Amphibious vision - Optical design model of the hooded merganser eye.

A comprehensive schematic eye model of the hooded merganser is introduced for the first time to advance the understanding of amphibious vision. It is comprised of two different configurations, the first one modeling its visual system in air (unaccommodated state) and the second one representing the case where the eye is immersed in water (accommodated state). The model was designed using available data of former studies, image analysis and the implementation of feasible assumptions that serve as starting values. An optimization process incorporating an optical design program is used to vary the starting values with the aim of finding the setup offering the best acuity. The image quality was measured using the root-mean-square radius of the focal spot formed on the retina. The resulting schematic eye model comprises all relevant optical specifications, including aspherical geometrical parameters for cornea and lens, distances between the surfaces, the gradient index distribution of the lens, the retinal specifications and the object distance in both media. It achieves a spot radius of 4.20 µm for the unaccommodated state, which meets the expectations derived by the mean ganglion cell density and comparison with other animals. In contrast, under water the determined spot radius of 11.48 µm indicates an acuity loss. As well as enhancing our understanding of the vision of the hooded merganser, the schematic eye model may also serve as a simulation basis for examing similar animal eyes, such as the cormorant or other fish hunting birds.

Employing Nanostructured Scaffolds to Investigate the Mechanical Properties of Adult Mammalian Retinae Under Tension.

Numerous eye diseases are linked to biomechanical dysfunction of the retina. However, the underlying forces are almost impossible to quantify experimentally. Here, we show how biomechanical properties of adult neuronal tissues such as porcine retinae can be investigated under tension in a home-built tissue stretcher composed of nanostructured TiO2 scaffolds coupled to a self-designed force sensor. The employed TiO2 nanotube scaffolds allow for organotypic long-term preservation of adult tissues ex vivo and support strong tissue adhesion without the application of glues, a prerequisite for tissue investigations under tension. In combination with finite element calculations we found that the deformation behavior is highly dependent on the displacement rate which results in Young's moduli of (760-1270) Pa. Image analysis revealed that the elastic regime is characterized by a reversible shear deformation of retinal layers. For larger deformations, tissue destruction and sliding of retinal layers occurred with an equilibration between slip and stick at the interface of ruptured layers, resulting in a constant force during stretching. Since our study demonstrates how porcine eyes collected from slaughterhouses can be employed for ex vivo experiments, our study also offers new perspectives to investigate tissue biomechanics without excessive animal experiments.

Glia of the human retina.

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.

Cone-to-Müller cell ratio in the mammalian retina: A survey of seven mammals with different lifestyle.

Mammalian retinal glial (Müller) 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 Müller 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-Müller cell ratio. To determine this ratio in the retinas of mammals with different lifestyle, we evaluated the local densities of cones and Müller 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 Müller 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-Müller cell ratios of above one, the local densities of cones and Müller 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 Müller 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 Müller cells may be coupled to S-cones, and 40% of S-cones may be not coupled to Müller cells. Among the species investigated, the density of Müller 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) Müller cells support high acuity vision by predominant guidance of red-green light to M-opsin expressing cones.

Comprehensive optical design model of the goldfish eye and quantitative simulation of the consequences on the accommodation mechanism.

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 µm which is below the inter cone distance of 5.5 µm. 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.

Comparison of cellular localisation of the Ca2+ -binding proteins calbindin, calretinin and parvalbumin in the retina of four different Macaca species.

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, Müller 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.

Radial glial elements in the cerebral cortex of the lesser hedgehog tenrec.

We investigated astroglial cells in several areas of the telencephalic cortex of the lesser hedgehog tenrec (Echinops telfairi). Compared to other mammals, the cortex of the tenrec has a relatively large paleocortex and a low encephalization index. We stained sections from tenrec forebrains with structural and functional glia markers focusing on selected cortical areas, the paleocortex, rhinal cortex, neocortex and the dentate gyrus of the hippocampal formation. We found that in all parts of the tenrec forebrain cortex, radial processes exist which are positive for glial fibrillary acidic protein (GFAP) although with differential localization: in the rhinal cortex and neocortical region radial glial fibers are located in the subventricular regions, whereas in the dentate gyrus and paleocortex they appear to arise from the cells in the respective granular layers. The relatively high abundance of the radial fibers in layer III of the paleocortex was very conspicuous. Only few of these radial processes were also co-labeled with doublecortin (DCX), yet most of the DCX-positive cells were negative for GFAP. The GFAP-positive radial fibers were in turn neither positive for glutamine synthetase, nor did they show immunoreactivity for the astroglia-specific water channel aquaporin-4 (AQP4). Star-shaped astrocytes, however, displayed the typical perivascular and subpial expression patterns for AQP4. We conclude that the radial glia in the adult tenrec represents an immature form of astroglia that persists in these animals throughout life.

Retinal adaptation to dim light vision in spectacled caimans (Caiman crocodilus fuscus): Analysis of retinal ultrastructure.

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.

Müller glial cells contribute to dim light vision in the spectacled caiman (Caiman crocodilus fuscus): Analysis of retinal light transmission.

In this study, we show the capability of Müller glial cells to transport light through the inverted retina of reptiles, specifically the retina of the spectacled caimans. Thus, confirming that Müller cells of lower vertebrates also improve retinal light transmission. Confocal imaging of freshly isolated retinal wholemounts, that preserved the refractive index landscape of the tissue, indicated that the retina of the spectacled caiman is adapted for vision under dim light conditions. For light transmission experiments, we used a setup with two axially aligned objectives imaging the retina from both sides to project the light onto the inner (vitreal) surface and to detect the transmitted light behind the retina at the receptor layer. Simultaneously, a confocal microscope obtained images of the Müller cells embedded within the vital tissue. Projections of light onto several representative Müller cell trunks within the inner plexiform layer, i.e. (i) trunks with a straight orientation, (ii) trunks which are formed by the inner processes and (iii) trunks which get split into inner processes, were associated with increases in the intensity of the transmitted light. Projections of light onto the periphery of the Müller cell endfeet resulted in a lower intensity of transmitted light. In this way, retinal glial (Müller) cells support dim light vision by improving the signal-to-noise ratio which increases the sensitivity to light. The field of illuminated photoreceptors mainly include rods reflecting the rod dominance of the of tissue. A subpopulation of Müller cells with downstreaming cone cells led to a high-intensity illumination of the cones, while the surrounding rods were illuminated by light of lower intensity. Therefore, Müller cells that lie in front of cones may adapt the intensity of the transmitted light to the different sensitivities of cones and rods, presumably allowing a simultaneous vision with both receptor types under dim light conditions.

The primate fovea: Structure, function and development.

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 Müller 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 "Müller cell cone" of the foveola comprises specialized Müller cells which do not support neuronal activity but may serve optical and structural functions. In addition to the "Müller cell cone", structural stabilization of the foveal morphology may be provided by the 'z-shaped' Müller 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 Müller cells. After widening of the foveal pit likely mediated by retracting astrocytes, Henle fibers are formed by horizontal contraction of Müller 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.

Electrophysiological characterization of Müller cells from the ischemic retina of mice deficient in the leukemia inhibitory factor.

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 Müller glial cells as a hallmark of reactive gliosis is suppressed during retinal degenerations. Here, we detected expression of LIF and its receptors in Müller cells of the murine retina. Moreover, electrophysiological alterations of Müller cells 7 days after transient retinal ischemia were studied by the patch-clamp technique. The amplitude of inward currents in Müller 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 Müller cells even in the absence of LIF.

Müller glial cells of the primate foveola: An electron microscopical study.

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.

Müller Cell-Derived PEDF Mediates Neuroprotection via STAT3 Activation.

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.

Early evolution of radial glial cells in Bilateria.

Bilaterians usually possess a central nervous system, composed of neurons and supportive cells called glial cells. Whereas neuronal cells are highly comparable in all these animals, glial cells apparently differ, and in deuterostomes, radial glial cells are found. These particular secretory glial cells may represent the archetype of all (macro) glial cells and have not been reported from protostomes so far. This has caused controversial discussions of whether glial cells represent a homologous bilaterian characteristic or whether they (and thus, centralized nervous systems) evolved convergently in the two main clades of bilaterians. By using histology, transmission electron microscopy, immunolabelling and whole-mount in situ hybridization, we show here that protostomes also possess radial glia-like cells, which are very likely to be homologous to those of deuterostomes. Moreover, our antibody staining indicates that the secretory character of radial glial cells is maintained throughout their various evolutionary adaptations. This implies an early evolution of radial glial cells in the last common ancestor of Protostomia and Deuterostomia. Furthermore, it suggests that an intraepidermal nervous system-composed of sensory cells, neurons and radial glial cells-was probably the plesiomorphic condition in the bilaterian ancestor.

The Retina of Asian and African Elephants: Comparison of Newborn and Adult.

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.

Two different mechanosensitive calcium responses in Müller glial cells of the guinea pig retina: Differential dependence on purinergic receptor signaling.

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 Müller 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 Müller 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 Müller cells with different kinetics in dependence on the distance from the stimulation site. Müller 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 Müller 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 Müller 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 Müller cells of the guinea pig retina release ATP which induces a propagating calcium wave in surrounding Müller 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 Müller 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.

Comparative electrophysiology of retinal Müller glial cells-A survey on vertebrate species.

Müller 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, Müller 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 Müller 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 Müller 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 Müller cells developed early in vertebrate evolution. GLIA 2017;65:533-568.

Stroke-induced blood-brain barrier breakdown along the vascular tree - No preferential affection of arteries in different animal models and in humans.

Stroke-induced blood-brain barrier breakdown promotes complications like cerebral edema and hemorrhagic transformation, especially in association with therapeutical recanalization of occluded vessels. As arteries, capillaries and veins display distinct functional and morphological characteristics, we here investigated patterns of blood-brain barrier breakdown for each segment of the vascular tree in rodent models of embolic, permanent, and transient middle cerebral artery occlusion, added by analyses of human stroke tissue. Twenty-four hours after ischemia induction, loss of blood-brain barrier function towards FITC-albumin was equally observed for arteries, capillaries, and veins in rodent brains. Noteworthy, veins showed highest ratios of leaky vessels, whereas capillaries exhibited the most and arteries the least widespread perivascular tracer extravasation. In contrast, human autoptic stroke tissue exhibited pronounced extravasations of albumin around arteries and veins, while the pericapillary immunoreactivity appeared only faint. Although electron microscopy revealed comparable alterations of the arterial and capillary endothelium throughout the applied animal models, structural loss of arterial smooth muscle cells was only observed in the translationally relevant model of embolic middle cerebral artery occlusion. In light of the so far available concepts of stroke treatment, the consideration of a differential vascular pathophysiology along the cerebral vasculature is likely to allow development of novel effective treatment strategies.

Role of Purines in Müller Glia.

Müller glia, the principal macroglia of the retina, express diverse subtypes of adenosine and metabotropic purinergic (P2Y) receptors. Müller cells of several species, including man, also express ionotropic P2X7 receptors. ATP is liberated from Müller cells after activation of metabotropic glutamate receptors and during osmotic and mechanical induction of membrane stretch; adenosine is released through equilibrative nucleoside transporters. Müller 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 Müller cell gliosis, for example, of cellular proliferation and downregulation of potassium channels, which are important for the homeostatic functions of Müller cells. Purinergic glial calcium waves may also promote the long-range propagation of gliosis and neuronal degeneration across the retinal tissue. The osmotic ATP release is inhibited under pathological conditions. Inhibition of the ATP release may result in osmotic Müller cell swelling and dysregulation of the water transport through the cells; both may contribute to the development of retinal edema. Suppression of the osmotic ATP release and upregulation of the ecto-apyrase (NTPDase1), which facilitate the extracellular degradation of ATP and the formation of adenosine, may protect neurons and photoreceptors from death due to overactivation of P2X receptors. Pharmacological inhibition of P2X7 receptors and stimulation of adenosine receptors may represent clinical approaches to prevent retinal cell death and dysregulated cell proliferation, and to treat retinal edema.

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

Water accumulation in retinal glial (Müller) 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 Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller 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-ß1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller 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 Müller 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-ß1 from Müller cells.