Wolfram syndrome 1 (WFS1) protein expression in retinal ganglion cells and optic nerve glia of the cynomolgus monkey

Wolfram syndrome (WFS1, OMIM 222300) is a rare genetic disorder associated with multiple organ abnormalities, most prominently optic nerve atrophy and diabetes. Mutations in the WFS1 gene coding for wolframin have been identified. The pathogenesis for optic nerve atrophy remains elusive. We here tested the hypothesis that wolframin is expressed in glial cells of the optic nerve and in retinal ganglion cells in the cynomolgus monkey. Paraffin sections through the retina and optic nerve were examined with immunohistochemistry using affinity-purified antibodies to wolframin. Retinal ganglion cells and optic nerve glial cells were found to be strongly labeled. Dual dysfunction of wolframin in optic nerve glial cells and retinal ganglion cells may explain the progressive optic nerve atrophy in Wolfram syndrome.     

Expression of the sodium-coupled monocarboxylate transporters SMCT1 (SLC5A8) and SMCT2 (SLC5A12) in retina

PURPOSE: Monocarboxylates are primary energy substrates in the retina. Recently, the authors identified two sodium-coupled monocarboxylate transporters (SMCTs), SMCT1 (a high-affinity transporter) and SMCT2 (a low-affinity transporter). Expression of SMCT1 and SMCT2 has been studied in several tissues; however, little is known about their expression in retina. In the present study, the authors asked whether SMCT1 and SMCT2 are also expressed in retina and, if so, in which particular retinal cell types. METHODS: SMCT1 and SMCT2 expression was analyzed in intact mouse retina and cultured retinal cells (ganglion, Müller, RPE) by RT-PCR, in situ hybridization, and immunofluorescence. Uptake assays were performed to demonstrate SMCT1 (RGC-5 and ARPE-19 cells) and SMCT2 (rMC-1 cells) expression at the functional level. RESULTS: SMCT1 mRNA and protein were detected in the ganglion cell layer, inner nuclear layer, inner/outer plexiform layers, photoreceptor inner segments, and RPE. In RPE, the expression of SMCT1 was restricted to the basolateral membrane. SMCT2 mRNA and protein were detected only in neural retina, with a pattern of protein localization consistent with labeling of Müller cells. In vitro studies confirmed the cell type-specific expression of SMCT1 and SMCT2. Uptake assays demonstrated Na(+)-coupled monocarboxylate transport in RGC-5, ARPE-19, and rMC-1 cells. CONCLUSIONS: These data provide the first evidence for the expression of SMCT1 and SMCT2 in the retina and for the cell-type specific distribution of these transporters within the retina. These studies suggest that SMCT1 and SMCT2 play a differential role in monocarboxylate transport in the retina in a cell type-specific manner.     

Human intraretinal myelination: Axon diameters and axon/myelin thickness ratios

Purpose:

Human intraretinal myelination of ganglion cell axons occurs in about 1% of the population. We examined myelin thickness and axon diameter in human retinal specimens containing myelinated retinal ganglion cell axons.

Materials and Methods:

Two eyes containing myelinated patches were prepared for electron microscopy. Two areas were examined in one retina and five in the second retina. Measurements were compared to normal retinal and optic nerve samples and the rabbit retina, which normally contains myelinated axons. Measurements were made using a graphics tablet.

Results:

Mean axon diameter of myelinated axons at all locations were significantly larger than unmyelinated axons (P ≤ 0.01). Myelinated axons within the patches were significantly larger than axons within the optic nerve (P < 0.01). The relationship between axon diameter/fiber diameter (the G-ratio) seen in the retinal sites differed from that in the nerve. G-ratios were higher and myelin thickness was positively correlated to axon diameter (P < 0.01) in the retina but negatively correlated to axon diameter in the nerve (P < 0.001).

Conclusion:

Intraretinally myelinated axons are larger than non-myelinated axons from the same population and suggests that glial cells can induce diameter changes in retinal axons that are not normally myelinated. This effect is more dramatic on intraretinal axons compared with the normal transition zone as axons enter the optic nerve and these changes are abnormal. Whether intraretinal myelin alters axonal conduction velocity or blocks axonal conduction remains to be clarified and these issues may have different clinical outcomes.     

Wolfram syndrome

Wolfram syndrome (WFS) is a rare diffuse neurodegenerative disorder characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness, and a wide variety of central nervous system abnormalities. Insulin-dependent diabetes mellitus with optic nerve atrophy is sufficient criteria for the diagnosis. WFS is a devastating disease for the patients and their families. This study emphasizes the need for careful evaluation of cases having insulin-dependent diabetes mellitus and optic atrophy.     

Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway

Cadmium (Cd), released from cigarette smoke and metal industrial activities, is known to accumulate in human body organs including retina and is particularly higher in retinal tissues of age-related macular degeneration (AMD) eyes compared to non-AMD eyes. We have determined the cytotoxic effects of Cd on human retinal pigment epithelial (RPE) cells. Upon Cd treatment, there was a dose- and time-dependent decline in ARPE-19 cell viability as well as early apoptotic changes such as altered mitochondrial membrane potential (MMP) and Cytochrome C release in cytosol. Depletion of GSH by buthionine-[S,R]-sulfoximine (BSO) resulted in increased Cd toxicity in ARPE-19 cells. Cadmium also caused reactive oxygen species (ROS) generation and activation of mitogen-activated protein kinases (MAPKs) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) significantly reduced Cd-induced toxicity. These results indicate that elevated ROS-induced activation of the MAPK signaling pathway could be associated with Cd-induced RPE cell apoptosis, one of the major contributing factors in AMD. The toxic effects of Cd on ARPE-19 cells indicate that environmental heavy metals such as Cd could be important potential factors in RPE cells death associated retinal diseases particularly related to smoking.     

Retention of retinal axon collateral is responsible for induced ipsilateral retinotectal projections in adult goldfish.

In normal goldfish, optic axons innervate only the contralateral optic tectum. When one eye was enucleated and the optic nerve of the other eye crushed, the regenerating optic axons innervated both optic tecta. We studied the presence of bilaterally projecting retinal ganglion cells by double retrograde cell labeling methods using Nuclear Yellow and True Blue dyes. About 10% of the retinal ganglion cells were double labeled and these cells were found throughout the retina. In addition, HRP application to the ipsilateral tectum revealed retrogradely-labeled retinal ganglion cells of all morphological types. These results suggest that induced ipsilateral projections are formed by regenerating axon collaterals and that all cell types are involved in the generation of normal mirror image typography.     

All-trans-retinol generated by rhodopsin photobleaching induces rapid recruitment of TIP47 to lipid droplets in the retinal pigment epithelium

PURPOSE: To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE). METHODS: Dark-adapted mouse eyes were exposed to intense flashes of light, and ARPE-19 cells were treated with all-trans-retinol. The two specimens were labeled with BODIPY493/503 for LDs and with antibodies for three LD proteins: adipocyte differentiation-related protein (ADRP), TIP47, and Rab18. The labeling intensity in fluorescence microscopy was quantified by image analysis. Localization of mutated TIP47 was also examined. Immunoelectron microscopy was performed for ADRP in mouse RPE. Expression of TIP47 in ARPE-19 cells was knocked down by RNA interference (RNAi), and its effect on retinyl ester storage was measured by HPLC. RESULTS: Both flashes of light on mouse eyes and all-trans-retinol on ARPE-19 cells caused rapid translocation of TIP47 from the cytosol to LDs, whereas ADRP distributed constitutively in LDs. The density of LDs did not show visible changes by any treatment. The localization of TIP47 to LDs was abolished when either the amino-terminal or the carboxyl-terminal half of the molecule was deleted, but was enhanced by a short deletion in the carboxyl terminus. Manipulation of TIP47 expression by RNAi or cDNA transfection did not affect the retinyl ester amounts in ARPE-19 cells significantly. CONCLUSIONS: All-trans-retinol generated by photobleaching in the retina induces rapid translocation of TIP47 to LDs in the RPE.     

Subretinal delivery of immunoglobulin G with gold nanoparticles in the rabbit eye

Purpose  To examine the feasibility of subretinal delivery of immunoglobulin G (IgG) adsorbed onto gold nanoparticles (GNPs) and its histologic distribution in the rabbit retina after the injection. Methods  Goat IgG was adsorbed onto GNPs electrostatically. Goat IgG-adsorbed GNPs or buffer with goat IgG was injected into the subretinal space of rabbit eyes and followed up for 3 months by examination of fundus photographs, immunohistochemistry against goat IgG, and transmission electron microscopy (TEM). Human retinal pigment epithelial cells (ARPE-19 cells) were cultured, and cell proliferation with or without GNPs was assayed. Results  At 1 week after the subretinal injection of goat IgG-adsorbed GNPs, retinal degeneration was observed in the outer retina, and goat IgG was immunolabeled in the retinal pigment epithelium (RPE) and the photoreceptor cells. TEM showed GNPs located in the outer segments and in the lysosomes in the RPE at 1 month and no apparent cytotoxicity of the RPE. There were no inhibitory effects of GNPs on proliferation of ARPE-19 cells. Conclusions  Goat IgG was successfully delivered into photoreceptor cells and RPE using GNPs, though retinal degeneration in the outer retina occurred in this model. This might be an alternative drug delivery method to photoreceptors and RPE.     

High intraocular pressure-induced ischemia and reperfusion injury in the optic nerve and retina in rats

• Purpose: The purpose of this paper is to describe the damage caused to the retina and the axons of the optic nerve by acute ischemia-reperfusion injury and the extent to which optic nerve damage correlates with the duration if ischemia due to high intraocular pressure (IOP). • Methods: Acute ischemia in the retina and optic disc was induced in albino rats by increasing the IOP to 110 mmHg for a period of 45–120 min. Thereafter, the eyes were reperfused at normal IOP after 7 days. The retina and optic nerve were examined by light and electron microscopy, and morphometrical counts of the optic nerve axons were performed. • Results: After 45 min of ischemia, electron microscopic examination revealed swelling of mitochondria and degeneration of neurotubules on axons in cross sections of the optic nerve. The axonal counts in eyes subjected to 45 min of ischemia were 29% lower than in control eyes. After 60 min of ischemia, there were distinct disruptions of mitochondria and degeneration of the axons. After 90 min of ischemia, numerous axons showed degeneration with disordered myelin sheaths. Neuronal cell death was seen in the retina, mainly in the ganglion cell layer. • Conclusion: Damage to the retinal ganglion cell layer and the optic nerve was evident after only 45 min of ischemia in normal eyes. This experiment suggests that seriously injured eyes must be protected from high IOP; if IOP elevation is required during vitrectomy, it is essential to reduce the duration of interruption of blood flow to a minimum.     

Heparan sulfate regulates intraretinal axon pathfinding by retinal ganglion cells

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.     

Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD

PURPOSE: To demonstrate structural and immunocytochemical changes associated with light-induced degeneration in albino rat retinas and human AMD retinas. METHODS: Retinas from Wistar rats aged 3, 6, or 10 months were examined by immunocytochemistry, with antibodies to neuronal and glial markers. Results were compared with human nonneovascular AMD retinas. RESULTS: In aging rat retinas, many photoreceptors were lost in response to normal ambient light exposure. Photoreceptor loss was preceded by loss of RPE cells. Müller cells extended processes through gaps in Bruch's membrane, into the choroid. immunolabeling for gamma-aminobutyric acid (GABA), the glycine transporter Glyt-1, and the rod bipolar cell marker PKC revealed the presence of numerous neuronal somata and processes that appeared to have migrated into the choroidal region. Processes of presumptive ganglion cells remodeled and stratified in the choroid, where strong labeling for synaptic vesicle antigens was present. Myelination of retinal ganglion cell axons was also observed, especially in the peripheral retina. In AMD retinas, glial rearrangement and displacement of neurons suggestive of their migration were also observed. CONCLUSIONS: In response to loss of RPE and photoreceptor cells, adult retinal neurons migrate out of the retina along remodeled processes of Müller cells. The presence of synaptic vesicle antigens suggests the formation of new synapses between migrating neurons. The myelination is probably due to the ingress of Schwann cells from the sclera. The presence of some similar changes in human AMD retinas suggests that these findings are of broad significance for determining the likely events in transplantation of neurons in the human retina and elsewhere.     

Evolving neurovascular relationships in the RCS rat with age

PURPOSE: To examine the course of development of vascular disorders in the Royal College of Surgeons (RCS) rat and how these may lead to retinal ganglion cell loss. METHODS: Whole-mount retinae from RCS rats were first stained for neurofilament protein and then for NADPH-diaphorase staining. A separate group of RCS rats was injected with Type II Peroxidase and the retinae were subsequently processed for peroxidase histochemistry. RESULTS: The first changes in the deep vascular plexus occur as the photoreceptor layer is lost and it comes into close proximity to the retinal pigment epithelial (RPE) cell layer. RPE cells migrate onto retinal vessels, and at such locations vascular complex develop. These are first found ventral to the optic nerve head and then gradually progress over most of the retina. The inner retinal vessels that supply the complexes cross the optic nerve fiber layer and appear to be under tension. They ligate axons, which leads to retinal ganglion cell loss. CONCLUSIONS: These observations show vascular changes can have secondary repercussions for neurons distant from the primary lesion.     

TNF-alpha-induced optic nerve degeneration and nuclear factor-kappaB p65

PURPOSE: To characterize a model of optic nerve axonal degeneration induced by tumor necrosis factor (TNF)-alpha and to determine the role of nuclear factor (NF)-kappaB p65 in axonal degeneration. METHODS: Groups of rats were euthanatized at 1 day, 1 or 2 weeks, or 1 or 2 months after intravitreal injection of TNF-alpha. Morphometric analyses of neurofilament- or Thy-1-positive cells, retinal ganglion cells (flat preparations stained with cresyl violet or retrograde labeling with a neurotracer), the number of axons, immunostaining for myelin basic protein, and TUNEL assays were performed. Levels of NF-kappaB p65 protein in retina and optic nerve were determined by Western blot analysis and immunohistochemistry. The effects of antisense oligodeoxynucleotide (AS ODN) against NF-kappaB p65 and helenalin, an inhibitor of NF-kappaB p65 activation, on TNF-alpha-induced optic nerve degeneration were determined by counting the number of axons. RESULTS: Intravitreal injections of TNF-alpha induced obvious axonal loss and extensive degeneration of the axons from 2 weeks to 2 months after injection, whereas significant retinal ganglion cell loss was noted only at 2 months after injection. NF-kappaB p65 was increased in the optic nerve but not in the retina and was found to colocalize with ED-1 and Iba1, markers of microglia. Inhibition of NF-kappaB p65 with AS ODN or helenalin significantly ameliorated the effects of TNF-alpha-mediated axonal loss. CONCLUSIONS: TNF-alpha causes axonal degeneration with probable delayed loss of retinal ganglion cell bodies. NF-kappaB p65 may play a pivotal role in axonal degeneration, with the possible involvement of microglial cells.     

Characteristics of Optic Nerve Damage Induced by Chronic Intraocular Hypertension in Rat

Purpose:To set up the Sharma‘s chronic intraocular hypertension model and investigate the intraocular pressure (IOP) as well as the optic nerve damage of this model in rat.Methods :The operations of the chronic intraocular hypertension model were performed as described by Sharma in 60 male Lewis albino rats. IOP was measured using the TonoPen XL immediately after surgery and then at 5 day, 2 week or 4 week intervals. Cresyl violet staining of whole-mounted retinas was used to label retinal ganglion cells (RGCs),then RGCs were counted. Paraphenylenediamine (PPD) staining was performed in the semi-thin cross sections of optic nerve of rat, in order to know whether the axons of optic nerve were degenerated or not.Results:There were 47 rats with higher IOP after the episcleral veins cauterized in 60 rats. The ratio of elevated IOP was 78.3%. The IOPs were stable in 4 weeks. After cresyl violet staining, the RGCs loss was 11.0% and 11.3% was found in the central and peripheral retina respectively after 2 weeks of increased IOP. After 4 weeks of increased IOP, the loss of RGCs was 17% for the central retina and 24.6% for the peripheral retina. In the retinas without higher IOP, there was no loss of RGCs. PPD staining showed that optic nerve of rat with about 5.3% damage of axons located at the superior temporal region. Region of affected optic nerve 1 mm posterior to the globe by light microscope showed evidence of damaged axons with axonal swelling and myelin debris.Conclusion:Sharma‘s chronic intraocular hypertension model is a reproducible and effective glaucoma model, which mimics human glaucoma with chronically elevation IOP and induced RGCs loss and damage of optic nerve.     

Identification of intracellular phospholipases A2 in the human eye: involvement in phagocytosis of photoreceptor outer segments

PURPOSE: To identify intracellular phospholipases A(2) (PLA(2)) in the human retina and to explore the role of these enzymes in human retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS). METHODS: PCR amplification and Western blot analysis were used to identify mRNA and protein expression of intracellular PLA(2) subtypes in the retinal pigment epithelial cell line ARPE-19. Immunohistochemical staining of normal human eye sections was performed to reveal the cellular location of the enzymes. A model of RPE phagocytosis of POS was used to explore the role of intracellular PLA(2) in phagocytosis. An activity assay was used to evaluate PLA(2) activity, and inhibitors of specific PLA(2) were applied to evaluate the role of PLA(2) in RPE phagocytosis. RESULTS: Genes encoding calcium-independent (i)PLA(2), group VIA; calcium-dependent cytosolic (c)PLA(2), groups IVA, IVB, and IVC; and iPLA(2), group VIB, were identified in the human RPE cell line ARPE-19. Furthermore, protein of iPLA(2)-VIA, cPLA(2)-IVA, and iPLA(2)-VIB were identified in ARPE-19 cells and in various parts of the normal human eye. iPLA(2)-VIA protein levels were upregulated during phagocytosis, and iPLA(2)-VIA activity was found to be specifically increased 12 hours after ARPE-19 cells were fed with POS. Finally, RPE phagocytosis was inhibited by the iPLA(2)-VIA inhibitor bromoenol lactone. CONCLUSIONS: Various intracellular PLA(2) subtypes are present in the human retina. iPLA(2)-VIA may play an important role in the regulation of RPE phagocytosis of POS and may also be involved in the regulation of photoreceptor cell renewal.     

Optic nerve degeneration in a murine model of juvenile ceroid lipofuscinosis

PURPOSE: To investigate optic nerve degeneration associated with CLN3 deficiency in a murine model of juvenile neuronal ceroid lipofuscinosis (Batten disease). METHODS: Using light and electron microscopy, the density and diameter of axons and the thickness of myelin in optic nerve were compared between age-matched cln3 knock-out (cln3-/-) and wild-type (129ev/TAC) mice. Western blot analysis was used to assay expression of Cln3 in mouse and primate retina and optic nerve. RESULTS: Morphologically identified mast cells were present in the meningeal sheaths surrounding the cln3-/- nerve and in the nerve itself. The cln3-/- optic nerve exhibited an overall loss of uniformity and integrity. Axon density in cln3-/- optic nerve was only 64% of that in wild-type optic nerve (P < 0.01). Accounting for differences in axon density, the diameter of axons in cln3-/- optic nerve was 1.2 times greater than in wild-type optic nerve (P < 0.01). Electron micrographs revealed large spaces between axons and 32% thinner myelin surrounding axons in cln3-/- mice than in wild type (P < 0.01). Western blot analysis demonstrated that Cln3 was expressed in retinas and optic nerves of mouse and primate. CONCLUSIONS: The presence of apparent mast cells in cln3-/- optic nerve suggests compromise of the blood-brain barrier. The absence of Cln3 causes loss of axons, axonal hypertrophy, and a reduction in myelination of retinal ganglion cells. Furthermore, expression of CLN3 in mouse and primate optic nerve links degeneration to loss of Cln3.     

The spatial and temporal expression patterns of netrin receptors, DCC and neogenin, in the developing mouse retina

Recently it has been demonstrated that the guidance of retinal ganglion cell (rgc) axons through the optic disc is dependent on the DCC/netrin-1 axonal guidance system. To gain further insight into the function of the netrin receptors, DCC and Neogenin, in retinal development we have studied the expression patterns of these receptors in the embryonic mouse retina. Neogenin mRNA was restricted to a single neural cell type, the rgc. However, strong Neogenin mRNA expression was observed in the extending fiber cells of the developing lens suggesting a role for Neogenin in the migration events shaping the early lens. Our studies demonstrated that DCC mRNA was expressed at high levels in chains of closely opposed neurons as they migrated towards the emerging mantle layer in the early retina (E12.5-E13.5) suggesting a role for DCC in the migration of neurons out of the ventricular zone. DCC protein expression was high on rgc axons as they actively navigated through the optic disc into the optic nerve. At birth, when the majority of rgc axons had projected through the optic disc, DCC protein was no longer detectable on the distal axonal segments within the optic nerve despite significant DCC protein expression on the proximal axonal membranes in the nerve fiber layer. These observations suggest that a localized down-regulation of DCC protein occurs on projecting axonal membranes once the DCC guidance function is no longer required. We also demonstrated that DCC mRNA and protein were expressed by amacrine cells and Müller glial cells while DCC mRNA was detected in horizontal cells. Taken together, these expression patterns suggest a role for DCC in axon outgrowth and/or pathfinding for a variety of retinal neurons and in the migration of newly born neurons within the developing retina.     

Representation of the temporal raphe within the optic tract of the cat

The retinal topography of the cat's optic tract was determined by means of injections of the enzyme horseradish peroxidase (HRP) into the tract. This analysis was accomplished by the subtraction of all HRP injection sites not labeling a defined retinal area from those injection sites which resulted in ganglion cell labeling (Venn diagram analysis). Using this method, the following correspondences were demonstrated for the ipsilateral and contralateral projections: superior retina represented in medial optic tract; inferior retina in lateral tract; and area centralis in a dorsocentral location (which was part of a larger area representing the visual streak). The temporal raphe was represented in the ipsilateral tract as a band curving from the area centralis region toward the dorsomedial border of the tract. Contralateral fibers from a region superior to the optic disc were found to be displaced with respect to the general retinal representation in the optic tract and this appeared to be related to retinal development. The ratio of contralateral to ipsilateral fibers was determined and found to be nonuniform within the tract. Injection of HRP into the optic tract of the cat also allowed the axons from labeled retinal ganglion cells to be traced within the retina and optic disc. Axons from ganglion cells lying temporal to the raphe curve around the area centralis enter the optic disc on the lateral and inferior aspects. Ganglion cells lying nasal to the raphe send their axons more directly to enter the optic disc on its superior aspect. A schema is proposed whereby the retina is mapped onto the optic tract.     

Optic Nerve Engraftment of Neural Stem Cells

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm² vs. 199 ± 58 RGCs/mm²; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.