Early postnatal Müller cell death leads to retinal but not optic nerve degeneration in NSE-Hu-Bcl-2 transgenic mice

Topographically localized over-expression of the human Bcl-2 protein in retinal glial Müller cells of a transgenic mice (line 71) leads to early postnatal apoptotic Müller cell death and retinal degeneration. Morphological, immunohistological and confocal laser microscopic examination of transgenic and wild-type retinas were achieved on paraffin retinal sections, postnatally. Apoptosis occurs two to three days earlier in the internal nuclear layer of transgenic retinae, than in wild-type littermates. In parallel there was a progressive disappearance of transgenic Hu-Bcl-2 over-expression, as well as of the Müller cell markers, cellular retinaldehyde-binding protein and glutamine synthetase. This phenomenon led to retinal dysplasia, photoreceptor apoptosis and then retinal degeneration and proliferation of the retinal pigment epithelium. The optic nerve, however, remains intact. Two complementary observations confirm the pro-apoptotic action of Bcl-2 over-expression in Müller cells: (i) in the peri-papillary and peripheral regions where the transgene Bcl-2 is not expressed, cellular retinaldehyde-binding protein or glutamine synthetase immunostaining persist and Müller glia do not die; and (ii) the retina conserves a normal organisation in these two regions inspite of total retinal degeneration elsewhere.We conclude that retinal dysplasia and degeneration are linked to primary Müller cell disruption. Besides its generally accepted anti-apoptotic function, over-expression of Bcl-2 also exerts a pro-apoptotic action, at least in immature Müller glia. One may suppose that Bcl-2 translocation resulting in its over-expression in retinal Müller cells could be a putative mechanism for early retinal degeneration.     

Expression of aquaporin-1 immunoreactivity by photoreceptor cells in the mouse retina

Aquaporin water channels play a crucial role in the maintenance of ionic and osmotic homeostasis in the neural tissue. In the sensory retina, aquaporin-4 is expressed by Müller glial cells, predominantly in the inner retina, while aquaporin-1 is expressed mainly in the outer retina. However, it is unknown whether aquaporin-1 expression occurs in Müller cells or photoreceptor cells. By using immunohistochemical staining of retinal slices from rds mice, we show that the immunoreactivity for aquaporin-1 disappears along with the photoreceptor cell degeneration. In suspensions of dissociated retinal cells from control mice, photoreceptor cells expressed aquaporin-1 immunoreactivity while Müller cells were largely devoid of staining. The data suggest that photoreceptor cells, but not Müller cells, express aquaporin-1 in the murine retina.     

Becoming glial in the neural retina.

During development of the vertebrate neural retina, multipotent stem cells give rise to retinal neurons as well as to Müller cells, the principal glial population in the retina. Recent studies have shed light upon the extracellular and intracellular signaling pathways that regulate Müller glial cell genesis. Emerging evidence demonstrates that activation of the Notch signaling pathway can play a role in regulating Müller cell development as well as gliogenesis in other parts of the central nervous system. Cyclin dependent kinase (CDK) inhibitors of the Cip/Kip subfamily are cell cycle regulators that can regulate progenitor proliferation during retinal development, but also regulate the proliferation of Müller glia when they become activated in response to stress or injury. Surprisingly this class of proteins can also promote the development of Müller glia. In this review we discuss the role of both Notch and the CDK inhibitors in regulating Müller cell development.     

Immunohistochemical demonstration of glial fibrillary acidic protein in normal rat Müller glia and retinal astrocytes

The presence of glial fibrillary acidic protein (GFA)-positive Müller glia and retinal astrocytes were studied immunohistochemically in normal rat retina. Using GFA antiserum both Müller glia and separate star-shaped cells were observed in spread-preparations as well as cryostat sections. The retinal astrocytes were also visualized using two different monoclonal GFA antibodies. These cells were found to be located in the nerve fiber and ganglion cell layers. In contrast, Müller glia were not normally visualized with any of the monoclonal GFA antibodies but could be stained 4 days after an optic nerve crush. Our results demonstrate that normal rat Müller glia expresses GFA-like immunoreactivity.     

Neuron-glia interactions in the rat retina infected by Borna disease virus

Summary.  Neuron-glia interactions in the Borna disease virus (BDV)-infected rat retina were investigated with emphasis on the ultrastructural characterization of degenerative alterations in the ganglion cell and photoreceptor layer. Immuno- and cytochemical techniques were applied to label microglia, macrophages and Müller (macroglial) cells. Four weeks after intracerebral infection of adult rats, the total thickness of the retina was considerably diminished, primarily due to the loss of photoreceptor segments and ganglion cells. A gradual reduction of both plexiform layers was also observed. There was a remarkable increase in the number of microglial cells, predominantly in the ganglion cell and the inner plexiform layers. Ultrastructural analysis confirmed that microglia, but also macrophages, were involved in phagocytosis accompanying severe neuronal degeneration in the ganglion cell and the photoreceptor layer. In contrast, Müller cells showed moderate morphological and cytochemical alterations, indicating that Müller cells play only a minor role in early stages of BDV-induced retinitis. Monitoring neuron-glia interactions in BDV-induced retinopathy, combined with the application of different protocols of immunosuppression effecting the BDV virus and/or the microglia, might help to establish specific strategies to suppress BDV-induced neuronal degeneration. Received October 21, 1998/Accepted June 23, 1999     

Localization of glial aquaporin-4 and Kir4.1 in the light-injured murine retina

Excessive light causes damage to photoreceptor and pigment epithelial cells, and a local edema in the outer retina. Since Müller glial cells normally mediate the osmohomeostasis in the inner retina (mainly via channel-mediated transport of potassium and water), we determined whether retinal light injury causes an alteration in the retinal localization of glial water (aquaporin-4) and potassium (Kir4.1) channels, and in the potassium conductance of Müller cells. Mice were treated with bright white light (intensity, 15,000lx) for 2h. Light treatment results in Müller cell gliosis as indicated by the enhanced staining of the glial fibrillary acidic protein and an increase in the cell membrane area reflecting cellular hypertrophy. In light-injured retinas, the immunostaining of the photoreceptor water channel aquaporin-1 disappeared along with the degeneration of the outer retina, and the outer nuclear layer contained large spherical bodies representing photoreceptor nuclei which were fused together. The immunostainings of the aquaporin-4 and Kir4.1 proteins were increased in the outer retina after light treatment. Since the amplitude of the potassium currents of Müller cells remained largely unaltered, the increase in the Kir4.1 immunostaining is supposed to be caused by a redistribution of the channel protein. The data indicate that Müller glial cells respond to excessive light with an alteration in the localization of Kir4.1 and aquaporin-4 proteins; this alteration is thought to be a response to the edema in the outer retina and may support the resolution of edema.     

Closer look at lactose-mediated support of retinal morphogenesis

We have previously shown in intact isolated eye rudiments from Xenopus laevis that lactose, but not mannose, permits the formation of organized photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of this study was to determine, using electron microscopic analysis, the key ultrastructural differences between healthy retinas, lactose-protected retinas, and retinas that developed aberrantly to reveal which subcellular structures were exclusively present in healthy retinas. Filamentous actin was also localized in retinas to determine its distribution under the various conditions. In healthy retinas, calycal processes extending approximately three-fourths of the length of the outer segment surrounded highly organized photoreceptor outer segments. Adherens junctions were localized between adjacent photoreceptors and Müller cells at the outer limiting membrane. In addition, Müller cells possessed apical processes that extended for a short distance beyond the adherens junctions. These fine cytoarchitectural details were missing in retinas that completed differentiation in the absence of the RPE; both calycal and apical processes were no longer present and adherens junctions were sparsely intermittent. Müller cells appeared atrophic. Similarly, mannose promoted none of the fine cytoarchitectural details of the retina. Lactose, however, supported the formation of the proper subcellular cytoarchitecture of both photoreceptor and Müller cells. These results suggest that these subcellular structures may be fundamental for the proper assembly and stability of organized outer segments and are necessary to allow for normal cytogenesis of the outer retina.     

Pigmentary degeneration indwed by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina

Pigmentary degeneration of the retina was induced by a single intraperitoneal Injection of 75mgkg of N-methyl-N-nitrosourea (MNU) In female Brown-Norway colored rats at 50 days of age, which were then observed at 24, 48 and 72 h and 7, 21,35 and 150 days after the treatment. MNU-treated rats showed selective destruction of the photoreceptor cells by an apoptotic mechanlsm 24 h after the treatment, and the destruction was completed by day 7. During the photoreceptor cell degeneration, proliferation of Miller cells and infiltratlon of macrophages was prominent 72h and 21 days aRttr the treatment, respectively. Müller cell proliferation and macrophage infiltratbn corresponded to degenerative photo-receptor cell phagocytosis, and prollferating Müller cell processes responded to stabilize the damaged retina. Pigment epithelial cell detachment from the Bruch's membrane was seen 72 h after the treatment, and migration within all layers of the retina was seen at day 7 when photoreceptor Cells were lost. At 21, 35 and 150 days after the treatment, lack of photoreceptor cells and deposition of pigment epithelial cells within the retina but not in contact to vascular endothe-lial cells were characteristic. MNU-induced photoreceptor apoptosis followed by Miiller cell and macrophage reaction then pigment epithellal cells deposition withln the retina partially resembles retinitis pigmentosa in humans.     

Spatial buffering of potassium by retinal Müller (glial) cells of various morphologies calculated by a model

In a previous study we found the morphometrical data of rabbit retinal Müller (radial glial) cells to vary greatly with their localization in various parts of the retina. The long cells of the central retina have thinner vitreal processes and smaller endfeet than the short cells of the retinal periphery. This configuration should impair the spatial buffering capacity of the central Müller cells for excess K+ ions. To test this hypothesis, we developed a simple modified model for the calculation of K+ clearance by spatial buffering, diffusion through the extracellular space, and co-operation of both processes. K+ clearance processes were demonstrated to depend greatly on the retinal geometry and Müller cell morphology in different parts of the retina. The efficiency of spatial buffering exhibited an obvious optimum for Müller cells of intermediate length, and decreased very steeply in longer cells. Some conclusions are drawn with respect to retinal physiology. In particular, it is suggested that very long and slender radial glia is unable to perform sufficient K+ clearance preventing long-lasting extracellular [K+] elevations after neuronal activity. Such [K+] elevations could depolarize these glial cells so as to enforce their mitotic division. This mechanism might lead to the perinatal transformation of embryonic radial glia into adult multipolar glia when neuronal activity commences in CNS tissues thicker than the maximal effective length of radial glial cells.     

Development of the neonatal rabbit retina in organ culture

Organ cultures from neonatal rabbit retinae grew well over periods of up to 2 weeks in vitro. Proliferation in vitro declined in parallel with the decline seen in vivo, although the rate of proliferation in the explants was slightly reduced. The proliferation of progenitor cells in vitro produced the same cell types produced postnatally in vivo. Postnatally generated cell clones, labeled by means of a retroviral vector, consisted mainly of rods and Müller cells. The layers of the retinae developed as in vivo; an outer plexiform layer occurreed after the first 2 days in vitro. Ultrastructurally, ribbon synapses (outer and inner plexiform layer) and conventional synapses (inner plexiform layer) were observed. The photoreceptor cells grew well-developed inner segments and cilia but no mature outer segments. The cultured retinae contained a well-developed, regular lattice of Müller cells expressing vimentin as in vivo. The neuron-to-Müller cell-ratios were essentially the same as in vivo, viz. about 15 to 16 neurons, among them about 10 to 11 (rod) photoreceptor cells per Müller cell. When the glia cell-specific toxin α-aminoadipic acid (αAAA) was applied, the pattern of vimentin-positive Müller cells became irregular, or even locally missing. In such cases, the tissue became disorganized as indicated by a local disappearance of the regular layering, and development of many rosettes. It is concluded that an intact lattice of Müller cells is necessary for the migration of young neurons, and for correct formation of retinal layers.     

Momordica charantia (bitter melon) attenuates high-fat diet-associated oxidative stress and neuroinflammation

Purpose

Microglia and Müller cells are prominent participants in retinal responses to injury and disease that shape eventual tissue adaptation or damage. This investigation examined how microglia and Müller cells interact with each other following initial microglial activation.

Methods

Mouse Müller cells were cultured alone, or co-cultured with activated or unactivated retinal microglia, and their morphological, molecular, and functional responses were evaluated. Müller cell-feedback signaling to microglia was studied using Müller cell-conditioned media. Corroborative in vivo analyses of retinal microglia-Müller cell interactions in the mouse retina were also performed.

Results

Our results demonstrate that Müller cells exposed to activated microglia, relative to those cultured alone or with unactivated microglia, exhibit marked alterations in cell morphology and gene expression that differed from those seen in chronic gliosis. These Müller cells demonstrated in vitro (1) an upregulation of growth factors such as GDNF and LIF, and provide neuroprotection to photoreceptor cells, (2) increased pro-inflammatory factor production, which in turn increased microglial activation in a positive feedback loop, and (3) upregulated chemokine and adhesion protein expression, which allowed Müller cells to attract and adhere to microglia. In vivo activation of microglia by intravitreal injection of lipopolysaccharide (LPS) also induced increased Müller cell-microglia adhesion, indicating that activated microglia may translocate intraretinally in a radial direction using Müller cell processes as an adhesive scaffold.

Conclusion

Our findings demonstrate that activated microglia are able to influence Müller cells directly, and initiate a program of bidirectional microglia-Müller cell signaling that can mediate adaptive responses within the retina following injury. In the acute aftermath following initial microglia activation, Müller cell responses may serve to augment initial inflammatory responses across retinal lamina and to guide the intraretinal mobilization of migratory microglia using chemotactic cues and adhesive cell contacts. Understanding adaptive microglia-Müller cell interactions in injury responses can help discover therapeutic cellular targets for intervention in retinal disease.     

An ultramicroscopic study on the distribution of Müller cell processes in the outer retinal layers of the zebrafish

The zebrafish, Brachydanio rerio, is a good model for studying the development of various organs. We have assayed the distribution pattern of Müller cell processes in zebrafish retinas by electron microscopy. In the outer nuclear (ON) layer, multiple layers of Müller cell processes were present along both sides of the large pyramidal endings of the synaptic terminals. We found that the inner segments (ISs) of the zebrafish photoreceptors (PRs), including the cones, double cones and rods, were arranged in different planes, and that the Müller cell processes formed multilayered sheaths around virtually all PR compartments except their outer segments. Thus, Müller cell processes beyond the outer limiting membrane (OLM) are more easily observed in zebrafish retina than in the retinas of other species. To our knowledge, this is the first study to show the exact ultrastructural distribution of Müller cell processes around the OLM and the PR layer in zebrafish retina.     

Evidence for a columnar organization of cones, Müller cells, and neurons in the retina of a cichlid fish

In the retina of many lower vertebrates, the arrangement of cells, in particular of cone photoreceptors, is highly regular. The data presented in this report show that in the retina of a cichlid fish (Astatotilapia burtoni) the regular arrangement is not restricted to cone photoreceptors and their synaptic terminals but can be found in elements of the inner retina as well. A variety of immunocytochemical and other markers was used in combination with confocal microscopy on whole-mount preparations and tangential sections. Nearest neighbor analysis was performed and density recovery profiles as auto- and cross-correlograms were generated. Cells displaying a regular arrangement of their synaptic processes in matching radial register to each other were identified for each major retinal neuronal cell type except ganglion cells (i.e. photoreceptors, horizontal cells, bipolar cells, and amacrine cells). The precise location of some of the corresponding cell bodies was not as regular but still non-random, however there was no spatial cross-correlation between cell bodies of different types. The radial processes of Müller glial cells displayed a distribution correlating to the arrangement of photoreceptors and neurons. Thus, for one Müller glial cell I found two PKC-positive cone bipolar cells, a spatially corresponding grid of parvalbumin-positive amacrine cell processes, one H1 horizontal cell, and two pairs of double cones. There was no evidence among ganglion cells matching this pattern, possibly due to the lack of suitable markers. Although many other cell types do not follow this matching regular mosaic arrangement, a basic columnar building block can be postulated for the retina at least in cichlid fish. This suggests a functional radial unit from photoreceptors to the inner plexiform layer.     

Reactive changes of retinal astrocytes and Müller glial cells in kainate-induced neuroexcitotoxicity

The aim of this study was to investigate reactive changes of astrocytes and Müller glial cells in rats subjected to kainate treatment, which leads to neuronal degeneration in the ganglion cell layer and the inner border of the inner nuclear layer as confirmed by labelling with Fluoro-Jade B, a marker for degenerating neurons and fibres. Both the astrocytes and the Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein and glutamine synthetase. A major finding was the induced expression of nestin together with glial fibrillary acidic protein beginning at 1 day post-injection of kainate. The marked nestin expression appeared to be most intense at 1 day and was sustained till 2 weeks as compared with the untreated/normal retina. Western blotting analysis confirmed a marked increase in expression of nestin, glial fibrillary acidic protein and glutamine synthetase as compared with untreated/normal retina. Double labelling study revealed that astrocytes and Müller glial cells expressed the radial glia marker nestin, and incorporated bromodeoxyuridine to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Indeed, with the acquisition of an immature molecular profile as manifested by the induced expression of brain lipid-binding protein and doublecortin in astrocytes and Müller glial cells, the potential of these cells to de-differentiate in retinal neurodegeneration is greatly amplified.     

Na+,K+-activated adenosine triphosphatase of isolated Müller cells from the rabbit retina shows a K+ dependence similar to that of brain astrocytes

Müller (glial) cells from the rabbit retina were isolated by means of papain and mechanical dissociation. Their Na+,K+-adenosine triphosphatase (ATPase) activity was measured using a radiochemical method, and its K+ dependence was determined. In contrast to that of photoreceptors (data from the literature), the Na+,K+-ATPase of Müller cells could be shown to increase its activity greatly when the [K+] was enhanced up to 10 mM. The functional implications of this behaviour for the K+ clearance in the retina are discussed.     

斑马鱼视网膜再生研究进展

哺乳动物视网膜损伤后仅有非常有限的自我修复能力。与哺乳动物不同,硬骨鱼类如斑马鱼的视网膜则具有旺盛的再生能力。斑马鱼视网膜损伤后,能够再生其丢失的所有类型的神经元和胶质细胞,并恢复大部分视力。研究表明,斑马鱼视网膜再生的细胞来源是Müller细胞。近年来,关于斑马鱼视网膜Müller细胞去分化及其增殖的分子机制和信号通路的研究取得了许多新的进展。我们就斑马鱼视网膜再生的研究历史、损伤模型和再生细胞来源以及调控的分子机制等做一综述。     

Heterogeneity of Müller cell endfeet in the rabbit retina as revealed by freeze-fracturing

The Müller cell membranes of the rabbit retina were investigated by means of freeze-fracturing. Orthogonal arrays of particles (OAP) were found almost exclusively where the Müller cell endfoot had direct contact with the vitreous body. In the center of the retina where the Müller cells are long and thin, the density of OAP was about 170/microns2. In the periphery where the Müller cells are shorter and thicker, the density of OAP was about 20/microns2. In this area, only half of the Müller cell endfeet were equipped with OAP. The results are discussed with respect to electrophysiological studies on the role of various K+ channels in glial cells. It is proposed that OAP might possibly represent specialized 'maxi'-K+ channels which are involved in K+ spatial buffering of the extracellular space.