Müller cells in vascular and avascular retinae: a survey of seven mammals.

Eight monoclonal antibodies were used to label Müller cells in four mammals that have vascular retinae (cats, dogs, humans, and rats) and in three with avascular retinae (echidnas, guinea pigs, and rabbits). Müller cells were found to have a fairly uniform retinal distribution in seven species, with a mean density of 8,000-13,000 cells mm-2. Müller cells in avascular retinae differ from their vascular counterparts in four respects. First, they are shorter than those in vascular retinae. This difference is mainly due to a reduction in the thickness of the outer nuclear layer. Second, the trunks of Müller cells in avascular retinae tend to be thicker, although those in echidnas are an exception to this trend. Third, Müller cell rootlets in avascular retinae follow a more tortuous course than those in vascular retinae, reflecting the fact that photoreceptor nuclei in the two types of retina have different shapes and stacking patterns. Fourth, due to a reduction in the density of photoreceptors in avascular retinae, there are fewer neurones per Müller cell. Although these four features may enable Müller cells to assist the nutrition of neurones in the inner layers of avascular retinae, they are unlikely to be morphological specializations that have evolved for that purpose. Rather, these features appear to be a direct consequence of the fact that avascular retinae are thinner and have a differently organised outer nuclear layer. These features aside, Müller cells in avascular retinae closely resemble their counterparts in vascular retinae.     

Electrophysiological properties of retinal Müller glial cells from myelin mutant rat

The structural and functional similarities between Müller cells and oligodendrocytes prompted the present study of the electrophysiological properties of Müller (glia) cells obtained from the retinae of control and myelin mutant taiep rats during the postnatal developmental period (P12-P180). The whole-cell configuration of the patch-clamp technique was used to characterize the general properties and the K+ currents from dissociated Müller cells. During the first 3 weeks of life, a decrease of the membrane resistance and an increase of the membrane potential were observed in Müller cells from both control and taiep rats. However, Müller cells from taiep rats never achieved the very negative membrane potential (-50 mV vs -80 mV) and the low membrane resistance characteristic for control cells. Furthermore, Müller cells displayed increased inward and outward K+ currents during postnatal development up to P30/60 in controls; however, in taiep rats, this increase ceased at P20/30, and low-amplitude currents persisted into adulthood. These results provide first evidence of physiological changes in retinal Müller cells as a consequence of a myelin mutation causing a progressive deterioration of the central nervous system (CNS) due to a disturbance of the microtubule network of oligodendrocytes. We hypothesize that the progressive dysmyelination process of the optic nerve, accompanied by functional deficits of retinal neurons (e.g., ganglion cells), induces physiological alterations of Müller cells.     

Ocular inflammation alters swelling and membrane characteristics of rat Müller glial cells

Ocular inflammation is a common cause of retinal edema that may involve swelling of Müller glial cells. In order to investigate whether endotoxin-induced ocular inflammation in rats alters the swelling and membrane characteristics of Müller cells, lipopolysaccharide (LPS; 0.5%) was intravitreally injected. At 3 and 7 days after treatment, hypotonic challenge induced swelling of Müller cell somata that was not observed in non-treated control eyes. Müller cells of LPS-treated eyes displayed a downregulation of inward K(+) currents and upregulation of A-type K(+) currents that was associated with a decreased expression of Kir4.1 protein in retinal slices. The data suggest that ocular inflammation induces alterations of both the swelling characteristics and the K(+) channel expression of Müller cells.     

Electrophysiological alterations and upregulation of ATP receptors in retinal glial Müller cells from rats infected with the Borna disease virus

Infection with the neurotropic Borna disease virus (BDV) causes an immune-mediated neurological disease in a broad range of species. In addition to encephalitis, BDV-infected Lewis rats develop a retinitis histologically characterized by the loss of most retinal neurons. By contrast, the dominating retinal macroglia, the Müller cells, do not degenerate. It is known from several models of neurodegeneration that glial cells may survive but undergo significant alterations of their physiological parameters. This prompted us to study the electrophysiology and ATP-induced changes of intracellular Ca(2+)-concentration ([Ca(2+)](i)) in Müller cells from BDV-infected rat retinae. Freshly isolated cells were used for whole-cell patch-clamp recordings. Whereas neither zero current potentials nor membrane resistances showed significant alterations, the membrane capacitance increased in cells from BDV-infected rats during survival times of up to 8 months. This process was accompanied by a decrease in K(+) current densities. Müller cells from BDV-infected rats were characterized by expression of a prominent fast-inactivating A-type K(+) current which was rarely found in control cells. Moreover, the number of cells displaying Na(+) currents was slightly increased after BDV-infection. ATP evoked increases in [Ca(2+)](i) in Müller cells within retinal wholemounts of both control and BDV-infected animals. However, the number of ATP-responding isolated cells increased from 24% (age-matched controls) to 78% (cells from animals > or =18 weeks after infection). We conclude that in BDV-induced retinopathy, reactive rat Müller cells change their physiological parameters but these changes are different from those in Müller cells during proliferative vitreoretinopathy in man and rabbit.     

Phagocytosis of latex beads by rabbit retinal Müller (glial) cells in vitro.

The ability of rabbit retinal Müller (glial) cells to perform phagocytosis was studied in vitro. Müller cells were feed with various kinds of latex beads either shortly after enzymatical isolation from adult retinae or in monolayer cell cultures derived from neonatal retinae and kept 14 days in vitro. Both types of Müller cell preparations showed intense phagocytosis of latex beads. Moreover, when entire retinae were isolated and exposed (sclerad side up) to latex beads in vitro for 30 min, Müller cells had picked up fluorescent beads and showed, after fixation, intense labeling in radial sections of such retinae. Effective phagocytosis by Müller cells was demonstrated 1.) by transmission electron microscopy, 2.) by bright-field light microscopy of unstained large beads (diameter 660 nm), or 3.) by fluorescence microscopy of small (diameter about 60 nm) and large latex beads labeled with rhodamine. These results suggest that both labeled and unlabeled latex beads are suitable tools to study the phagocytotic activity of retinal glial cells in vitro, thus providing information on important processes occurring in situ during ontogenesis, physiological renewal of retinal receptor cells, and pathological events. We found that movements of cells or cytoplasmic excrescences, and cell-cell interactions, play important roles in removal of foreign particles out of the fluid environment. Engulfed latex beads move through the elongated cells with velocities similar to slow axoplasmic transport.     

Development of the rabbit retina: II. Müller cells

Müller (glial) cells of the rabbit retina were stained with antibodies against the intermediate filament protein vimentin in retinal wholemounts from various developmental stages. Both the density of stained profiles and the mean diameter of these profiles were measured, with the microscope focus in the inner plexiform layer of the retinae. Within this retinal layer, every Müller cell possesses one stout vitread process; thus counts of the stained profiles allow an estimation of their number. After postnatal day (P) 9, the total number of stained cells was slightly above 4 million per retina; for the adult rabbit retina, this agrees well with earlier data obtained by our group based on another method, as well as with published data from other groups. We suggest that after P 9, only Müller cells are stained, and this population is numerically stable. In contrast, neonatal retinae contained significantly more stained profiles. This indicates that either the total number of Müller cells is reduced by "physiological cell death" or that additional cells are stained neonatally. We discuss why we favour the second possibility. After P 9, two peculiarities occur in the Müller cell population: (1) their density decreases gradually, to a greater extent in the retinal periphery than in the center (i.e., in the "visual streak"), and (2) Müller cell diameters increase, again more in the periphery than in the center. We argue that differential retinal expansion leads to dispersion of the pre-existing cell population and allows for widening of the Müller cell processes. We conclude that Müller cells can be used postnatally in the rabbit retina as "landmarks" of expansion.     

The effects of gabaculine in vivo on the distribution of GABA-like immunoreactivity in the rat retina

gamma-Aminobutyric acid (GABA) is an inhibitory transmitter found in the retinae of mammals largely within certain amacrine cells. In previous studies from this laboratory, subcutaneous administration to rats of gabaculine, an enzyme-activated irreversible inhibitor of gamma-aminobutyric acid (GABA)-transaminase, produced large, rapid and long-lasting increases in levels of retinal GABA. We employed immunocytochemistry to determine whether such changes in the levels of retinal GABA are accompanied by changes in the cellular distribution of GABA. Using a recently developed antiserum to a GABA-protein conjugate, and the peroxidase-antiperoxidase method, we examined retinae from control rats and from rats 2 or 8 h after administration of 10 mg/kg gabaculine. From previous work, retinal levels of GABA were respectively elevated 3- or 6-fold at those postgabaculine times. In the present study, marked changes in the distribution of GABA-like immunoreactivity (GABA-LIR) were apparent by 2 h after injection of gabaculine, and were more striking at 8 h postgabaculine. The pattern of staining for GABA-LIR strongly suggested that much of the GABA in gabaculine-treated retinae was within Müller glial cells. That observation provides evidence for the importance of those cells in the uptake and degradation of GABA after its release from retinal neurons.     

Müller (glial) cell development in vivo and in retinal explant cultures: morphology and electrophysiology, and the effects of elevated ammonia

Retinal explant cultures have been established as a useful tool to study both the normal development of the mammalian retina and the effects of pathogenic agents. We used such cultures as a model for the (ammonia-induced) hepatic retinopathy, earlier observed in humans with chronical liver failure, and ascribed to a breakdown of Müller (glial) cell function. In the explant cultures, one day exposure to elevated (7 mM) ammonia was sufficient to cause Müller cell reactivity as indicated by increasing immunopositivity for glial fibrillary acidic protein. After 4 days in elevated ammonia, the Müller cells were severely deformed, the layered structure of the retinae became disorganized, and significant neuronal cell death occurred. Using whole-cell voltage-clamp recordings, the expression of K+ channels was compared in Müller cells isolated from retinae of rabbits at postnatal days 9 to 12 and from neonatal explants cultured for 9 to 12 days, respectively. Müller glial cells grown both in vivo and in vitro express the same set of K+ channels in their membranes: (i) inwardly rectifying K+ (K(IR)) channels which were selectively blocked by Ba2+ ions; (ii) large-conductance, Ca2+-activated K+ (BK(Ca)) channels which were blocked by iberiotoxin and were activated by phloretin; and (iii) delayed rectifying voltage-gated K+ channels. The presence of K(IR) channels indicates successful differentiation of the Müller cells grown in vitro, as these channels are not expressed in cells from neonatal animals. Four days of elevated ammonia in the culture medium caused a complete loss of K(IR) channels in Müller cell membranes, and a significant decrease of the membrane potential. The results indicate that in hepatic retinopathy, the well-known morphological and enzymatical alterations of Müller glial cells may be accompanied by changes in their membrane permeability for K+.     

Müller cells in adult rabbit retinae: morphology, distribution and implications for function and development

We describe the morphology and distribution of Müller cells in wholemounts of rabbit retinae labelled with either monoclonal antibodies (anti-Vimentin, 3H3, 4D6, and 4H11), or intracellular horseradish peroxidase. Several new features of Müller cell organization are noted. First, Müller cells appear to compose a single morphological class and their morphology varies systematically with retinal thickness. Second, in contrast to other retinal glia, Müller cells have a neuronlike distribution, with a peak density of 10,700-15,000 cells per mm2 at the visual streak and a minimum density of 4,400-6,000 per mm2 at both the superior and inferior retinal edges. There are 4.2 +/- 0.5 x 10(6) Müller cells per retina. Third, unlike in other species, rabbit Müller cells do not contact blood vessels, suggesting that they do not participate in the transfer of metabolites or in the blood:retinal barrier. Fourth, each Müller cell has a vitread endfoot about 20-40 microns in diameter composed of numerous fimbriae. The fimbriae from a single Müller cell generally contact several axon fascicles in the nerve fibre layer, and at each point along its length each fascicle is enclosed by the overlapping fimbriae from several Müller cells. Fifth, in the inner and outer plexiform layers, numerous filamentous branchlets extend 20 microns or more from the radial trunk, interweaving with branchlets from nearby Müller cells to form dense and continuous strata. In the ganglion cell layer and outer nuclear layer, Müller cell processes completely wrap neuronal somata, whereas in the inner nuclear layer they partially wrap somata. We discuss the functional and developmental implications of these observations.     

Adult rat retinal glia in vitro: effects of in vivo crush-activation on glia proliferation and permissiveness for regenerating retinal ganglion cell axons

The effects of optic nerve crush on adult rat retinal glia activation were studied in vitro. In adult rats the optic nerves were crushed and the corresponding retinae were explained 5 to 7 days later and cultured in vitro. The glial response of retinae with precrushed optic nerves was compared to the glial response of retinae without prior optic nerve crush. As a consequence of crush-axotomy more glial cells migrated out from retinal explants and covered significantly larger areas of the substratum than glia from noncrushed retinae. Migration of immunohistochemically distinguishable Vimentin-positive Müller cells and glial fibrillary acidic protein-positive astrocytes could be observed in both types of cultures. Astrocytes as well as Müller cells incorporated bromodeoxyuridine after explantation. In noncrushed retinal explants Thy 1.1-immunopositive flat cells were much more frequent and the relative proportion of glial cells was much lower than in crush-activated cultures. In a second set of experiments the ability of adult rat retinal glia to support retinal ganglion cell regeneration was examined. Normal retinal explants (without optic nerve crush) which usually do not substantially regenerate axons were cultured on retinal glia from normal and crush-activated explants. Both glia preparations supported axon growth from retinal explants after 3 days in vitro. Neuritic growth was significantly better when retinal explants from normal adult rats were cultured on crush-activated retinal glia as compared to glia derived from noncrushed retinae. It is concluded that activated adult rat retinal glia, unlike adult glia found in other brain regions, support adult rat retinal ganglion cell regeneration in vitro.     

Electrophysiological properties of rat retinal Müller (glial) cells in postnatally developing and in pathologically altered retinae

Retinal glial Müller cells are characterized by dominant K(+) conductances. The cells may undergo changes of their membrane currents during ontogeny and gliosis as described in rabbit and man. Although the rat retina is often used in physiological experiments, the electrophysiology of rat Müller cells is less well studied. The aim of the present study was to characterize their membrane currents in postnatal development and in two models of retinal degeneration. Freshly isolated cells were subjected to whole-cell patch clamp recordings. During the first 4 weeks after birth of rats, their Müller cells displayed an increase in all membrane currents, particularly in the inward currents elicited at hyperpolarizing potentials. The decrease of the membrane resistance from more than 760 MOmega to less than 50 MOmega was accompanied by a shift of the zero current potential from about -20 mV to -80 mV, similar as earlier observed in developing rabbit Müller cells. These developmental changes were found in pigmented Brown Norway rats as well as in rats with inherited retinal dystrophy (RCS rats). Moreover, an infection of Lewis rats with the Borna disease virus caused substantial neuroretinal degeneration but did not result in a strong reduction of inward currents and of the zero current potential of the Müller cells. Thus, rat Müller cells fail to change their basic membrane properties in two different models of retinal pathology. This is in contrast to human and rabbit Müller cells, which have been shown to undergo dramatic changes of their membrane physiology in response to retinal diseases and injuries.     

VEGF release by retinal glia depends on both oxygen and glucose supply

Isolated retinae or isolated Müller cells were cultured in vitro, and vascular endothelial growth factor (VEGF) was assayed as protein (by ELISA) and as mRNA (by semi-quantitative RT-PCR). In both types of cultures, hypoxia (5% O2) resulted in an upregulated VEGF release. While the unstimulated VEGF secretion was virtually independent of glucose (0.125 - 25 mM), elevated glucose concentrations (10 - 25 mM) blocked most of the stimulatory effect of hypoxia on VEGF mRNA synthesis (determined in Müller cell cultures) as well as on VEGF release (in both retina and Müller cell cultures). It is concluded that in retinal glial (Müller) cells, being responsible for retinal VEGF synthesis (and, thus, for undesirable neovascularization), the metabolic effects of hypoxia can be compensated by a surplus of glucose.     

Müller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina

We characterized morphological effects of the endogenous excitotoxin, glutamate in ex vivo retinal segments prepared from 30-day-old rats. Initial changes induced by glutamate consisted of reversible, sodium-dependent Müller cell swelling. This glial swelling was mimicked by glutamate transport substrates but not by ionotropic glutamate receptor agonists. Only very high concentrations of exogenous glutamate (3,000 microM) produced excitotoxic neuronal damage. The neuronal damage was accompanied by severe glial swelling and was blocked by an antagonist of non-N-methyl-D-aspartate (NMDA) receptors but not by an NMDA receptor antagonist. Because glutamate uptake can be influenced by changes in cellular energy levels, we studied the effects of oxidative and glycolytic energy depletion on glutamate-mediated Müller cell swelling. Oxygen deprivation produced little morphological change and did not alter either glutamate-mediated Müller cell swelling or glutamate-induced excitotoxicity. In contrast, inhibition of glycolysis by iodoacetate produced severe neuronal damage without Müller cell swelling. In the presence of iodoacetate, exogenous glutamate failed to cause glial swelling. The neuronal damage produced by iodoacetate was inhibited by pyruvate, a substrate that sustains oxidative energy pathways. In the presence of iodoacetate plus pyruvate, glutamate failed to cause Müller cell swelling but became neurotoxic at low concentrations through activation of non-NMDA receptors. These results indicate that glycolytic energy metabolism plays a critical role in sustaining ionic balances required for Müller cell glutamate uptake and glial uptake helps to prevent glutamate-mediated excitotoxicity.     

Response of Müller cells to growth factors alters with time in culture.

We have developed an explant culture technique, using the retinae of newborn guinea pigs, that reliably yields cultures of Müller cells showing uniform morphology and phenotype. Since the guinea pig retina is avascular and lacks astrocytes, Müller cells are the only glial cell-type and the only vimentin-positive population present. Virtually all passaged cells (greater than 98%) contain vimentin-positive intermediate filaments and no glial fibrillary acidic protein (GFAP) has been detected using a range of GFAP antibodies known to label astrocytes in the guinea pig optic nerve. Most vimentin-positive cells were also labeled with an antibody to carbonic anhydrase II, an enzyme which in the retina is specific for Müller cells. Proliferating Müller cells were identified within the inner nuclear layer of retinal fragments as early as 2 days in culture using bromodeoxyuridine (BrdU) and vimentin double labeling. Cultured Müller cells change their growth characteristics with successive passaging. The length of the cell cycle increases from 25.4 h for cells at first passage, to 66.7 h for cells at fourth passage. Altered responses to mitogens were also observed with passaging. First-passage cultures responded to basic fibroblast growth factor (bFGF) but not to several other factors tested including interleukin-2 (IL-2). In contrast, older cultures were highly responsive to IL-2 but showed a minimal response to bFGF. The altered responsiveness to mitogens observed in vitro may be relevant to changes in growth control of Müller cells in the developing and mature retina. The guinea pig retina provides an ideal mammalian tissue for generating Müller cell cultures that are free of astrocytes, endothelial cells, and pericytes, the most frequent contaminants of retinal glial cultures. The monolayers obtained show a high degree of homogeneity and are well suited for studies of Müller cell function.     

Retinal regions shape human and murine Müller cell proteome profile and functionality

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.     

Quantitative and qualitative morphology of rabbit retinal glia. A light microscopical study on cells both in situ and isolated by papaine

Rabbit retinal glia was studied by light microscopy of both stained sections of frozen retinae and enzymatically isolated cells. In the vast majority of this tissue, except for a small region around the optic nerve head, the glia consists solely of radial glia, i.e. Müller cells whose morphology was found to depend markedly on their topographic localization within the retina. Müller cells in the periphery are short and have thick vitreal processes bearing a single large endfoot. Central Müller cells are long and slender; through the thickening nerve fibre layer they send vitreal processes which are subdivided into several fine branches ending with multiple small endfeet. Müller cells in the retinal centre are far more closely packed than those in the periphery; everywhere, however, a constant ratio of Müller cells: neurons of about 1:15 was found, except for the juxta-optic nerve head region where this ratio is slightly reduced. Where the central retina reaches a thickness requiring Müller cell lengths of more than 130 micron, additional non-radial glial cells occur within the nerve fibre layer. The majority of these cells seem to be astrocytes. Their number per retinal area increases with the thickening of both the whole retina and the nerve fibre layer. The occurrence of these non-radial glial cells leads to an enhancement of the glia:neuron index in the retinal centre. Possible mechanisms of physiological control of gliogenesis are discussed.     

ATP-evoked calcium responses of radial glial (Müller) cells in the postnatal rabbit retina

Here we show that rabbit Müller cell differentiation from radial glial progenitor cells is accompanied by a decreasing capability to respond to specific stimuli (depolarization and extracellular adenosine 5'-triphosphate [ATP]) with an elevation of intracellular calcium. Intracellular free calcium was recorded in retinal wholemounts from young (postnatal days [P] 2 to 31) and adult rabbits. Images were taken from the nerve fiber/ganglion cell layers where the endfeet of radial glial/ Müller cells can be identified after selective uptake of calcium-sensitive dyes. The area of responding endfeet was determined as the percentage of the total area occupied by Müller cell endfeet, as an estimate of the percentage of responding cells. In response to depolarization (50 mM potassium), an increase of intracellular free calcium occurred in 19% of cells from young postnatal retinae (P2-31) but only in 2% from adults. This depolarization-induced calcium rise was caused both by a calcium influx from extracellular space and by an intracellular calcium release. The latter response was inhibited by the P2 receptor blocker pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), indicating that extracellular calcium-independent ATP release into the extracellular space occurs during retinal depolarization. When extracellular ATP (200 microM) was applied, calcium responses were recorded in 83% of cells from young postnatal retinae (P2-6); in the course of further development, both the percentage of responding cells (7% in retinae from adult rabbits) and the amplitude of the calcium responses decreased. It is concluded that during the differentiation of immature radial glia into mature Müller cells, stimulus-evoked intracellular calcium signaling mechanisms change.     

Diversity of Kir channel subunit mRNA expressed by retinal glial cells of the guinea-pig

One of the main functions of Müller glial cells is the performance of retinal K+ homeostasis which is thought to be primarily mediated by K+ fluxes through inwardly rectifying K+ (Kir) channels expressed in Müller cell membranes. Until now, there is limited knowledge about the types of Kir channel subunits expressed by Müller cells. Using RT-PCR, we investigated the expression of mRNA encoding different Kir channel subunits in the retina of the guinea pig. In order to verify expression by Müller cells, primary cultures of guinea pig Müller cells were also investigated. Both retinae and cultured Müller cells express mRNA for a diversity of Kir channel subtypes which include members of at least four channel subfamilies: Kir2.1, Kir2.2, Kir2.4, Kir3.1, Kir 3.2, Kir4.1, Kir6.1, and Kir6.2. mRNAs for the following Kir channel subtypes were not detected in Müller cells: Kir1.1, Kir2.3, Kir3.3, Kir3.4, Kir4.2, and Kir5.1. It is concluded that the spatial buffering of extracellular K+ by Müller cells may be mediated by cooperation of different subtypes of Kir channels, and that the distinct Kir channel types involved in this function may change depending on the physiological or metabolic state of the retina.     

Ultrastructure of rabbit retinal nerve fibre layer--neuro-glial relationships, myelination, and nerve fibre spectrum

The ultrastructure of the rabbit retinal nerve fibre layer was studied both in retinal centre and periphery. The central nerve fibre layer was found to contain large masses of--mostly myelinated--nerve fibres, somata and processes of astrocytes and oligodendrocytes, vitreal processes of Müller cells, and blood vessels. Astrocyte and Müller cell processes could be discriminated both by their direction and by the thickness of their intermediate filaments which was about 7 nm in Müller cells and about 10 nm in astrocytes. Some peculiarities of nuclei and cytoplasmic organelles of rabbit retinal astrocytes and oligodendrocytes are described. Myelin sheaths are demonstrated to be derived from oligodendrocytes; in some cases, two axons were found within a common myelin sheath. In the retinal periphery, only sparse thin bundles of unmyelinated axons were found in between a thick row of big Müller cell endfeet; astrocytes, oligodendrocytes, and blood vessels were missing here. In both retinal regions, node-like membrane specializations of optic axons were found; these were always surrounded by a corona of fine glial processes arising from astrocytes as well as from Müller cells. The features of myelination within the rabbit nerve fibre layer were quantified, and compared with recent literature data. A hypothesis is offered relating the production of myelin to the release of diffusable substance(s) by active axons. This hypotheses allows to account for the striking finding that relatively thick axons remain unmyelinated in the nerve fibre layer of most mammalian retinae like in the rabbit retinal periphery whereas they become myelinated in the central rabbit retina like in central nervous system in general.     

Müller cell changes precede vascularization of the pigment epithelium in the dystrophic rat retina

In the Royal College of Surgeons rat with inherited retinal dystrophy, photoreceptor cell degeneration is accompanied by retinal pigment epithelial (RPE) cell alterations and Müller cell changes such as increased expression of glial fibrillary acidic protein (GFAP). Vascular changes such as vascularization of the RPE, vascular proliferation, and formation of vitreoretinal membranes (VRMs) are observed later. To study the relationship of Müller cell changes to the vascular alterations in the dystrophic retina, we used immunoperoxidase techniques and antibodies against GFAP and vimentin. Our study showed that during photoreceptor degeneration, Müller cells expressed small amounts of GFAP. As degeneration progressed, GFAP expression increased and morphological alterations occurred in Müller cells. Müller cell apical processes extended and proliferated in the subretinal space and contacted the apical surface of duplicated RPE cells. Later, GFAP reactive fibers surrounded retinal vessels apposed to the RPE. As the vessels became enmeshed within the RPE, the GFAP-positive perivascular processes disappeared. Eventually, the RPE-associated vessels became displaced into the inner retina where VRMs were sometimes observed. Immunoblots showed increased GFAP in dystrophic as compared with control retinas. Studies of vimentin distribution in the dystrophic retina showed results similar to the GFAP study. Moreover, the vimentin study suggested increased number of Müller cell processes in the dystrophic as compared with control retinas. The close temporal and anatomical relationships among Müller cell, RPE, and vascular changes in the dystrophic rat suggest a role for Müller cells in retinal neovascularization and proliferative retinopathy.