Histochemical studies on hyaluronic acid in the developing human retina

Changes in the distribution of hyaluronic acid in the developing human retina were investigated histochemically with alcian blue staining and theStreptomyces hyaluronidase digestion method using 56 human embryos and fetuses ranging from 5 to 41 weeks of gestational age. Hyaluronic acid was first detected in the inner layer of the retina at 12 weeks. The site of accumulation extended towards the outer layer by 20 weeks. At the neonatal stage, longitudinal fibers, possibly the processes of Müller cells, were proved to contain hyaluronic acid. These findings suggest that Müller cells produce hyaluronic acid transiently from 12 weeks' gestation to the neonatal stage.     

Evidence for retinal pathology following interruption of neural regulation of choroidal blood flow: Müller cells express GFAP following lesions of the nucleus of Edinger-Westphal in pigeons

Choroidal blood flow in pigeons is regulated by the medial part of the nucleus of Edinger-Westphal (EW) via the ipsilateral ciliary ganglion. Interruption of this circuit by unilateral lesions of EW results in pathological modifications in the morphology of retinal photoreceptors in the ipsilateral eye in pigeons housed under 12hr light (400 lux)/12hr dark conditions. In the present study, we examined the effects of unilateral EW lesions on glial fibrillary acidic protein (GFAP) expression by retinal Müller cells in pigeons housed under the same lighting conditions. Since Müller cells in the retina of land vertebrates express increased GFAP during conditions of retinal pathology or stress (e.g. inflammation or hypoxia), this study would enable us to further evaluate the effects of disruption in the neural regulation of choroidal blood flow on the retina. We found that following EW lesions, retinal Müller cells expressed GFAP, with the precise intracellular location of the GFAP dependent on the amount of time elapsed following the lesion. One week after the EW lesions, GFAP labelling was restricted to the Müller cell endfeet in the nerve fiber layer and ganglion cell layer. By two-three weeks, the labelling had extended outward (or sclerad) into the portions of the Müller cells spanning the inner plexiform layer. Finally, by six weeks post-lesion, the entire extent of the Müller cell from the nerve fiber layer to the outer limiting membrane contained GFAP. No GFAP immunoreactivity in Müller cells was observed in the eyes contralateral to the EW lesions or in eyes in which the pupil had been fixed and dilated by lesions of the pretectal region. Our results suggest that the retina is in a state of physiological stress following interruption of the neural regulation of choroidal blood flow by EW lesions. Although the precise mechanisms by which altered choroidal blood flow regulation affects Müller cell GFAP production require elucidation, the results nonetheless highlight the importance of intact neural regulation of choroidal blood flow for retinal health.     

Muller cell expression of glutamate cycle related proteins and anti-apoptotic proteins in early human retinal development

AIMS: The distribution of glutamate cycle related proteins (glutamine synthetase (GS) and GLAST) and anti-apoptotic proteins (Bcl-2 and Bcl-X) was investigated in Müller cells during early human retinal development, relative to the onset of expression of synaptophysin, a presynaptic vesicle protein. METHODS: Using frozen sections of human fetal eyes (13-22 weeks gestation) (n = 10), Bcl-2, Bcl-X, GS, GLAST, and synaptophysin immunoreactivities (IR) were imaged using fluorescence microscopy and plotted as a function of eccentricity from the incipient fovea. Frozen sections of adult human retina (n = 4) were immunolabelled with antibodies to Bcl-2 and Bcl-X. RESULTS: Müller cell immunoreactivity for GS, GLAST, and Bcl-2 was initially detected in the incipient fovea, and then at more peripheral locations with increasing age. Synaptophysin-IR appeared earlier than all other target proteins. Within the synaptophysin-IR region, mature (differentiated) Müller cells expressed both Bcl-2 and Bcl-X-IR from 13 weeks gestation, ahead of GS-IR and GLAST-IR that were first seen at 14 weeks gestation. Additionally, from as early as 13 weeks gestation, ganglion cells and immature neuronal progenitor cells across the entire retina expressed Bcl-2-IR and Bcl-X-IR, respectively. In adult retina, ganglion cells and some bipolar cells expressed Bcl-X but not Bcl-2. CONCLUSION: Müller cells express Bcl-2 and Bcl-X after synaptogenesis has commenced, but before the onset of GS and GLAST expression, suggesting a protective role for these proteins in Müller cells during the onset of glutamatergic transmission in early human retinal development.     

Increased Müller cell density during diabetes is ameliorated by aminoguanidine and ramipril

Background: The Müller cell, the major glial cell in the retina, may be important in diabetes. The purpose of this project was to examine the localisation of glutamine synthetase in control and diabetic Müller cells and to determine whether the number of Müller cells is altered during diabetes. We also examined whether two experimental treatments of diabetes, aminoguanidine and ramipril, ameliorated these changes. Methods: Normal Sprague‐Dawley rats rendered diabetic by a single injection of streptozotocin (50 mg/kg) were treated with either aminoguanidine, ramipril or standard water. Following 12 weeks, animals were sacrificed, their eyes removed and the retinae processed for glutamine synthetase immunocytochemistry. The level of glutamine synthetase was quantified in control and diabetic animals and the number of Müller cells counted for each of the treatment groups. Results: In all retinae examined, glutamine synthetase labelled Müller cells along their entire cellular extent and endfeet were more intensely labelled. Following 12 weeks of diabetes, there was a small increase in the level of glutamine synthetase labelling in somata and endfeet compared with controls (ANOVA, P < 0.05). The number of Müller cells was increased following 12 weeks of diabetes (ANOVA, P < 0.0001). This effect was ameliorated by treatment with ramipril and aminoguanidine. Conclusions: These data suggest that Müller cells are altered in number following 12 weeks of diabetes. Moreover, the two experimental treatments were beneficial in preventing this change in Müller cells. Further work is required to establish the mechanisms underlying the change to Müller cells during diabetes.     

Diabetes-induced dysfunction of the glutamate transporter in retinal Müller cells

PURPOSE: A decrease in the ability of Müller cells to remove glutamate from the extracellular space may play a critical role in the disruption of glutamate homeostasis that occurs in the diabetic retina. Because this amino acid is toxic to retinal neurons and is likely to exacerbate oxidative stress, elucidation of the mechanisms by which glutamate levels are elevated in diabetes may help in the understanding of the pathogenesis of diabetic retinopathy. This study tested the hypothesis that the function of the glutamate transporter in Müller cells of the diabetic retina is compromised by a mechanism involving oxidation. METHODS: Müller cells were freshly isolated from normal rats and those made diabetic by streptozotocin injection. The activity of the Müller cell glutamate transporter, which is electrogenic, was monitored by the perforated-patch configuration of the patch-clamp technique. RESULTS: Four weeks after the onset of hyperglycemia, a significant dysfunction of the Müller cell glutamate transporter was detected. After 13 weeks of streptozotocin-induced diabetes, the activity of this transporter was decreased by 67%. Consistent with oxidation's causing this dysfunction, exposure to a disulfide-reducing agent rapidly restored the activity of the glutamate transporter in Müller cells of diabetic retinas. CONCLUSIONS: Early in the course of diabetic retinopathy, the function of the glutamate transporter in Müller cells is decreased by a mechanism that is likely to involve oxidation.     

Müller glial cells of the goldfish retina are phagocytic in vitro but not in vivo.

The role of Müller glial cells in the process of degeneration and regeneration of the goldfish retina is poorly understood. One potential role is phagocytosis of neuronal debris in degenerating retinas. We investigated the phagocytic capacity of Müller glial cells of the goldfish retina both in vitro and in vivo. Müller glial cells from primary or first passage cultures were incubated with latex beads to assess their phagocytic ability, and acridine orange staining was used to identify phagolysosomes in living Müller glial cells. These experiments showed that Müller glial cells are phagocytic in culture. Cell identity was verified with an antibody raised against glial fibrillary acidic protein (GFAP). For the in vivo experiments fluorescent latex beads alone or in combination with the metabolic poison ouabain were injected into the posterior chamber. At various intervals (4 days to 8 weeks) after injection the retinas were prepared for immunocytochemistry. Polyclonal anti-GFAP and NN-1, a monoclonal antibody which recognizes macrophages and microglia within the goldfish retina, were used to identify the phagocytic cells. When the beads were injected into the eye, they were phagocytosed by macrophages/microglia cells but not by Müller cells.     

Rabbit retinal Müller cells undergo antigenic changes in response to experimentally induced proliferative vitreoretinopathy.

Experimental proliferative vitreoretinopathy (PVR) was induced in the rabbit eye by injecting mitotically active Müller cells into the vitreal chamber. Two weeks after the initiation of PVR, the retina and the epiretinal membrane that formed were examined to ascertain the antigenic expression of Müller cells in the retina and in the epiretinal membrane. Examination of various regions of the retina from the experimental PVR eye demonstrated that vimentin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde binding protein (CRALBP), and beta-amyloid precursor protein (beta-APP), which were present in the Müller cells of the retina from the control eye, increased their expression, while the antigenicity of glutamine synthetase (GS), did not change; these proteins were also present in the cells contained within the experimentally induced epiretinal membrane. Alpha smooth muscle actin (alpha-SMA), a cytoskeletal protein that is associated with migration and tractional forces in many cell types, was not only present in the cells embedded within the epiretinal membrane, but was also present in the Müller cells underlying the epiretinal membrane. However, Müller cells that were in the inferior portion of the retina, where epiretinal membrane pathology was absent, did not express alpha-SMA. Although this protein is not normally found in Müller cells, they do express it de novo when they are maintained in culture. This suggests that a localized mechanism associated with epiretinal membrane formation induces the expression of alpha-SMA in Müller cells while the increased expression of GFAP, beta-APP, vimentin, and CRALBP are probably regulated via a more general mechanism.     

Angiotensin II and its receptor subtypes in the human retina

PURPOSE: To quantify and evaluate the distribution of angiotensin II (Ang II) and its receptors in the human retina. METHODS: Donor eyes were obtained within 12 hours postmortem and classified as hypertensive or normotensive and diabetic or nondiabetic, based on the donors' medical histories. Ang II in retina and vitreous was quantified by RIA. Ang II receptors were characterized and quantified by competitive membrane-binding assays. Ang II, its heptapeptide metabolite Ang-(1-7), and AT1 and AT2 receptors were localized by immunohistochemistry and confocal imaging. RESULTS: Levels of Ang II in the retina were significantly higher than in vitreous (P < 0.05). Ang II in the diabetic retina had a higher median compared with that in the nondiabetic retina. Ang II and Ang-(1-7) colocalized in retinal Müller cells. The retina had the highest levels of Ang II receptors that were significantly higher than the optic nerve, retinal pigment epithelium-choroid complex, and ciliary body-iris complex (P < 0.05). AT1 receptors were more abundant than AT2 receptors in the retina. Immunoreactivity for AT1 was detected in Müller cells and on blood vessels. AT2 receptors were localized throughout the Müller cells and nuclei of ganglion cells and neurons in the inner nuclear layer. CONCLUSIONS: In the human retina, identification of Ang II and its bioactive metabolite Ang-(1-7) in Müller cells suggests that these glial cells are able to produce and process Ang II. Ang receptors were localized in the blood vessels and neural cells. Local Ang II signaling may thus allow for autoregulation of neurovascular activity. Such an autonomous system could modulate the onset and severity of retinovascular disease.     

Myelin-associated glycoprotein in the developing human retina

The immunohistochemical presence of myelin-associated glycoprotein (MAG) in Müller cells of the developing human retina was examined with rat monoclonal antibodies to MAG and the peroxidase antiperoxidase (PAP) method of Sternberger. Retinas of various developmental stages ranging between 9-31 gestational weeks were stained. There was no staining in the retinas of 9-12-week embryos. Between 13-16 gestational weeks the staining was faint and located mostly in the inner and middle portion of the retina, primarily around the optic nerve head. After midterm, Müller cells invariably stained through all retinal layers. The staining increased gradually up to the twenty-third gestational week, when it reached the level found in the retinas of newborn children.     

Characterization of two spontaneously generated human muller cell lines from donors with type 1 and type 2 diabetes

PURPOSE: Müller cells are the principal glial cells of the retina. They span the entire thickness of the neural retina, and they are in close contact with neurons. Müller cells grow very slowly, and they undergo senescence with increasing passages. Moreover, successful primary cultures of Müller cells can be obtained only with donors no older than 35 years. These limitations of primary cultures motivated the characterization of cell lines. The purpose of this study was thus to compare normal human Müller cells (NHMCs) with two spontaneously generated human Müller cell lines from donors with type 1 and 2 diabetes (HMCLs). METHODS: Both cell lines were investigated for the expression of known markers of Müller cells as well as epithelial and endothelial cells by immunofluorescence and Western blot analyses. RT-PCR was also performed with growth factors that are typical of human Müller cells. RESULTS: In contrast to the typical fibroblast-like morphology of Müller cells, HMCLs showed an epithelial shape. Immunofluorescence analyses and Western blot showed that both NHMCs and HMCLs express the known markers of Müller cells. In addition, HMCLs express cytokeratins K8 and K18 as well as typical growth factors for NHMCs. Finally, HMCLs have reached 30 passages until now without any change in their morphology or expression of markers, whereas NHMCs cannot typically be passed beyond small number of passages. HMCLs are the only human Müller cells lines that have a normal karyotype. CONCLUSIONS: HMCLs can be used as a model to improve the understanding of Müller cells in the context of chronic diabetes.     

Müller cell production of insulin-like growth factor-binding proteins in vitro: modulation with phenotype and growth factor stimulation

PURPOSE: Müller cells are present in diabetic fibrocontractive ocular tissues and generate tractional forces in response to insulin-like growth factors. Recent studies indicate that diabetes-associated increases in vitreous insulin-like growth factor activity are, in part, attributable to changes in insulin-like growth factor binding proteins (IGFBPs). The objectives of this study were to evaluate Müller cells as a source of IGFBPs and characterize changes associated with cell phenotype and growth factor stimuli known to be present in diabetic vitreous. METHODS: Müller cells isolated from normal porcine retina were maintained in culture for 1 and 5 weeks, yielding phenotypes described as proliferative and myofibroblastic. RNA preparations from porcine liver, retina, and Müller cell cultures were evaluated by RT-PCR and Northern blot analysis. IGFBP production was verified by Western ligand and Western blot analysis of Müller-cell-conditioned media and detergent-extracted proteins. RESULTS: Molecular biological analyses of RNA from normal retina and from proliferative and myofibroblastic Müller cells did not detect message for IGFBP-1, but revealed progressive increases in message abundance for IGFBP-2, -3, -4, and -6. IGFBP-5 message was detected in all samples, but was least abundant in myofibroblastic Müller cells. Stimulation of myofibroblastic Müller cells by IGF-I and -II, but not PDGF, further increased message abundance and production of IGFBP-2, -4, -5, and -6, but not IGFBP-3. CONCLUSIONS: Müller cell production of IGFBPs changes with phenotype and, in most cases, is highest in the cells most likely to participate in fibrocontractive retinal disease. IGFBP production by these cells is further increased by IGF-I and -II, growth factors known to be present and active in proliferative vitreoretinal disorders, suggesting that Müller cells represent a potential source of vitreous IGFBPs in disorders involving this cell type.     

Ultrastructural lesions of retinal pericapillary Müller cells in streptozotocin-induced diabetic rats

Seventeen Wistar inbred rats were made diabetic by a single injection of streptozotocin (50 mg/kg body weight) and were killed after periods of 3, 6, 9 or 12 months. Pathological changes in pericapillary Müller cells of the retina were studied using electron microscopy. Basement membrane-like material proliferated in the intercellular space of the Müller cell network and occasionally appeared to insinuate into the Müller cell cytoplasm far from capillary pericytes and endothelial cells. The part of the Müller cell that was enveloped by proliferating basement membrane-like material showed partial necrosis which was thought to contribute to the widening of the capillary wall. A breakdown of the retinal framework, which leads to capillary dilatation, was also thought to be associated with partial necrosis of Müller cells. Highly electron-dense bodies accumulated in the Müller cell cytoplasm which surrounded the retinal capillaries. Ultrastructurally, these dense bodies resembled lysosomes. Their increased number might reflect the altered metabolism of the diabetic retina.     

Glial reactivity, an early feature of diabetic retinopathy

PURPOSE: To characterize early structural gliotic reactions in retinal Müller cells, astrocytes, and microglia in experimentally induced diabetes. METHODS: Rats were rendered diabetic by streptozotocin injection and killed after 2, 4, 12, or 20 weeks. Cell densities were determined in flatmounted retinas or transverse semithin sections. Expression of glial fibrillary acidic protein (GFAP) was localized on frozen sections or flatmounts by immunofluorescence and confocal microscopy, and GFAP content was evaluated by Western blot analysis. Microglial cells were visualized by binding of isolectin B4 or staining with antibodies to phosphotyrosine residues. The integrity of the blood-retinal barrier was assessed by intravenous injection of Evans blue. RESULTS: The density of Müller cells and microglia was significantly increased at 4 weeks of diabetes compared with nondiabetic controls. GFAP expression in Müller cells was not detected at 4 weeks but was prominent at 12 weeks. The number of astrocytes was significantly reduced at 4 weeks in the peripapillary and far peripheral retina. Shape changes of microglial cells indicated functional activation. Leakage of the blood-retinal barrier was observed at 2 weeks of hyperglycemia, the earliest time point investigated. CONCLUSIONS: The leakage of the blood-retinal barrier before glial reactivity suggests that glia are early targets of vascular hyperpermeability. The individual glial cell types react differentially to the diabetic state. Müller cells undergo hyperplasia preceding GFAP expression, and microglial cells are activated, whereas astrocytes regress. This glial behavior may contribute decisively to the onset and development of neuropathy in the diabetic retina.     

Nitric oxide synthase expression in ischemic rat retinas

PURPOSE: To investigate the expression of nitric oxide synthase (NOS) in the ischemic retina. METHODS: Retinal ischemia was induced in rats by bilateral common carotid artery occlusion (BCCAO) for various lengths of time. Using the retina after BCCAO, expression of neuronal NOS (nNOS) and inducible NOS (iNOS) and identification of their positive cells were studied by histological and immunohistochemical examinations. RESULTS: Histological examinations revealed significant reduction in the thickness of the inner plexiform layer and the outer plexiform layer of the retina. Expression of nNOS was detected in retinal ganglion cells, amacrine cells, and Müller cells after BCCAO. The expression of nNOS and iNOS detected in Müller cells became stronger and persisted long after BCCAO. CONCLUSIONS: In the ischemic retina, Müller cells and retinal ganglion cells expressed nNOS and iNOS. These phenomena may be involved in the ischemic damage to the retina.     

Atrophy of Müller glia and photoreceptor cells in chick retina misexpressing cNSCL2.

PURPOSE: To investigate whether and how the basic helix-loop-helix (bHLH) gene cNSCL2 is involved in retinal development. METHODS: cNSCL2, the chick homologue of human NSCL2, was isolated and sequenced. In situ hybridization was used to examine its spatial and temporal expression pattern in the retina. Replication-competent retrovirus RCAS was used to drive cNSCL2 misexpression in the developing chick retina, and the effect of the misexpression was analyzed. RESULTS: Expression of cNSCL2 in the retina was restricted. Its mRNA was detected in amacrine and horizontal cells, but not in photoreceptor, bipolar, or ganglion cells. Retroviral-driven misexpression of cNSCL2 in the developing chick retina resulted in missing photoreceptor cells and gross deficits in the outer nuclear layer (ONL). These deficits were probably not because of decreased photoreceptor production, in that the ONL appeared normal in early developmental stages. TUNEL+ cells were detected in the ONL, indicating that photoreceptor cells underwent apoptosis in retinas misexpressing cNSCL2. Müller glial cells were far fewer in the experimental retina than in the control, indicating that cNSCL2 also caused Müller glia atrophy. The onset of Müller glia disappearance preceded that of photoreceptor degeneration. CONCLUSIONS: Expression of cNSCL2 in the chick retina was restricted to amacrine and horizontal cells. Misexpression of cNSCL2 caused severe retinal degeneration, and photoreceptor cells and Müller glia were particularly affected.     

Müller cell endfeet at the inner surface of the retina: light microscopy

Using fractions of the protein spectrum of the cat retina as immunogens, we have generated antibodies with substantial specificity for the Müller cells of the retina of cat, rabbit, guinea pig, and rat. The antibodies appear to bind to the filamentous components of the Müller cells and allow demonstration of the pattern of Müller cell endfeet at the inner surface of the retina, best seen in wholemount preparations. In sections and at the edge of wholemount preparations the somas and processes of the cells can be observed. Müller cells are more evenly distributed over the retina than ganglion cells, indicating that their proliferation continues during the differential growth of retina which continues into postnatal life. The morphology and distribution of the endfeet varies with the structures present at the inner surface of the retina. Where the axon bundles are thick, the endfeet are relatively small and are confined to narrow rows between bundles. Müller cell endfeet are also separated widely by large blood vessels. In both situations, it seems likely that Müller cells and astrocytes both contribute, perhaps competitively, to form the glia limitans of the inner surface of the retina. Where the somas of neurones are densely packed in the ganglion cell layer, the endfeet are small and numerous, forming rings around the somas. Where axon bundles, vessels, and somas are sparse, the endfeet appear largest and form a regular array.     

Synthesis of retinoic acid from retinol by cultured rabbit Müller cells.

Previous observations have shown that Müller glial cells of the vertebrate retina contain cellular retinoid-binding proteins, that the retina contains retinoic acid, and that cellular retinoic acid-binding protein is present in amacrine neurons (and, in some species, Müller cells) within the retina. These findings led to the suggestion that Müller cells may synthesize retinoic acid and release it for use by other retinal cells. To test this possibility, we cultured Müller cells from adult rabbit retinas, incubated the cultures with radioactive retinol, and identified and quantified the resultant radioactive retinoids by HPLC. Retinaldehyde was rapidly synthesized from retinol, reaching a plateau of 1-2 pmol mg-1 cell protein by 30 min. Retinoic acid initially accumulated more slowly, but by 30 min constituted most of the synthesized retinoid. While the retinaldehyde remained within the cells, retinoic acid was rapidly released into the medium; extracellular retinoic acid exceeded the intracellular amount after 30 min of incubation. Smaller amounts of retinyl esters were also synthesized and retained by the cells. These results are consistent with the suggestion that Müller glia are a source of retinoic acid in the retina. The synthesis of retinoic acid by these cells, and the presence of retinal neurons that contain cellular retinoic acid-binding protein, raise the possibility that retinoic acid plays a role in the retina, although this role is not presently known. Furthermore, these results may have implications for other parts of the adult nervous system. Adult brain contains retinol- and retinoic acid-binding proteins, and, therefore, may also be a site of retinoic acid metabolism. Because of the relatively simple cellular organization of the retina and its demonstrated capacity to synthesize retinoic acid, the retina may be a system of choice for further studies of the synthesis and function of retinoic acid in adult neural tissue.     

Changes of GABA metabolic enzymes in acute retinal ischemia.

It is reported that GABA accumulates in Müller cells in ischemic and diabetic rat retina. To investigate the mechanism of GABA accumulation in Müller cells, we localized GABA and glutamate in ischemic rat retina and measured the activity of GAD and GABA-T, enzymes involved in GABA metabolism. Using general anesthesia, we incised the bulbar conjunctiva of the rat around the limbus and clamped the left optic nerve. A sham operation was performed on the right eyes. Ocular ischemia was sustained for 30, 60 and 90 minutes. Rat eyes were enucleated immediately after ischemia and prepared for immunohistochemistry and enzyme activity measurement. Glutamate-like immunoreactivity (Glu-IR) in the sham-operated rat retina was observed in all retinal layers, showing intense staining in the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL). Glu-IR increased in the outer plexiform layer (OPL) and outer nuclear layer (ONL) in an ischemic time-dependent manner. GABA-like immunoreactivity (GABA-IR) in sham-operated rat retina was observed in NFL, GCL, IPL and inner nuclear layer (INL). When the ischemic time was extended, GABA-IR intensely stained Müller cells. GAD activity was not changed in ischemic rat retina as compared to normal rat retina, but GABA-T activity was significantly decreased in ischemic rat retina. These results suggested that glutamate was induced by ischemia and was converted to GABA by GAD activity. Increased GABA was not metabolized because GABA-T activity was decreased. GABA accumulation in Müller cells progressed during the change in activity of these metabolic enzymes.     

An immunoperoxidase study of early phase experimental retinitis induced by herpes simplex virus

Herpes simplex virus type 1 (HSV-1) was intravitreally inoculated in domesticated rabbits, and retinitis was observed with the ABC-method using peroxidase-conjugated antibody. Trypsin digestion was used for antigen visualization. Three days after inoculation, although cell degeneration was not found in the retina, DAB was deposited on the inner limiting membrane and the nerve fiber layer, which corresponded to the basal side of Müller cells. These findings indicated that viral antigen was already present in the retina at this early stage. Four or five days after inoculation, numerous variously sized exudative retinal lesions were seen. In the small exudative foci, DAB deposition was restricted to only in the nuclei of the nerve cells of the inner nuclear layer, while it extended to the outer and inner retinal layer in the large exudative foci. These results suggested that when HSV-1 was injected intravitreally it invaded from the basal side of Müller cells to the inner nuclear layer, where it replicated. The virus spread to the outer nuclear layer and ganglion cells as infection proceeded.     

Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish

Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells. During adult growth, Müller glial nuclei undergo sporadic, asymmetric, self-renewing mitotic divisions in the inner nuclear layer to generate a rod progenitor that migrates along the radial fiber of the Müller glia into the outer nuclear layer, proliferates, and differentiates exclusively into rod photoreceptors. When retinal neurons are destroyed, Müller glia in the immediate vicinity of the damage partially and transiently dedifferentiate, re-express retinal progenitor and stem cell markers, re-enter the cell cycle, undergo interkinetic nuclear migration (characteristic of neuroepithelial cells), and divide once in an asymmetric, self-renewing division to generate a retinal progenitor. This daughter cell proliferates rapidly to form a compact neurogenic cluster surrounding the Müller glia; these multipotent retinal progenitors then migrate along the radial fiber to the appropriate lamina to replace missing retinal neurons. Some aspects of the injury-response in fish Müller glia resemble gliosis as observed in mammals, and mammalian Müller glia exhibit some neurogenic properties, indicative of a latent ability to regenerate retinal neurons. Understanding the specific properties of fish Müller glia that facilitate their robust capacity to generate retinal neurons will inform and inspire new clinical approaches for treating blindness and visual loss with regenerative medicine.