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The human retina contains three types of glial cells: microglia and two types of macroglia, astrocytes and Müller cells. Macroglia provide homeostatic and metabolic support to photoreceptors and neurons required for neuronal activity. The fovea, the site of the sharpest vision which is astrocyte- and microglia-free, contains two populations of Müller glia: cells which form the Müller cell cone in the foveola and z-shaped Müller cells of the foveal walls. Both populations are characterized by morphological and functional differences. Müller cells of the foveola do not support the activity of photoreceptors and neurons, but provide the structural stability of the foveal tissue and improve the light transmission through the tissue to the photoreceptors. This article gives overviews of the glia of the human retina and the structure and function of both Müller cell types in the fovea, and describes the contributions of astrocytes and Müller cells to the ontogenetic development of the fovea.

To further extent our understanding of aquatic vision, we introduce a complete optical model of a goldfish eye, which comprises all important optical parameters for the first time. Especially a spherical gradient index structure for the crystalline lens was included, thus allowing a detailed analysis of image quality, regarding spot size, and wavelength dependent aberration. The simulation results show, that our realistic eye model generates a sufficient image quality, with a spot radius of 4.9 mm which is below the inter cone distance of 5.5 mm. Furthermore, we optically simulate potential mechanical processes of accommodation and compare the results with contradictory findings of previous experimental studies. The quantitative simulation of the accommodation capacity shows that the depth of field is strongly dependent on the resting position and becomes significantly smaller when shorter resting positions are assumed. That means, to enable an extended depth perception with high acuity for the goldfish an adaptive, lens shifting mechanism would be required. In addition, our model allows a clear prediction of the expected axial lens-shift, which is necessary to ensure a sufficient resolution over a large object range.

Ca2+ -binding proteins are differentially expressed in the nervous system; their functional role often remains unclear. This immunohistochemical study aimed at characterising and comparing the expression pattern of the Ca2+ -binding proteins calbindin (Calb), calretinin (Calr) and parvalbumin (Parv) in the retina of four species of macaque monkeys: Macaca fascicularis (cynomolgus macaque), M. mulatta (rhesus macaque), M. thibetana (Tibetan macaque) and M. fuscata (Japanese macaque). Calb was found in cone photoreceptors and in a subset of bipolar cells. Calr was expressed in a subpopulation of amacrine cells. Parv was present in horizontal and ganglion cells. In addition, Muller cells were stained using antibodies against the specific marker cellular retinaldehyde-binding protein (CRALBP). Immunostainings were used for calculation of the density of different cell populations. The expression pattern was similar between the examined species and between retinal regions.

It has been shown that mammalian retinal glial (Müller) cells act as living optical fibers that guide the light through the retinal tissue to the photoreceptor cells (Agte et al., 2011; Franze et al., 2007). However, for nonmammalian species it is unclear whether Müller cells also improve the transretinal light transmission. Furthermore, for nonmammalian species there is a lack of ultrastructural data of the retinal cells, which, in general, delivers fundamental information of the retinal function, i.e. the vision of the species. A detailed study of the cellular ultrastructure provides a basic approach of the research. Thus, the aim of the present study was to investigate the retina of the spectacled caimans at electron and light microscopical levels to describe the structural features. For electron microscopy, we used a superfast microwave fixation procedure in order to achieve more precise ultrastructural information than common fixation techniques. As result, our detailed ultrastructural study of all retinal parts shows structural features which strongly indicate that the caiman retina is adapted to dim light and night vision. Various structural characteristics of Müller cells suppose that the Müller cell may increase the light intensity along the path of light through the neuroretina and, thus, increase the sensitivity of the scotopic vision of spectacled caimans. Müller cells traverse the whole thickness of the neuroretina and thus may guide the light from the inner retinal surface to the photoreceptor cell perikarya and the Müller cell microvilli between the photoreceptor segments. Thick Müller cell trunks/processes traverse the layers which contain light-scattering structures, i.e., nerve fibers and synapses. Large Müller cell somata run through the inner nuclear layer and contain flattened, elongated Müller cell nuclei which are arranged along the light path and, thus, may reduce the loss of the light intensity along the retinal light path. The oblique arrangement of many Müller cell trunks/processes in the inner plexiform layer and the large Müller cell somata in the inner nuclear layer may suggest that light guidance through Müller cells increases the visual sensitivity. Furthermore, an adaptation of the caiman retina to low light levels is strongly supported by detailed ultrastructural data of other retinal parts, e.g. by (i) the presence of a guanine-based retinal tapetum, (ii) the rod dominance of the retina, (iii) the presence of photoreceptor cell nuclei, which penetrate the outer limiting membrane, (iv) the relatively low densities of photoreceptor and neuronal cells which is compensated by (v) the presence of rods with long and thick outer segments, that may increase the probability of photon absorption. According to a cell number analysis, the central and temporal areas of the dorsal tapetal retina, which supports downward prey detection in darker water, are the sites of the highest diurnal contrast/color vision, i.e. cone vision and of the highest retinal light sensitivity, i.e. rod vision.

Background/ Aims: This study was performed to reveal signaling pathways exploited by pigment epithelium-derived factor (PEDF) derived from retinal (glial) Müller cells to protect retinal ganglion cells (RGCs) from cell death. Methods: The survival of RGCs was determined in the presence of conditioned culture media (MCM) from or in co-cultures with Müller cells. The significance of PEDF-induced STAT3 activation was evaluated in viability assays and using Western blotting analyses and siRNA-transfected cells. Results: Secreted mediators of Müller cells increased survival of RGCs under normoxia or hypoxia to a similar degree as of PEDF- or IL-6-exposed cells. PEDF and MCM induced an increased STAT3 activation in RGCs and R28 cells, and neutralization of PEDF in MCM attenuated STAT3 activation. Inhibition of STAT3 reduced PEDF-promoted survival of RGCs. Similar to IL-6, PEDF induced STAT3 activation, acting in a dose-dependent manner via the PEDF receptor (PEDF-R) encoded by the PNPLA2 gene. Ablation of PEDF-R attenuated MCM-induced STAT3 activation and compromised the viability of PEDF-exposed R28 cells. Conclusions: Müller cells are an important source of PEDF, which promotes RGC survival through STAT3 activation and, at least in part, via PEDF-R. Enhancing the secretory function of Müller cells may be useful to promote RGC survival in retinal neurodegenerative diseases.

Elephants are precocial mammals that are relatively mature as newborns and mobile shortly after birth. To determine whether the retina of newborn elephants is capable of supporting the mobility of elephant calves, we compared the retinal structures of 2 newborn elephants (1 African and 1 Asian) and 2 adult animals of both species by immunohistochemical and morphometric methods. For the first time, we present here a comprehensive qualitative and quantitative characterization of the cellular composition of the newborn and the adult retinas of 2 elephant species. We found that the retina of elephants is relatively mature at birth. All retinal layers were well discernible, and various retinal cell types were detected in the newborns, including Müller glial cells (expressing glutamine synthetase and cellular retinal binding protein; CRALBP), cone photoreceptors (expressing S-opsin or M/L-opsin), protein kinase Cα-expressing bipolar cells, tyrosine hydroxylase-, choline acetyltransferase (ChAT)-, calbindin-, and calretinin-expressing amacrine cells, and calbindin-expressing horizontal cells. The retina of newborn elephants contains discrete horizontal cells which coexpress ChAT, calbindin, and calretinin. While the overall structure of the retina is very similar between newborn and adult elephants, various parameters change after birth. The postnatal thickening of the retinal ganglion cell axons and the increase in ganglion cell soma size are explained by the increase in body size after birth, and the decreases in the densities of neuronal and glial cells are explained by the postnatal expansion of the retinal surface area. The expression of glutamine synthetase and CRALBP in the Müller cells of newborn elephants suggests that the cells are already capable of supporting the activities of photoreceptors and neurons. As a peculiarity, the elephant retina contains both normally located and displaced giant ganglion cells, with single cells reaching a diameter of more than 50 µm in adults and therefore being almost in the range of giant retinal ganglion cells found in aquatic mammals. Some of these ganglion cells are displaced into the inner nuclear layer, a unique feature of terrestrial mammals. For the first time, we describe here the occurrence of many bistratified rod bipolar cells in the elephant retina. These bistratified bipolar cells may improve nocturnal contrast perception in elephants given their arrhythmic lifestyle.

Muller cells are the dominant macroglial cells in the retina of all vertebrates. They fulfill a variety of functions important for retinal physiology, among them spatial buffering of K+ ions and uptake of glutamate and other neurotransmitters. To this end, Muller cells express inwardly rectifying K+ channels and electrogenic glutamate transporters. Moreover, a lot of voltage- and ligand-gated ion channels, aquaporin water channels, and electrogenic transporters are expressed in Muller cells, some of them in a species-specific manner. For example, voltage-dependent Na+ channels are found exclusively in some but not all mammalian species. Whereas a lot of data exist from amphibians and mammals, the results from other vertebrates are sparse. It is the aim of this review to present a survey on Muller cell electrophysiology covering all classes of vertebrates. The focus is on functional studies, mainly performed using the whole-cell patch-clamp technique. However, data about the expression of membrane channels and transporters from immunohistochemistry are also included. Possible functional roles of membrane channels and transporters are discussed. Obviously, electrophysiological properties involved in the main functions of Muller cells developed early in vertebrate evolution. GLIA 2017;65:533-568.

Tractional forces or mechanical stimulation are known to induce calcium responses in retinal glial cells. The aim of the study was to determine the characteristics of calcium responses in Muller glial cells of the avascular guinea pig retina induced by focal mechanical stimulation. Freshly isolated retinal wholemounts were loaded with Mitotracker Deep Red (to fill Muller cells) and the calcium-sensitive dye Fluo-4/AM. The inner retinal surface was mechanically stimulated with a micropipette tip for 10 ms. Stimulation induced two different cytosolic calcium responses in Muller cells with different kinetics in dependence on the distance from the stimulation site. Muller cells near the stimulation site displayed an immediate and long-lasting calcium response with high amplitude. This response was mediated by calcium influx from the extracellular space likely triggered by activation of ATP-insensitive P2 receptors. More distant Muller cells displayed, with a delay of 2.4 s, transient calcium responses which propagated laterally in a wave-like fashion. Propagating calcium waves were induced by a calcium-independent release of ATP from Muller cells near the stimulation site, and were mediated by a release of calcium from internal stores triggered by ATP, acting in part at P2Y1 receptors. The data suggest that mechanically stimulated Muller cells of the guinea pig retina release ATP which induces a propagating calcium wave in surrounding Muller cells. Propagating calcium waves may be implicated in the spatial regulation of the neuronal activity and homeostatic glial functions, and may transmit gliosis-inducing signals across the retina. Mechanical stimulation of guinea pig Muller cells induces two calcium responses: an immediate response around the stimulation site and propagating calcium waves. Both responses are differentially mediated by activation of purinergic receptors. GLIA 2016 GLIA 2017;65:62-74.

Water accumulation in retinal glial (Muller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Muller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Muller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Muller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Muller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Muller cells.

Retinal glial (Müller) cells possess an endogenous purinergic signal transduction cascade which normally prevents cellular swelling in osmotic stress. The cascade can be activated by osmotic or glutamate receptor-dependent ATP release. We determined whether activation of this cascade is altered in Müller cells of transgenic rats that suffer from a slow photoreceptor degeneration due to the expression of a truncated human cilia gene polycystin-2 (CMV-PKD21/703 HA). Age-matched Sprague-Dawley rats served as control. Retinal slices were superfused with a hypoosmotic solution (60 % osmolarity). Müller cells in retinas of PKD21/703 rats swelled immediately in hypoosmotic stress; this was not observed in control retinas. Pharmacological blockade of P2Y1 or adenosine A1 receptors induced osmotic swelling of Müller cells from control rats. The swelling induced by the P2Y1 receptor antagonist was mediated by induction of oxidative-nitrosative stress, mitochondrial dysfunction, production of inflammatory lipid mediators, and a sodium influx from the extracellular space. Exogenous VEGF or glutamate prevented the hypoosmotic swelling of Müller cells from PKD21/703 rats; this effect was mediated by activation of the purinergic signaling cascade. In neuroretinas of PKD21/703 rats, the gene expression levels of P2Y1 and A1 receptors, pannexin-1, connexin 45, NTPDases 1 and 2, and various subtypes of nucleoside transporters are elevated compared to control. The data may suggest that the osmotic swelling of Müller cells from PKD21/703 rats is caused by an abrogation of the osmotic ATP release while the glutamate-induced ATP release is functional. In the normal retina, ATP release and autocrine P2Y1 receptor activation serve to inhibit the induction of oxidative-nitrosative stress, mitochondrial dysfunction, and production of inflammatory lipid mediators, which otherwise will induce a sodium influx and cytotoxic Müller cell swelling under anisoosmotic conditions. Purinergic receptors may represent a target for the protection of retinal glial cells from mitochondrial oxidative stress.

Mammalian retinal glial (Muller) cells are known to guide light through the inner retina to photoreceptors (Franze et al., 2007; Proc Natl Acad Sci U S A 104:8287-8292). It was shown that Muller cells transmit predominantly red-green and less violet-blue light (Labin et al., 2014; Nat Commun 5:4319). It is not known whether this optical function is reflected in the cone-to-Muller cell ratio. To determine this ratio in the retinas of mammals with different lifestyle, we evaluated the local densities of cones and Muller cells in the retinas of guinea pigs, rabbits, sheep, red deer, roe deer, domestic pigs, and wild boars. Retinal wholemounts were labeled with peanut agglutinin to mark cones and anti-vimentin antibodies to identify Muller cells. Wholemounts of guinea pig and rabbit retinas were also labeled with anti-S-opsin-antibodies. With the exceptions of guinea pig and pig retinas that had cone-to-Muller cell ratios of above one, the local densities of cones and Muller cells in the retinas of the species investigated were roughly equal. Because the proportion of S-cones is usually low (for example, 5.3% of all cones in the dorsal guinea pig retina expressed S-opsin), it is suggested that Muller cells are mainly coupled to M-cones. Exceptions are the ventral peripheries of guinea pig and rabbit retinas which are specialized areas with high S-cone densities. Here, up to 50% of Muller cells may be coupled to S-cones, and 40% of S-cones may be not coupled to Muller cells. Among the species investigated, the density of Muller cells in the central retina was inversely correlated with the axial length of the eyes. It is suggested that (with the exception of specialized S-cone areas) Muller cells support high acuity vision by predominant guidance of red-green light to M-opsin expressing cones.

Leukemia inhibitory factor (LIF) is a cytokine that exerts different effects in the nervous system. It is involved in neuronal injuries and diseases and is assumed to be neuroprotective and to regulate reactive gliosis. In LIF-deficient (LIF-/-) mice, expression of glial fibrillary acidic protein in retinal Muller glial cells as a hallmark of reactive gliosis is suppressed during retinal degenerations. Here, we detected expression of LIF and its receptors in Muller cells of the murine retina. Moreover, electrophysiological alterations of Muller cells 7 days after transient retinal ischemia were studied by the patch-clamp technique. The amplitude of inward currents in Muller cells from the postischemic retina was reduced to 51% in wild type and to 70% in LIF-/- mice. This demonstrates that decrease of inward currents takes place in reactive Muller cells even in the absence of LIF.

Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition.The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging.Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks.SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Several scleral cross-linking (SXL) methods were suggested to increase the biomechanical stiffness of scleral tissue and therefore, to inhibit axial eye elongation in progressive myopia. In addition to scleral cross-linking and biomechanical effects caused by riboflavin and light irradiation such a treatment might induce tissue damage, dependent on the light intensity used. Therefore, we characterized the damage threshold and mechanical stiffening effect in rabbit eyes after application of riboflavin combined with various blue light intensities. Adult pigmented and albino rabbits were treated with riboflavin (0.5 %) and varying blue light (450 ± 50 nm) dosages from 18 to 780 J/cm2 (15 to 650 mW/cm2 for 20 min). Scleral, choroidal and retinal tissue alterations were detected by means of light microscopy, electron microscopy and immunohistochemistry. Biomechanical changes were measured by shear rheology. Blue light dosages of 480 J/cm2 (400 mW/cm2) and beyond induced pathological changes in ocular tissues; the damage threshold was defined by the light intensities which induced cellular degeneration and/or massive collagen structure changes. At such high dosages, we observed alterations of the collagen structure in scleral tissue, as well as pigment aggregation, internal hemorrhages, and collapsed blood vessels. Additionally, photoreceptor degenerations associated with microglia activation and macroglia cell reactivity in the retina were detected. These pathological alterations were locally restricted to the treated areas. Pigmentation of rabbit eyes did not change the damage threshold after a treatment with riboflavin and blue light but seems to influence the vulnerability for blue light irradiations. Increased biomechanical stiffness of scleral tissue could be achieved with blue light intensities below the characterized damage threshold. We conclude that riboflavin and blue light application increased the biomechanical stiffness of scleral tissue at blue light energy levels below the damage threshold. Therefore, applied blue light intensities below the characterized damage threshold might define a therapeutic blue light tolerance range.

Vimentin (Vim) and glial fibrillary acidic protein (GFAP) are important components of the intermediate filament (IF) (or nanofilament) system of astroglial cells. We conducted full-field electroretinogram (ERG) recordings and found that whereas photoreceptor responses (a-wave) were normal in uninjured GFAP(-/-)Vim(-/-) mice, b-wave amplitudes were increased. Moreover, we found that Kir (inward rectifier K(+)) channel protein expression was reduced in the retinas of GFAP(-/-)Vim(-/-) mice and that Kir-mediated current amplitudes were lower in Müller glial cells isolated from these mice. Studies have shown that the IF system, in addition, is involved in the retinal response to injury and that attenuated Müller cell reactivity and reduced photoreceptor cell loss are observed in IF-deficient mice after experimental retinal detachment. We investigated whether the lack of IF proteins would affect cell survival in a retinal ischemia-reperfusion model. We found that although cell loss was induced in both genotypes, the number of surviving cells in the inner retina was lower in IF-deficient mice. Our findings thus show that the inability to produce GFAP and Vim affects normal retinal physiology and that the effect of IF deficiency on retinal cell survival differs, depending on the underlying pathologic condition.

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Intravitreal administration of neurotrophins such as brain-derived neurotrophic factor (BDNF) is known to promote survival of retinal neurons. Here, we show that exogenous BDNF inhibits the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices or freshly isolated cells with a hypoosmotic solution containing barium ions. BDNF also inhibited the osmotic swelling of bipolar cell somata in retinal slices, but failed to inhibit the osmotic soma swelling of freshly isolated bipolar cells. The inhibitory effect of BDNF on Müller cell swelling was mediated by activation of tropomyosin-related kinase B (TrkB) and transactivation of fibroblast growth factor receptors. Exogenous basic fibroblast growth factor (bFGF) fully inhibited the osmotic swelling of Müller cell somata while it partially inhibited the osmotic swelling of bipolar cell somata. Isolated Müller cells displayed immunoreactivity of truncated TrkB, but not full-length TrkB. Isolated rod bipolar cells displayed immunoreactivities of both TrkB isoforms. Data suggest that the neuroprotective effect of exogenous BDNF in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. While BDNF directly acts on Müller cells by activation of TrkB, BDNF indirectly acts on bipolar cells by inducing glial release of factors like bFGF that inhibit bipolar cell swelling.

A fovea is a pitted invagination in the inner retinal tissue (fovea interna) that overlies an area of photoreceptors specialized for high acuity vision (fovea externa). Although the shape of the vertebrate fovea varies considerably among the species, there are two basic types. The retina of many predatory fish, reptilians, and birds possess one (or two) convexiclivate fovea(s), while the retina of higher primates contains a concaviclivate fovea. By refraction of the incoming light, the convexiclivate fovea may function as image enlarger, focus indicator, and movement detector. By centrifugal displacement of the inner retinal layers, which increases the transparency of the central foveal tissue (the foveola), the primate fovea interna improves the quality of the image received by the central photoreceptors. In this review, we summarize ‒ with the focus on Muller cells of the human and macaque fovea ‒ data regarding the structure of the primate fovea, discuss various aspects of the optical function of the fovea, and propose a model of foveal development. The "Muller cell cone" of the foveola comprises specialized Muller cells which do not support neuronal activity but may serve optical and structural functions. In addition to the "Muller cell cone", structural stabilization of the foveal morphology may be provided by the 'z-shaped' Muller cells of the fovea walls, via exerting tractional forces onto Henle fibers. The spatial distribution of glial fibrillary acidic protein may suggest that the foveola and the Henle fiber layer are subjects to mechanical stress. During development, the foveal pit is proposed to be formed by a vertical contraction of the centralmost Muller cells. After widening of the foveal pit likely mediated by retracting astrocytes, Henle fibers are formed by horizontal contraction of Muller cell processes in the outer plexiform layer and the centripetal displacement of photoreceptors. A better understanding of the molecular, cellular, and mechanical factors involved in the developmental morphogenesis and the structural stabilization of the fovea may help to explain the (patho-) genesis of foveal hypoplasia and macular holes.

Previous studies on the ultrastructure of the primate foveola suggested the presence of an inverted cone-like structure which is formed by 25-35 specialized Müller cells overlying the area of high photoreceptor density. We investigated the ultrastructure of the Müller cells in the foveola of a human and macaque retina. Sections through the posterior poles of an eye of a 40 years-old human donor and an eye of an adult cynomolgus monkey (Macaca fascicularis) were investigated with transmission electron microscopy. The foveola consisted of an inner layer (thickness, 5.5-12 μm) which mainly contained somata (including nuclei) and inner processes of Müller cells; this layer overlaid the central Henle fibers and outer nuclear layer. The inner layer contained numerous watery cysts and thin lamelliform and tubular Müller cell processes which spread along the inner limiting membrane (ILM). The cytoplasm of the outer Müller cell processes became increasingly dispersed and electron-lucent in the course towards the outer limiting membrane. The ILM of the foveola was formed by a very thin basal lamina (thickness,40 nm) while the basal lamina of the parafovea was thick (0.9-1 μm). The data show that there are various conspicuous features of foveolar Müller cells. The numerous thin Müller cell processes below the ILM may smooth the inner surface of the foveola (to minimize image distortion resulting from varying light refraction angles at an uneven retinal surface), create additional barriers to the vitreous cavity (compensating the thinness of the ILM), and provide mechanical stability to the tissue. The decreasing density of the outer process cytoplasm may support the optical function of the foveola.

A fovea, the retinal pitted invagination in the area centralis, is found in many vertebrates such as fish, reptiles, birds and in higher primates including humans. Because of the refractive index step between the retinal tissue and vitreous, the fovea defines an optically effective interface with aspherical characteristics, which influences the ray propagation to the receptor layer buried about 250 μm within the retinal layers [1]. In principle, two basic foveal shapes can be distinguished [2]. On the one hand, the convexiclivate type of fovea is a deep, funnel-shaped depression with a convex slope of the side walls and is observed in birds, some lizards, and certain deep-sea teleosts. On the other hand, the concaviclivate type of fovea is shallow and gradually shaped; the human fovea is of this type. Previous examinations on the influence of the fovea on the image-forming process have mainly been focused on the steep foveae types in which increasing magnification and directional-dependent distortion effects were predicted [3], [4], [5], [6], [7]. These investigations have been restricted to simple geometrical descriptions of the foveal shape, such as spheres and paraboloids. Real geometrical foveal structures from living creatures have not yet been examined. Moreover, the ray-trace considerations have been partly reduced to a few selected rays approaching the vitreo-retinal interface, and very limited numbers of incidence directions and wavelength-dependent effects have been investigated. Studies on the refracting characteristics of human foveae types are even more rare [2], [8]. C. Ross analyzed the optical functionality of the foveae of tarsiers, neglecting the influence of the anterior part of the eye and regarding only position shifts of three single rays. This contribution aims to answer the question on whether a comprehensive human eye model, which takes all optical components and their aberrations into account, leads to more detailed findings. In particular, field- and wavelength-dependent influences on spot sizes and positions, based on actual ray-bundle-cones are considered, which allow the estimation of aberration effects. With the current investigation, we address this issue by combining a realistic anatomical model of the anterior parts of the eye with real human fovea geometries. For the anterior eye, the human eye model of Liou and Brennan [9], with a gradient index profile of the crystalline lens, was used. To investigate the influence of actual fovea geometries on image formation, two selected and different foveal shapes (average and extraordinary), based on in vivo optical coherence tomography (OCT) measurements of a human eye were used. We analyzed the ray-optical behavior and implemented additional considerations that involve the variation of the observed field position, pupil diameter, chromatic effects, and especially the imaging behavior of peripheral colors in the receptor plane when the primary color is focused on this theoretical surface. These simulations validate and expand the knowledge about the correlation between aperture size and real and theoretical spot size, the effect of the foveal shape on either spot size and axial spot position, and, finally, the lateral spot position and color dependency of the incident angle.

In our eyes, as in the eyes of all vertebrates, images of the environment are projected on­to an inverted retina, where photons must pass through most of the retinal layers before being captured by the light-sensitive cells. Light scattering in these retinal layers must decrease the signal-to-noise ratio of the im­ages and thus interfere with clear vision. Sur­prisingly however, our eyes display splendid visual abilities. This apparent contradiction could be resolved if intraretinal light scatter­ing were to be minimized by built-in opti­cal elements that facilitate light transmission through the tissue. Indeed, we were able to show that one function of radial glial (Mül­ler) cells is to act as effective optical fibers in the living retina, bypassing the light-scatter­ing structures in front of the light-sensitive cells. Each Müller cell serves as a ‘private’ light cable, providing one individual cone photo­receptor cell with its appropriate pixel of the environmental image, thus optimizing spe­cial resolution and visual acuity.

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium-containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post-ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75(NTR) , and was prevented by blockers of metabotropic glutamate, P2Y1 , adenosine A1 , and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line-derived neurotrophic factor and transforming growth factor-β1, but not epidermal growth factor and platelet-derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75(NTR) and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling. Cytotoxic cell swelling contributes to retinal neurodegeneration. Nerve growth factor (NGF) inhibits the osmotic swelling of glial cells by acting at TrkA, release of bFGF, and opening of K(+) and Cl(-) channels. The NGF-induced glial release of cytokines like bFGF inhibits the osmotic swelling of bipolar cells, suggesting that the neuroprotective effect of NGF is in part mediated by prevention of cytotoxic cell swelling.

Neovascularization is a sight-threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)-β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Muller) cells produce TGF-β2 under physiological and post-ischemic conditions. To characterize glial cell-derived mediators of angiogenesis regulation in glial-endothelial interactions in the retina, we co-cultured primary Muller cells and bovine microvascular retinal endothelial cells (BRECs). Muller cell-derived TGF-β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF-β receptors, and stimulated TGF-β-dependent anti-proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF-β2 as well as by the presence of Muller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti-angiogenic action of Muller cell-derived TGF-β2: (i) binding of TGF-β2 to BRECs is mediated by the type-II TGF-β receptor, leading to (ii) activation and phosphorylation of receptor-activated Smads; (iii) Muller cell-derived TGF-β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal-regulated kinase (ERK)-1/-2. Neutralizing TGF-β or TGF-β type-II receptor or blocking the activation of Smads partially abrogated the effect of Muller cell-conditioned media on BRECs. Together, our data suggest that Muller cells release TGF-β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context-dependent action of TGF-β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti-angiogenic role of TGF-β therein.

The biomechanical properties of Müller glial cells may have importance in understanding the retinal tissue alterations after retinal surgery with removal of the inner limiting membrane and during the ontogenetic development, respectively. Here, we compared the viscoelastic properties of Müller cells from man and monkey as well as from different postnatal developmental stages of the rat. We determined the complex Young's modulus E = E' + iE″ in a defined range of deforming frequencies (30, 100, and 200 Hz) using a scanning force microscope, where the real part E' reflects the elastic property (energy storage or elastic stiffness) and the imaginary part E″ reflects the viscous property (energy dissipation) of the cells. The viscoelastic properties were similar in Müller cells from man, monkey, and rat. In general, the elastic behavior dominated over the viscous behavior (E'E″). The inner process of the Müller cell was the softest region, the soma the stiffest (Einnerprocess(')Eendfoot(')Esoma(')). Neuronal somata were stiffer than somata of glial cells (Eneuron(')Eglia(')). These relations were also observed during the postnatal development of the rat. It is concluded that, generally, retinal cells display mechanics of elastic solids. In addition, the data indicate that the rodent retina is a reliable model to investigate retinal mechanics and tissue alterations after retinal surgery. During retinal development, neuronal branching and synaptogenesis might be particularly stimulated by the viscoelastic properties of Müller cell processes in the inner plexiform layer.

Osmotic swelling of retinal neurons and glial cells is an important pathogenic factor of retinal edema formation. Here, we show that the neuroprotective factor osteopontin (OPN), which is released from retinal glial (Müller) cells after stimulation of the cells with glial cell line-derived neurotrophic factor (Del Río et al., 2011, Glia 59:821-832), inhibits the swelling of rat Müller cells induced by hypoosmotic exposure of retinal slices in the presence of barium ions and H₂O₂, respectively, and in slices of postischemic retinas. OPN did not inhibit the hypoosmotic swelling of bipolar cells in slices of control and postischemic retinas. The inhibitory effect of OPN on Müller cell swelling was dose-dependent, with a half-maximal effect at ∼0.6 ng/ml. The effect of OPN was abrogated in the presence of pharmacological blockers of vascular endothelial growth factor (VEGF) receptor-2, metabotropic glutamate receptors, and purinergic receptors (P2Y₁, adenosine A1 receptors), as well as of a neutralizing anti-VEGF antibody. The data suggest that OPN induces the release of VEGF, glutamate, ATP, and adenosine from Müller cells. The effect of OPN was also prevented by blockers of voltage-gated sodium channels (tetrodotoxin), T-type voltage-gated calcium channels (kurtoxin), potassium channels (clofilium), and chloride channels 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The swelling-inhibitory effect of OPN was dependent on intracellular calcium signaling, activation of phospholipase C and protein kinase C, and vesicular exocytosis of glutamate. In retinal slices, Müller glial cells display immunoreactivity of OPN. The data suggest that Müller cell-derived OPN has (in addition to the effects on photoreceptors and retinal neurons) autocrine effects. The neuroprotective effects of OPN may be in part mediated by the prevention of cytotoxic Müller cell swelling and the release of VEGF and adenosine from Müller cells.

Regulation of cellular volume is of great importance to avoid changes in neuronal excitability resulting from a decrease in the extracellular space volume. We compared the volume regulation of retinal glial (Müller) and neuronal (bipolar) cells under hypoosmotic and glutamate-stimulated conditions. Freshly isolated slices of the rat retina were superfused with a hypoosmotic solution (60% osmolarity; 4 min) or with a glutamate (1 mM)-containing isoosmotic solution (15 min), and the size changes of Müller and bipolar cell somata were recorded. Bipolar cell somata, but not Müller cell somata, swelled under hypoosmotic conditions and in the presence of glutamate. The hypoosmotic swelling of bipolar cell somata might be mediated by sodium flux into the cells, because it was not observed under extracellular sodium-free conditions, and was induced by activation of metabotropic glutamate receptors and sodium-dependent glutamate transporters. The glutamate-induced swelling of bipolar cell somata was mediated by sodium chloride flux into the cells induced by activation of NMDA- and non-NMDA glutamate receptors, glutamate transporters, and voltage-gated sodium channels. The glutamate-induced swelling of bipolar cell somata was abrogated by adenosine and γ-aminobutyric acid, but not by vascular endothelial growth factor and ATP. The data may suggest that Müller cells, in contrast to bipolar cells, possess endogenous mechanisms which tightly regulate the cellular volume in response to hypoosmolarity and prolonged glutamate exposure. Inhibitory retinal transmission may regulate the volume of bipolar cells, likely by inhibition of the excitatory action of glutamate.

Muller cells, the major type of glial cells in the retina, are responsible for the homeostatic and metabolic support of retinal neurons. By mediating transcellular ion, water, and bicarbonate transport, Muller cells control the composition of the extracellular space fluid. Muller cells provide trophic and anti-oxidative support of photoreceptors and neurons and regulate the tightness of the blood-retinal barrier. By the uptake of glutamate, Muller cells are more directly involved in the regulation of the synaptic activity in the inner retina. This review gives a survey of recently discoved new functions of Muller cells. Muller cells are living optical fibers that guide light through the inner retinal tissue. Thereby they enhance the signal/noise ratio by minimizing intraretinal light scattering and conserve the spatial distribution of light patterns in the propagating image. Muller cells act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as soft substrate required for neurite growth and neuronal plasticity. Muller cells release neuroactive signaling molecules which modulate neuronal activity, are implicated in the mediation of neurovascular coupling, and mediate the homeostasis of the extracellular space volume under hypoosmotic conditions which are a characteristic of intense neuronal activity. Under pathological conditions, a subset of Muller cells may differentiate to neural progenitor/stem cells which regenerate lost photoreceptors and neurons. Increasing knowledge of Muller cell function and responses in the normal and diseased retina will have great impact for the development of new therapeutic approaches for retinal diseases.

Muller glia, the principal macroglia of the retina, express diverse subtypes of adenosine and metabotropic purinergic (P2Y) receptors. Muller cells of several species, including man, also express ionotropic P2X7 receptors. ATP is liberated from Muller cells after activation of metabotropic glutamate receptors and during osmotic and mechanical induction of membrane stretch; adenosine is released through equilibrative nucleoside transporters. Muller cell-derived purines modulate the neuronal activity and have autocrine effects, for example, induction of glial calcium waves and regulation of the cellular volume. Glial calcium waves induced by neuron-derived ATP mediate functional hyperemia in the retina. Purinergic signaling contributes to the induction of Muller cell gliosis, for example, of cellular proliferation and downregulation of potassium channels, which are important for the homeostatic functions of Muller cells. Purinergic glial calcium waves may also promote the long-range propagation of ...

Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Muller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Muller cell-RGC-co-cultures we observed that activity of Muller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Muller cells towards RGC.

We aimed to determine the ultrastructural changes of collagen fibrils and cells in the rabbit sclera after scleral crosslinking using riboflavin and blue light of different intensities. Scleral crosslinking is known to increase scleral stiffness and may inhibit the axial elongation of progressive myopic eyes.The equatorial parts of the sclera of one eye of six adult albino rabbits were treated with topical riboflavin solution (0.5 %) followed by irradiation with blue light (200, 400, 650 mW/cm(2)) for 20 min. After 3 weeks, the ultrastructure of scleral cells and the abundance of small- (10-100 nm) and large-diameter (100 nm) collagen fibrils in fibril bundles of different scleral layers were examined with electron microscopy.In the scleral stroma of control eyes, the thickness of collagen fibrils showed a bimodal distribution. The abundance of small-diameter collagen fibrils decreased from the inner towards the outer sclera, while the amount of large-diameter fibrils and the scleral collagen content did not differ between different stroma layers. Treatment with riboflavin and blue light at 200 mW/cm(2) did not induce ultrastructural changes of cells and collagen fibrils in the scleral stroma. Treatment with blue light of higher intensities induced scleral cell activation in a scleral layer-dependent manner. In addition, outer scleral layers contained phagocytes that engulfed collagen fibrils and erythrocytes. Blue light of the highest intensity induced a reduction of the scleral collagen content, a decreased abundance of large-diameter collagen fibrils, and an increased amount of small-diameter fibrils in the whole scleral stroma.The data indicate that in rabbits, scleral crosslinking with riboflavin and blue light of 200 mW/cm(2) for 20 min is relatively safe and does not induce ultrastructural alterations of scleral cells and of the collagen composition of the scleral stroma. Irradiation with blue light of intensities between 200 and 400 mW/cm(2) induces scleral cell activation, which may contribute to scleral scarring and stiffening. Higher intensities cause scleritis.

We determined the mechanisms of glutamate and ATP release from murine retinal glial (Müller) cells by pharmacological manipulation of the vascular endothelial growth factor (VEGF)- and glutamate-induced inhibition of cellular swelling under hypoosmotic conditions. It has been shown that exogenous glutamate inhibits hypoosmotic swelling of rat Müller cells via the induction of the release of ATP (Uckermann et al. in J Neurosci Res 83:538-550, 53). VEGF was shown to inhibit hypoosmotic swelling of rat Müller cells by inducing the release of glutamate (Wurm et al. in J Neurochem 104:386-399, 55). The swelling-inhibitory effect of VEGF in murine Müller cells was blocked by an inhibitor of vesicular exocytosis, by a modulator of the allosteric site of vesicular glutamate transporters, and by inhibitors of phospholipase C and protein kinase C. The swelling-inhibitory effect of glutamate in murine Müller cells was prevented by inhibitors of connexin hemichannels. The effects of both VEGF and glutamate were blocked by tetrodotoxin and by an inhibitor of T-type voltage-gated calcium channels. Murine Müller cells display connexin-43 immunoreactivity. The data suggest that Müller cells of the murine retina may release glutamate by vesicular exocytosis, whereas ATP is released through connexin hemichannels.

Osmotic swelling of retinal glial (Müller) cells may contribute to the development of edema in diabetic retinopathy. Here, we tested whether oxidative stress and mitochondrial dysfunction are pathogenic factors involved in the osmotic swelling of Müller cells in retinal slices from control and streptozotocin-injected hyperglycemic rats. Hypotonic challenge did not change the size of Müller cell somata from control animals but induced soma swelling in Müller cells of diabetic animals. Administration of a reducing agent blocked the osmotic swelling of Müller cell somata. In retinal tissues from control animals, administration of the reducing agent blocked also the swelling-inducing effects of antagonists of P2Y₁ and adenosine A₁ receptors. In tissues from diabetic animals, inhibition of xanthine oxidase decreased the soma swelling by approximately 50% while inhibition of NADPH oxidase and nitric oxide synthase had no effects. Blockade of mitochondrial oxidative stress by perindopril, as well as of mitochondrial permeability transition by cyclosporin A or minocycline, attenuated the swelling. In addition, activation of mitochondrial K(ATP) channels by pinacidil fully prevented the swelling. The data suggest that oxidative stress produced by xanthine oxidase, as well as the mitochondria, are implicated in the induction of osmotic swelling of Müller cells from diabetic rats.

Signalling of sphingosine-1-phosphate (S1P) via G-protein-coupled receptors of the Endothelial Differentiation Gene family differentially regulates cellular processes such as migration, proliferation and morphogenesis in a variety of cell types. Proliferation and migration of retinal Muller glial cells are involved in pathological events such as proliferative vitreoretinopathy and proliferative diabetic retinopathy. Investigation of possible functional roles of S1P receptors might thus open new insights into Muller cell pathophysiology. Here we show that cultured Muller cells from the guinea pig retina respond to application of S1P with an increase in the intracellular calcium content in a concentration-dependent manner (EC50 11 nM). This calcium increase consists of two components; an initial fast peak and a slow plateau component. The initial transient is caused by a release of calcium from intracellular stores and is suppressed by U-73122, a selective phospholipase C inhibitor. The slow plateau component is caused by a calcium influx. These results suggest that the S1P-induced calcium response in Muller cells partially involves signalling via G-protein-coupled receptors. Moreover, S1P slightly induced Muller cell migration but no proliferation. Thus, the data indicate that Muller cells might be involved in S1P signalling in the retina.

In the postnatal and mature retina, many processes are controlled by the action of nucleotides. Their effects are partly mediated via activation of metabotropic P2Y receptors. However, little is known about the developmental regulation and cellular localization of P2Y receptor subtypes. Combining immunohistochemical and neurophysiological methods, we investigated the developmental expression of P2Y receptors on Muller cells, the principal macroglial cells of the retina. The P2Y1 and the P2Y4 receptors, but no other subtypes, were unequivocally localized on Muller cells. P2Y1 was expressed from postnatal day 5 (P5) on and mediated a calcium response to ATP in Muller cells as well as a volume regulatory signaling cascade preventing Muller cells from swelling under hypotonic conditions. Differentiation of Muller cells was accompanied by a change of the calcium response pattern; the calcium responses in Muller cell endfeet persisted, but ATP responsiveness of Muller cell somata disappeared. P2Y4 immunoreactivity was observed in Muller cell endfeet and synaptic terminals of rod bipolar cells from P20 on. Activated protein kinases were detected by immunohistochemistry; p-ERK occurred in Muller cells and amacrine cells, whereas p-Akt was detected in bipolar cells. Our data indicate that purinergic signaling via P2Y1 and P2Y4 receptors might contribute to differentiation processes in the postnatal retina. © 2009 Wiley-Liss, Inc.

Müller cells are active players in normal retinal function and in virtually all forms of retinal injury and disease. Reactive Müller cells protect the tissue from further damage and preserve tissue function by the release of antioxidants and neurotrophic factors, and may contribute to retinal regeneration by the generation of neural progenitor/stem cells. However, Müller cell gliosis can also contribute to neurodegeneration and impedes regenerative processes in the retinal tissue by the formation of glial scars. This article provides an overview of the neuroprotective and detrimental effects of Müller cell gliosis, with accounts on the cellular signal transduction mechanisms and factors which are implicated in Müller cell-mediated neuroprotection, immunomodulation, regulation of Müller cell proliferation, upregulation of intermediate filaments, glial scar formation, and the generation of neural progenitor/stem cells. A proper understanding of the signaling mechanisms implicated in gliotic alterations of Müller cells is essential for the development of efficient therapeutic strategies that increase the supportive/protective and decrease the destructive roles of gliosis.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, gamma-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.

Intracellular calcium responses are a characteristic of glial activation upon neuronal activity. In acutely isolated preparations of the guinea pig retina, Müller glial cells displayed cytosolic calcium rises in response to repetitive light stimulation. The calcium rises consisted of two components, a slowly developing immediate response that occurred simultaneously over the whole length of all Müller cell fibers and a delayed fast response that originated in the ganglion cell layer and spread as a wave through the bodies of some Müller cells toward the outer processes in the photoreceptor layer. The slow calcium response was evoked by photoreceptor-to-glia signaling, resulting in a glutamate transporter- and zinc-mediated alteration in the membrane potential and an influx of calcium from the extracellular space. The fast calcium response was evoked by a release of calcium from intracellular stores, probably after activation of purinergic receptors. The data suggest that light stimulation of the retina causes glial activation by alterations in both the membrane potential and receptor-mediated mechanisms. The former may be implicated in glial support of the neuronal signal transfer from photoreceptors to ganglion cells (glial forward signaling), whereas the latter may constitute a glial feedback signaling from ganglion cells to photoreceptors.

A decrease in the expression of inwardly rectifying potassium (Kir) currents is a characteristic feature of retinal glial (Müller) cells in various retinopathies, e.g., after transient retinal ischemia. We used short-term retinal organ cultures to investigate whether similar physiological alterations can be induced under in vitro conditions. During 4 days in vitro, Müller cells displayed a decrease in Kir currents and an increase in transient A-type potassium currents which was similar to the alterations in membrane physiology during ischemia-reperfusion in vivo. In addition, gliosis of Müller cells both in vivo and in organ cultures was associated with cellular hypertrophy and an alteration in osmotic swelling characteristics. Whereas Müller cells in control retinae did not swell under hypotonic stress, cells in postischemic retinae and in organ cultures swelled upon hypotonic challenge. Therefore, Müller cells in organ cultures can be used to investigate distinct aspects of ischemia-induced Müller cell gliosis. Both the decrease in Kir currents and the alteration in osmotic swelling may reflect a dysfunction of Müller cells regarding the control of the ionic and osmotic homeostasis in the retina.

Glial cells are proposed to play a major role in the ionic and osmotic homeostasis in the CNS. Swelling of glial cells contributes to the development of edema in neural tissue under pathological conditions such as trauma and ischemia. In this study, we compared the osmotic swelling characteristics of murine hippocampal astrocytes, cerebellar Bergmann glial cells, and retinal Müller glial cells in acutely isolated tissue slices in response to hypoosmotic stress and pharmacological blockade of Kir channels. Hypoosmotic challenge induced an immediate swelling of somata in the majority of Bergmann glial cells and hippocampal astrocytes investigated, whereas Müller cell bodies displayed a substantial delay in the onset of swelling and hippocampal astroglial processes remained unaffected. Blockade of Kir channels under isoosmotic conditions had no swelling-inducing effect in Müller cell somata but caused a swelling in brain astrocytic somata and processes. Blockade of Kir channels under hypoosmotic conditions induced an immediate and strong swelling in Müller cell somata, but had no cumulative effect to brain astroglial somata. No regulatory volume decrease could be observed in all cell types. The data suggest that Kir channels are differently implicated in cell volume homeostasis of retinal Müller cells and brain astrocytes and that Müller cells and brain astrocytes differ in their osmotic swelling properties.

Excessive light causes damage to photoreceptor and pigment epithelial cells, and a local edema in the outer retina. Since Muller 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 Muller cells. Mice were treated with bright white light (intensity, 15,000 lx) for 2 h. Light treatment results in Muller 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 Muller 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 Muller 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.

Glial cells control the water homeostasis in the neural retina, in part via water transport through aquaporin (AQP) water channels. We investigated whether the immunolocalization of two water channels, AQP1 and AQP4, alters in the rat retina during experimental diabetes. Wistar rats were rendered diabetic by a single dose of streptozotocin, and retinal tissues were immunostained following 4 and 6 months. In control tissues, immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina and by distinct amacrine cells. In diabetic retinas, additional strong expression of AQP1 was found in glial cells located in the innermost retinal layers. The superficial retinal vessels were surrounded by AQP4 in control retinas, and by AQP1 in diabetic retinas. A similar alteration in the localization of AQP1 and AQP4 has been described in the rat retina after transient ischemia. The data suggest that the glial cell-mediated water transport in the retina of diabetic animals is altered especially at the superficial vessel plexus.

Under normal conditions, Muller cells support neuronal activity and the integrity of the blood-retinal barrier, whereas gliotic alterations of Muller cells under pathological conditions may contribute to retinal degeneration and edema formation. A major function of Muller cells is the fluid absorption from the retinal tissue, which is mediated by transcellular water transport coupled to currents through potassium channels. Alterations of retinal Muller cells under pathological conditions were investigated by immunohistochemistry and recording their behavior under osmotic stress. In animal models of various retinopathies, e.g., retinal ischemia, ocular inflammation, retinal detachment, and diabetes, it was found that Muller cells decrease the expression of their major potassium channel (Kir4.1). This alteration is associated with an impairment of the rapid water transport across Muller cell membranes, as recognizable in the induction of cellular swelling under hypoosmolar conditions. Osmotic swelling of Muller cells is also induced by oxidative stress and by inflammatory mediators such as arachidonic acid and prostaglandins. The data suggest that a disturbed fluid transport through Muller cells is (in addition to vascular leakage) a pathogenic factor contributing to the development of retinal edema. Pharmacological re-activation of the retinal water clearance by Muller cells may represent an approach to the development of new edema-resolving drugs. Triamcinolone acetonide, which is clinically used to resolve edema, prevents osmotic swelling of Muller cells as it induces the release of endogenous adenosine and subsequent A1 receptor activation which results in the opening of ion channels. Apparently, triamcinolone resolves edema by both inhibition of vascular leakage and stimulation of retinal fluid clearance by Muller cells.

Pigment epithelium-derived factor (PEDF), a glycoprotein with pleiotropic functions, is naturally occuring in the eye and considered as crucial to prevent pathological angiogenesis. Since retinal glial (Müller) cells produce PEDF, the authors have studied its impact on glial-endothelial cellular interactions. Bovine retinal endothelial cells were cultured in the presence of culture media originating from primary Müller cells, and endothelial proliferation as well as phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinases (ERK)-1/-2 were investigated. The concerted activity of Müller-cell derived soluble mediators attenuated endothelial proliferation and ERK-1/-2 activation, regardless of whether the Müller cells were preincubated under normoxia or hypoxia, and even though the endothelial cells were stimulated by vascular endothelial growth factor-A (VEGF). This inhibitory activity was no longer demonstrable if high levels of basic fibroblast growth factor or VEGF were supplied, suggesting that in cases of pathological neovascularization, overproduction of proangiogenic mediators overrides the "antiangiogenic background" provided by Müller cells. However, neutralizing the activity of PEDF partially restored endothelial cell proliferation and resulted in increased ERK-1/-2 activation, which is in concordance with findings demonstrating that exogenously applied PEDF is able to suppress VEGF-induced ERK-1/-2 phosphorylation. PEDF production by Müller cells is not only regulated by retinal oxygen but also by the activity of soluble factors released from retinal endothelial cells. For instance, PEDF levels were significantly elevated in glial (Müller)-endothelial cell cocultures as compared with bovine retinal endothelial cell-free Müller cell cultures. These results have implications for the pathogenesis of retinal neovascularization since the Müller cell may be regarded as a central control element which modulates retinal PEDF levels and, thus, is of critical importance for adjusting the balance between proangiogenic and antiangiogenic mediators.

Muller cells, the principal glial cells of the retina of all vertebrates, were discovered already in 1851 but it was only in the past decades that it became evident that these cells are essential players in vision. Muller cells constitute the ‘core’ of columnar units of clonally and functionally related groups of neurons. Their primary function is to support neuronal functioning (by increasing the signal-to-noise ratio of information processing) and survival (by maintaining a metabolic ‘symbiosis’ with the neurons). It has been shown that Muller cells increase the signal-to-noise ratio of retinal information processing by, for instance, (i) guiding the light towards the photoreceptor cells, (ii) removing excess neurotransmitter molecules from extracellular space, and (iii) performing an efficient clearance of excess extracellular potassium ions after neuronal excitation. The latter two functions are also crucial for neuronal survival – to prevent excitotoxic effects of glutamate – and are coupled to water clearance which is equally important for neuronal survival. As another case of glial homeostasis, the maintenance of appropriate microenvironmental biomechanics has recently been described. Normal glial cells are softer than the neurons, and thus provide a suitable substrate for neurite growth during development, and for functional plasticity. Finally it should be pointed out that Muller cells are capable of ‘sensing’ neuronal activity. They respond to physiological light stimulation of their adjacent photoreceptors with two distinct types of intracellular calcium rises. These calcium rises then may trigger the release of signal substances, incl. so-called ‘gliotransmitters’, from Muller cells which thus even may modify neuronal signal processing in the retina (e.g., in cases of adaptation to bright light, as another mechanism to increase the signal-to-noise ratio of information processing in retina). In cases of reactive Muller cell gliosis, the dominant potassium conductance of the membrane is down-regulated and an increased expression of intermediate filaments is associated with increasing stiffness of the glial cell processes, such that all above-mentioned glial functions are impaired.

Osmotic swelling of neurons and glial cells contributes to retinal edema and neurodegeneration. BDNF, a major neuroprotectant in the retina, was shown to inhibit osmotic swelling of glial (Muller) and bipolar cells in the rat retina; the effect of BDNF on the bipolar cell swelling is mediated by inducing a release of neuroprotective cytokines from Muller cells (Berk et al., Neuroscience 295:175–186, 2015). We determined whether BDNF-mediated cell volume regulation was altered after transient retinal ischemia. Retinal slices from the eyes of rats that underwent a 1-h pressure-induced retinal ischemia and from control eyes were superfused with a hypoosmotic solution. Exogenous BDNF prevented osmotic swelling of Muller cells in both control and post-ischemic retinal slices. BDNF also prevented osmotic swelling of bipolar cells in the control retina, but not in the ischemic retina. On the other hand, exogenous bFGF prevented the swelling of both Muller and bipolar cells in the ischemic retina. Freshly isolated Muller cells of control retinas displayed immunoreactivity of truncated but not full-length TrkB. In contrast, Muller cells of post-ischemic retinas displayed immunoreactivity of both TrkB isoforms. Bipolar cells isolated from control and post-ischemic retinas were immunolabeled for both TrkB isoforms. The data may suggest that the ischemic abrogation of the BDNF effect in bipolar cells is related to altered BDNF receptor expression in Muller cells. Glial upregulation of full-length TrkB may support the survival of Muller cells in the ischemic retina, but may impair the BDNF-induced release of neuroprotective cytokines such as bFGF from Muller cells.

Water accumulation in retinal glial (Muller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (σ) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective σ receptor agonist ditolylguanidine, and the selective σ1 receptor agonist PRE-084, inhibit the osmotic swelling of Muller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of σ receptor agonists on the Muller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that σ receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Muller cell swelling. The swelling-inhibitory effect of 17β-estradiol was prevented by the σ1 receptor antagonist BD1047, suggesting that the effect is mediated by σ1 receptor activation. The data may suggest that the neuroprotective effect of σ receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells.

Objective: The current study introduced OCT as a novel tool in clinical veterinary ophthalmology in a much wider variety of free-living avian species than hitherto studied. Methods: OCT was tested and performed in 39 free-living birds (21 species of 12 families) and compared to direct ophthalmoscopy. Birds were examined with combinations of different restraints (manual restraint or fixation on a holding device) and different anaesthesia regimes (none, sedation, general anaesthesia). Inter- and intra-species specific variations of the general procedure, the restraint methods and the clinical findings were evaluated. Results: OCT was possible in all examined avian species (from 40 g up to 7720 g) and superseded direct ophthalmoscopy in quality and quantity of ophthalmological findings. All restraint methods enabled OCT examination, however combination of general anaesthesia and a holding device provided the most rapid and subjectively the least stressful examination technique. Stability, stress reduction, head angle and distance from the OCT device were important factors influencing volume scanning quality. Sixteen, out of 39 birds, presented ocular abnormalities detected by OCT (compared to only five birds when using direct ophthalmoscopy). OCT with included fundus images offered an objective assessment of retinal changes. Retinal abnormalities included changes of fundus pigmentation, drusenoid changes and severe retinal and choroidal degenerations. Species-specific variations of retinal layer dimensions and of foveal structures were evident. Conclusion: OCT is a promising, non-invasive method, which significantly compliments standard techniques. OCT is applicable to a wide variety of avian species; it provides high quality cross-sectional images of the retina, enabling accurate and improved diagnosis and prognosis of therapies. An assessment of the visual capabilities of traumatised birds is a major factor for their rehabilitation and survival in the wild. Finally, this method is an excellent tool in interdisciplinary retinal research, providing novel insights into the diversity of very specialized structural adaptations of avian retina.

Glial cells control the retinal osmohomeostasis, in part via mediation of water fluxes through aquaporin (AQP) water channels. By using immunohistochemical staining, we investigated whether ischemia-reperfusion of the rat retina causes alterations in the distribution of AQP1 and AQP4 proteins. Transient ischemia was induced in retinas of Long-Evans rats by elevation of the intraocular pressure for 60 min. In control retinas, immunoreactive AQP1 was expressed in the outer retina and by distinct amacrine cells, and AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina. After ischemia, retinal glial cells in the nerve fiber/ganglion cell layers strongly expressed AQP1. The perivascular staining around the superficial vessels altered from AQP4 in control retinas to AQP1 in postischemic retinas. The data suggest that the glial cell-mediated water transport in the retina is altered after ischemia especially at the superficial vessel plexus.

Aquaporins are involved in the maintenance of ionic and osmotic balance in the central nervous system and in the eye. Whereas the expression of aquaporin-9 immunoreactivity in the brain has been described for catecholaminergic neurons and glial cells, the expression of aquaporin-9 in the neural retina is unclear. We examined the distribution of aquaporin-1 and -9 immunoreactivities in retinas of the rat. Aquaporin-9 immunoreactivity was expressed exclusively by tyrosine hydroxylase (TH) positive amacrine cells, while aquaporin-1 immunoreactivity was expressed by photoreceptor cells and by TH negative amacrine cells. The staining pattern of aquaporin-9 did not change up to 4 weeks after pressure-induced transient retinal ischemia. It is concluded that catecholaminergic, putatively dopaminergic, amacrine cells of the retina express aquaporin-9.