ON-RBCs in different retinal regions. The whole-cell patch clamping technique was used to record the electrophysiological properties of ON-RBCs.There was a significant loss of RCS-ON-RBCs compared with CTR (p0.01) at Pn60d. Loss of the RCS-ON-RBCs differed by region. From Pn60d onwards, the loss was more severe in the peripheral retinal regions (p0.01). From Pn21d, the ectopic neurites from the RCS-ON-RBCs reached the outer and inner nuclear layers. At Pn60d, terminal branches of RCS-ON-RBCs axons vanished and ectopic neurites from the RCS-ON-RBCs became entwined. The resting membrane potential, input resistance and outward membrane current amplitude of RCS-ON-RBCs were significantly higher than those of the ON-RBCs of CTR rats at Pn60d (p0.05).Our results indicate that more RCS-ON-RBCs survived in the central retinal area near cone clusters, potentially as a result of ectopic neuritis. Meanwhile the surviving RCS-ON-RBCs remained immature and had no normal electrophysiological characteristics.

Retinitis pigmentosa leads to progressive visual loss and blindness. Until now there has been no effective therapy. Experimental studies have shown that transplantation of olfactory ensheathing cells into CNS lesions reorganizes the glial scar and stimulates axon regeneration. We now report that cultured syngeneic adult rat olfactory ensheathing cells transplanted into the subretinal space of pigmented Royal College of Surgeon rats survived for a minimum of 8weeks, migrated into surrounding space, and suppressed the gliotic injury response of the Muller cells. Further investigation of intraretinal transplantation of olfactory ensheathing cells may suggest a possible future route for protection of the retina in retinitis pigmentosa.

The aim of this study was to evaluate the efficacy of subretinal transplantation of rat retinal stem cell when combined with Brain-derived neurotrophic factor (BDNF) in a rat model of retinal degeneration - Royal College of Surgeons (RCS) rats. Retinal stem cells were derived from embryonic day 17 Long-Evans rats and pre-labeled with fluorescence pigment-DiI prior to transplant procedures. RCS rats received injections of retinal stem cells, stem cells+BDNF, phosphate buffered saline or BNDF alone (n = 3 eyes for each procedure). At 1, 2 and 3 months after transplantation, the electroretinogram (ERG) was assessed and the outer nuclear layer thickness measured. The eyes receiving retinal stem cell and stem cell+BDNF transplants showed better photoreceptor maintenance than the other groups (P0.01) at all time points. One month after retina transplantation, the amplitudes of rod-ERG and Max-ERG b waves were significantly higher the eyes with stem cells+BDNF (P0.01), however, this difference was not seen at two and three months post transplantation. BDNF treatment alone group (without transplanted cells) had no effect when compared to buffer injections. The present results indicate that BDNF can enhance the short-term efficacy of the retinal stem cell transplantation in treating retinal degenerative disease.

Müller cells can be activated and included in different functions under many kinds of pathological conditions, however, the status of Müller cells in retinitis pigmentosa are still unknown. Using immunohistochemisty, Western blots and co-culture, we found that Müller cells RCS rats, a classic model of RP, could be activated during the progression of retinal degeneration. After being activated at early stage, Müller cells began to proliferate and hypertrophy, while at later stages, they formed a local 'glial seal' in the subretinal space. As markers of Müller cells activation, the expression of GFAP and ERK increased significantly with progression of retinal degeneration. Co-cultures of normal rat Müller cells and mixed RCS rat retinal cells show that Müller cells significantly increase GFAP and ERK in response to diffusable factors from the degenerting retina, which implies that Müller cells activation is a secondary response to retinal degeneration.

Background: Myelin-associated molecules are major impediments to axon regeneration after optic nerve injury. Intravitreal injection of α-crystallin can protect axons from optic nerve degeneration after crushing in rats. Objectives: Our purpose was to investigate whether α-crystallin could counteract the inhibitory effect of myelin and promote neurite growth. Methods: Newborn rat retinal neurons were cultured on myelin-coated dishes with DMEM containing α-crystallin (10–4 g/l) or bovine serum albumin. The density of neurons with neurites and the length of the longest neurite of the cells were analyzed on days 1, 3 and 5. Results: Cultures containing α-crystallin had significantly higher neurite-containing cell densities, and the neurites were significantly longer compared with cultures containing bovine serum albumin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors and stimulate neurite growth.

We aimed to design and manufacture a novel low-cost polyimide microelectrode array (MEA) chip for visual prosthesis research and to evaluate its biological features. A microelectrode array was developed, based on Flexible Printed Circuit Board (FPC) technology which enables electrical stimulation of the cortex. In an in vitro experiment, rat visual cortex cells were co-cultured with the chip and examined using scanning electron microscopy. Trypan blue exclusion and methyl blue tetrazolium tests showed that cell viability and survival rates (90-98%) did not significantly differ between the co-cultured chip group and the control group. In an in vivo experiment HE/Nissl staining performed to investigate the possibility of brain tissue degeneration around implanted MEAs showed no negative effects of the chip on visual cortical cells after 1 month in situ. The good functional characteristics and biocomptability suggest that such a low-cost device could have widespread application, particularly in countries with a large blind population and limited financial resources.

PURPOSE. Transplantation of retinal stem cells (RSCs) is a potential therapy for retinal degeneration. However, success critically depends on whether RSCs can differentiate into fully functional daughter cells. METHODS. Whole-cell patch clamp techniques were used to characterize the postinduction developmental profile of membrane potentials and ionic currents in RSCs cultured from embryonic day 17 Long Evans rats. These results were compared to recordings obtained from cultured cells obtained from postnatal day 1 rat pups. RESULTS. Outward rectifying potassium currents (I K+ ) were observed in approximately 15% of cells at day 3 and in all cells by day 7. Voltage-dependent sodium currents took longer to emerge (day 7), and TTX-sensitive channels were not fully mature until day 15. Broad excitatory potentials, characteristic of immature action potentials, could be reliably stimulated by day 10, and spontaneous action potentials were recorded at day 25, which were indistinguishable, in terms of width and amplitude, from the comparison group of developing retinal neurons. CONCLUSIONS. The maturation of presumptive retinal ganglion cell electrophysiological properties appears to take at least 15 days under these culture conditions. Knowledge of the timing of voltage-dependent ion channel development could improve the success rate in future transplantation protocols.

To screening genes associated with termination of the critical period of neuroplasticity in visual cortex and investigate the molecular mechanism.To construct a cDNA library using visual cortex of dark rearing rats and normal light rearing 1 week after dark rearing 60 days rats with suppression subtractive hybridization technique. PCR, reverse Northern hybridization, sequencing and homology search were used to analysis the differential expression genes fragments.The cDNA library of termination the critical period was set up successfully. After screening, sequencing and homology search 14 sequences were obtained. Among of the genes 13 were known genes and one was novel gene fragment. They were up-regulated genes in visual cortex when the critical period termination.Through construction the cDNA library, we screened a crop of candidate genes related to termination the critical period. The results provided important foundation to further illuminate the molecular mechanism of termination the critical period of neuroplasticity in visual cortex.

Purpose: Inhibition of microglial activation has become an important strategy to attenuate neurotoxic damage to the central nervous system. We evaluated the effects of α-crystallin on the production of cytokines in lipopolysaccharides (LPS) and optic nerve injury-activated retinal microglia. Methods: Microglia were collected from retinas of newborn rats, cultured and treated with LPS in vitro. Microglia were also activated by an optic nerve crush in vivo. Pretreatments with and without α-crystallin were performed in cultured cells, and by intravitreal injection in adult rats. Expression of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and inducible NOS synthase (iNOS) were measured by RT-PCR, ELISA, Western blot and the nitrate reductase method. Results: Activated microglia significantly upregulated TNF-α and iNOS mRNA expression and protein production in vitro. An optic nerve crush also increased expression of retinal iNOS and TNF-α protein. Treatment with α-crystallin in vitro and in vivo downregulated their expression. Conclusion: The protective effect of α-crystallin may be due to its effect on microglia via a downregulation in the expression and release of 2 key immune regulatory and inflammatory molecules: TNF-α and iNOS.