Search

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 P2Y 1 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., (© 2016 Wiley Periodicals, Inc.)

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 ET A and ET B receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y 1 , and adenosine A 1 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 Müller and bipolar cells displayed immunoreactivities of ET A and ET B 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-β1 from Müller cells.

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., (© 2014 International Society for Neurochemistry.)

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., (© 2013 International Society for Neurochemistry.)

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(')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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Vitrectomy is a frequently performed surgical intervention in ophthalmology to remove vitreous traction and opacities or to treat complicated retinal detachments and diabetic changes. However, there is lack of information about cellular responses in retinal tissue after a surgical intervention such as vitrectomy. Microglia cells, the immune competent cells of neuronal tissue, are involved in nearly all neuropathological changes and are additionally activated by neurosurgical interventions. For most neurodegenerative changes, it is described that microglia activation is generally accompanied by a reactive gliosis of macroglial cells. However, it is not known whether microglial cell activation is necessarily associated with macroglial cell gliosis or whether these processes are regulated separately. Furthermore, there is an ongoing debate about possible detrimental consequences of microglial cell activation for neurons in central neural and retinal tissue., Methods: Using immunohistochemistry and whole-cell patch clamp experiments in a rabbit model of partial pars plana vitrectomy, we investigated micro- and macroglial cell reactivity after this intervention., Results: Partial vitrectomy induced a massive microglia response characterized by morphological changes, intraretinal migration and proliferation of retinal microglial cells, respectively. Microglial cell reactivity was observed 2 days after the operation and was down-regulated after 7 days. Microglia reactivity was associated with neither a general Müller cell gliosis nor an obvious neuronal cell loss. Electrophysiological examinations revealed no significant changes of whole-cell currents and membrane potentials of Müller cells from healthy and vitrectomized eyes up to 3 weeks after operation. Only a small number of individual Müller glial cells expressed GFAP or reduced their inward currents as a sign of Müller cell gliosis., Conclusion: Vitrectomy induced a massive response of microglial cells. However, microglia activation and deactivation are effectively regulated and are not necessarily associated with macroglial (Müller) cell reactivity and with obvious detrimental effects to neurons., (© 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.)

Background/aims: Osmotic swelling of Müller cells is a common phenomenon in animal models of ischemic and diabetic retinopathies. Müller cells possess a swelling-inhibitory purinergic signaling cascade which can be activated by various receptor ligands including vascular endothelial growth factor (VEGF) and glutamate. Here, we investigated whether deletion of P2Y1 (P2Y1R) and adenosine A1 receptors (A1AR), and of inositol-1,4,5-trisphosphate-receptor type 2 (IP3R2), in mice affects the inhibitory action of VEGF and glutamate on Müller cell swelling., Methods: The cross-sectional area of Müller cell somata was recorded after a 4-min superfusion of retinal slices with a hypoosmotic solution., Results: Hypoosmolarity induced a swelling of Müller cells from P2Y1R(-/-), A1AR(-/-) and IP3R2(-/-) mice, but not from wild-type mice. Swelling of wild-type Müller cells was induced by hypoosmotic solution containing barium chloride. Whereas VEGF inhibited the swelling of wild-type Müller cells, it had no swelling-inhibitory effect in cells from A1AR(-/-) and IP3R2(-/-) mice. Glutamate inhibited the swelling of wild-type Müller cells but not of cells from P2Y1R(-/-), A1AR(-/-) and IP3R2(-/-) animals., Conclusion: The swelling-inhibitory effects of VEGF and glutamate in murine Müller cells is mediated by transactivation of P2Y1R and A1AR, as well as by intracellular calcium signaling via activation of IP3R2., (Copyright © 2013 S. Karger AG, Basel.)

Calcium acts as a prominent second messenger in virtually every cell type and modulates a plethora of cell functions. Thus, Ca(2+) microfluorimetry became a valuable tool to assess information about mechanisms involved in the regulation of the intracellular calcium level in research on living tissues. Here we offer insight into distinct approaches to detect changes in calcium levels specifically in Müller cells, the principal macroglial cells of the retina.

Various ependymoglial cells display varying degrees of process specialization, in particular processes contacting mesenchymal borders (pia, blood vessels, vitreous body), or those lining the ventricular surface. Within the neuropil, glial morphology, cellular contacts, and interaction partners are complex. It appears that glial processes contacting neurons, specific parts of neurons, or mesenchymal or ventricular borders are characterized by specialized membranes. We propose a concept of membrane domains in addition to the existing concept of ependymoglial polarity. Such membrane domains are equipped with certain membrane-bound proteins, enabling them to function in their specific environment. This review focuses on Müller cells and astrocytes and discusses exemplary the localization of established glial markers in membrane domains. We distinguish three functional glial membrane domains based on their typical molecular arrangement. The domain of the endfoot specifically displays the complex of dystrophin-associated proteins, aquaporin 4 and the potassium channel Kir4.1. We show that the domain of microvilli and the peripheral glial process in the Müller cell share the presence of ezrin, as do peripheral astrocyte processes. As a third domain, the Müller cell has peripheral glial processes related to a specific subtype of synapse. Although many details remain to be studied, the idea of glial membrane domains may permit new insights into glial function and pathology.

Purpose: To study physiologic characteristics of human Müller cells from healthy and pathologically altered eyes., Methods: Human tissue was used from organ donors and from patients affected with uveal melanoma. Several melanoma eyes also showed retinal detachment. Incubation of freshly prepared slices with a commercial vital dye preferentially stained Müller cells. The Müller cell response to hypotonic stress was observed by recording the cross-sectional area of cell somata. Electrophysiologic properties were investigated in parallel in whole-cell patch-clamp experiments., Results: Inward K+ currents mediated by inwardly rectifying Kir channels were significantly decreased in Müller cells from eyes with uveal melanoma compared with healthy controls. This was accompanied by a decrease of the membrane potential. Both effects were stronger in cells from eyes where the melanoma had caused a widespread retinal detachment. Application of a hypotonic solution did not affect Müller cells from healthy organ donors. By contrast, Müller cells from some melanoma eyes increased their soma size in response to hypotonic solution. This effect was aggravated in cells from eyes with widespread retinal detachment. The inflammatory mediator, arachidonic acid, could induce Müller cell swelling, whereas anti-inflammatory substances reduced the swelling., Conclusions: The experiments with human tissue confirm earlier data from animal models for retinal pathologies about typical alterations of reactive Müller cells. Hypotonic stress induced Müller cell swelling preferentially in cells from melanoma-affected eyes that displayed decreased inward current amplitudes. Widespread melanoma-associated retinal detachment potentiated the pathologic alterations of Müller cells.

Glial cells release molecules that influence brain development, function, and disease. Calcium-dependent exocytosis has been proposed as potential release mechanism in astroglia, but the physiological relevance of "gliotransmission" in vivo remains controversial. We focused on the impact of glial exocytosis on sensory transduction in the retina. To this end, we generated transgenic mice to block exocytosis by Cre recombinase-dependent expression of the clostridial botulinum neurotoxin serotype B light chain, which cleaves vesicle-associated membrane protein 1-3. Ubiquitous and neuronal toxin expression caused perinatal lethality and a reduction of synaptic transmission thus validating transgene function. Toxin expression in Müller cells inhibited vesicular glutamate release and impaired glial volume regulation but left retinal histology and visual processing unaffected. Our model to study gliotransmission in vivo reveals specific functions of exocytotic glutamate release in retinal glia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites, astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca(2+) signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca(2+) -dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer's disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review not only sheds new light on the brain operation in health and disease, but also points to many unknowns., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

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.

Purines (in particular, ATP and adenosine) act as neuro- and gliotransmitters in the sensory retina where they are involved in bidirectional neuron-glia signaling. This review summarizes the present knowledge about the expression and functional importance of P1 (adenosine) and P2 (nucleotide) receptors in Müller glial cells of the mammalian retina. Mammalian Müller cells express various subtypes of adenosine receptors and metabotropic P2Y receptors. Human Müller cells also express ionotropic P2X(7) receptors. Müller cells release ATP upon activation of metabotropic glutamate receptors and/or osmotic membrane stretching. The osmotic mechanism is abrogated under conditions associated with ischemia-hypoxia and inflammation, resulting in swelling of the Müller cells when the extracellular milieu is hypoosmotic. However, exogenous glutamate, which induces the release of ATP and adenosine, and thus activates P2Y(1) and A(1) adenosine receptors, respectively, prevents such osmotic swelling under pathological conditions, suggesting unimpaired receptor-induced release of ATP. In addition to the inhibition of swelling, which is implicated in regulating the volume of the extracellular space, purinergic signaling is involved in mediating neurovascular coupling. Furthermore, purinergic signals stimulate the proliferation of retinal precursor cells and Müller cells. In normal retinal information processing, Müller cells regulate the synaptic activity by the release of ATP and adenosine. In retinopathies, abrogation of the osmotic release of ATP, and the upregulation of ecto-apyrase (NTPDase1), may have neuroprotective effects by preventing the overactivation of neuronal P2X receptors that are implicated in apoptotic cell death. Pharmacological modulation of purinergic receptors of Müller cells may have clinical importance, e.g., for the clearance of retinal edema and for the inhibition of dysregulated cell proliferation in proliferative retinopathies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Transient retinal ischemia-reperfusion is associated with neuronal degeneration and activation of Müller glial cells. Reactive gliosis may impede the homeostatic functions of Müller cells. A viable animal model for human ischemic events should display similarities in eye size and retinal blood supply. Therefore, pigs were used in this investigation of physiological alterations in Müller cells after ischemia-reperfusion., Methods: Transient retinal ischemia was induced in young adult pigs by high intraocular pressure in one eye for 1 hour. After 3 days of reperfusion, the retinal tissue and isolated Müller cells were used for osmotic swelling recordings, whole-cell patch-clamp experiments, Ca(2+) microfluorimetry, and immunohistochemistry., Results: Müller cells in retinal slices from postischemic eyes but not control cells displayed a significant swelling of the somata when osmotic stress was applied by hypotonic extracellular solution. The amplitude of K(+) inward currents was significantly reduced (∼60% of the control value). This decrease was accompanied by a depolarization of the cell membrane. The number of Müller cell end feet displaying a Ca(2+) increase after application of adenosine 5'-triphosphate was increased in the ischemic retina. Moreover, reactive Müller cell gliosis was characterized by an (increased) expression of vimentin, glial fibrillary acidic protein, the phosphorylated mitogen-activated protein kinases extracellular signal-related kinase (ERK) 1 and 2, and the transcription factor c-fos., Conclusions: The alterations of reactive Müller cells after transient ischemia of the pig eye were similar to those found in rat and rabbit models, demonstrating that the porcine retina is a suitable model for the investigation of ischemic injury.

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP(-/-)Vim(-/-) mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration.

High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the effects of hypoosmolarity, ammonia, and glutamine on the induction of glial cell swelling in freshly isolated slices of the rat retina. Glutamine, but not ammonia or hypoosmolarity per se, evoked a rapid (within one minute) swelling of retinal glial (Müller) cell bodies under hypoosmotic conditions. Under isoosmotic conditions, glutamine evoked a delayed swelling after 10 min of exposure. The effect of glutamine was concentration-dependent, with half-maximal and maximal effects at ∼ 0.1 and 0.5 mM. Glutamine in hypoosmotic solution induced a dissipation of the mitochondrial membrane potential. The effects on the mitochondrial membrane potential and the glial soma size were reduced by (i) agents which inhibit the transfer of glutamine into mitochondria and its hydrolysis there, (ii) inhibition of the mitochondrial permeability transition, (iii) inhibitors of oxidative-nitrosative stress, and (iv) inhibitors of phospholipase A(2) and cyclooxygenase. Glutamine-induced glial swelling was also prevented by ATP and adenosine, acting at adenosine A(1) receptors. The data suggest that hypoosmolarity accelerates the swelling-inducing effect of glutamine on retinal glial cells, and that swelling induction by glutamine is mediated by inducing oxidative-nitrosative stress, inflammatory lipid mediators, and mitochondrial dysfunction., (© 2010 Wiley-Liss, Inc.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The glial water channel aquaporin-4 (AQP4) is implicated in the control of ion and osmohomeostasis in the sensory retina. Using retinal slices from AQP4-deficient and wild-type mice, we investigated whether AQP4 is involved in the regulation of glial cell volume under altered osmotic conditions. Superfusion of retinal slices with a hypoosmolar solution induced a rapid swelling of glial somata in tissues from AQP4 null mice but not from wild-type mice. The swelling was mediated by oxidative stress, inflammatory lipid mediators, and sodium influx into the cells and was prevented by activation of glutamatergic and purinergic receptors. Distinct inflammatory proteins, including interleukin-1 beta, interleukin-6, and inducible nitric oxide synthase, were up-regulated in the retina of AQP4 null mice compared with control, whereas cyclooxygenase-2 was down-regulated. The data suggest that water flux through AQP4 is involved in the rapid volume regulation of retinal glial (Müller) cells in response to osmotic stress and that deletion of AQP4 results in an inflammatory response of the retinal tissue. Possible implications of the data for understanding the pathophysiology of neuromyelitis optica, a human disease that has been suggested to involve serum antibodies to AQP4, are discussed., ((c) 2010 Wiley-Liss, Inc.)

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 Müller 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 Müller cell pathophysiology. Here we show that cultured Müller cells from the guinea pig retina respond to application of S1P with an increase in the intracellular calcium content in a concentration-dependent manner (EC(50) 11nM). 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 Müller cells partially involves signalling via G-protein-coupled receptors. Moreover, S1P slightly induced Müller cell migration but no proliferation. Thus, the data indicate that Müller cells might be involved in S1P signalling in the retina., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postischemic and diabetic retinas. Progesterone, testosterone, estriol, and 17beta-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 microM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y1, and adenosine A1 receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.

Retinal Müller glial cells are involved in K+ ion homeostasis of the tissue. Inwardly rectifying K(+) (Kir) channels play a decisive role in the process of spatial K+ buffering. It has been demonstrated that Kir-mediated currents of Müller cells are downregulated in various cases of retinal neurodegeneration. However, this has not yet been verified for any murine animal model. The aim of the present study was to investigate Müller cells after transient retinal ischemia in mice. High intraocular pressure was applied for 1h; the retina was analysed 1 week later. We studied protein expression in the tissue by immunohistochemistry, and membrane currents of isolated cells by patch-clamp experiments. We found the typical indicators of reactive gliosis such as upregulation of glial fibrillary acidic protein. Moreover, the membrane capacitance of isolated Müller cells was increased and the amplitudes of Kir-mediated currents were slightly, but significantly decreased. This murine high intraocular pressure model of transient retinal ischemia is proposed as a versatile tool for further studies on Müller cell functions in retinal degeneration., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Edema in the ischemic neural tissue develops by increased vascular permeability associated with extravasation of albumin, and by glial swelling. Here, we show that bovine serum albumin acutely administered to slices of the rat retina causes swelling of glial somata under hypoosmotic conditions. The effect of albumin was dose-dependent, with half-maximal and maximal effects at 10 nM and 1 microM, respectively, and was mediated by activation of transforming growth factor-beta receptor type II, oxidative stress, and the production of arachidonic acid and prostaglandins. Albumin-induced glial swelling was prevented by glutamate and purinergic receptor agonists. The data suggest that serum albumin may induce glial swelling in the presence of osmotic gradients., (2010 Elsevier B.V. All rights reserved.)

Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume.

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 Müller cells, the principal macroglial cells of the retina. The P2Y(1) and the P2Y(4) receptors, but no other subtypes, were unequivocally localized on Müller cells. P2Y(1) was expressed from postnatal day 5 (P5) on and mediated a calcium response to ATP in Müller cells as well as a volume regulatory signaling cascade preventing Müller cells from swelling under hypotonic conditions. Differentiation of Müller cells was accompanied by a change of the calcium response pattern; the calcium responses in Müller cell endfeet persisted, but ATP responsiveness of Müller cell somata disappeared. P2Y(4) immunoreactivity was observed in Müller cell endfeet and synaptic terminals of rod bipolar cells from P20 on. Activated protein kinases were detected by immunohistochemistry; p-ERK occurred in Müller cells and amacrine cells, whereas p-Akt was detected in bipolar cells. Our data indicate that purinergic signaling via P2Y(1) and P2Y(4) receptors might contribute to differentiation processes in the postnatal retina., ((c) 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.

Purpose: Osmotic swelling of Müller glial cells has been suggested to contribute to retinal edema. We determined the role of adenosine signaling in the inhibition of Müller cell swelling in the murine retina., Methods: The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution. Retinal tissues were freshly isolated from wild-type mice and mice deficient in A(1) adenosine receptors (A(1)AR(-/-)), or cultured as whole-mounts for three days. The potassium conductance of Müller cells was recorded in isolated cells, and retinal slices were immunostained against Kir4.1., Results: Hypotonic exposure for 4 min induced a swelling of Müller cell bodies in retinal slices from A(1)AR(-/-) mice but not wild-type mice. Pharmacological inhibition of A(1) receptors or of the ecto-5'-nucleotidase induced hypoosmotic swelling of Müller cells from wild-type mice. Exogenous adenosine prevented the swelling of Müller cells from wild-type but not A(1)AR(-/-) mice. The antiinflammatory corticosteroid, triamcinolone acetonide, inhibited the swelling of Müller cells from wild-type mice; this effect was blocked by an antagonist of A(1) receptors. The potassium conductance of Müller cells and the Kir4.1 immunolabeling of retinal slices were not different between A(1)AR(-/-) and wild-type mice, both in freshly isolated tissues and retinal organ cultures., Conclusions: The data suggest that autocrine activation of A(1) receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina. The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling.

Prevention of osmotic swelling of retinal glial (Müller) cells is required to avoid detrimental decreases in the extracellular space volume during intense neuronal activity. Here, we show that glial cells in slices of the wildtype mouse retina maintain the volume of their somata constant up to approximately 4 min of perfusion with a hypoosmolar solution. However, calcium chelation with BAPTA/AM induced a rapid swelling of glial cell bodies. In glial cells of retinas from inositol-1,4,5-trisphosphate-receptor type 2-deficient (IP(3)R2(-/-)) mice, hypotonic conditions caused swelling of the cell bodies without delay. Exogenous ATP (acting at P2Y(1) receptors) prevented the swelling of glial cells in retinal slices from wildtype but not from IP(3)R2(-/-) mice. Müller cells from IP(3)R2(-/-) mice displayed a strongly reduced amplitude of the ATP-evoked calcium responses as compared to cells from wildtype mice. It is concluded that endogenous calcium signaling mediated by IP(3)R2 is required for the osmotic volume regulation of retinal glial cells.

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.

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.

Purpose: Several eye diseases are accompanied by inflammatory processes. The authors examined the expression of the proinflammatory chemokine CXCL8 and the corresponding receptors in healthy human retinas, in cellular membranes from patients with proliferative vitreoretinopathy (PVR) or human glial cell cultures and in an animal model of PVR in rabbit eyes., Methods: The authors used immunohistochemical methods, Western blotting, RT-PCR, and real time RT-PCR to characterize the expression of CXCL8, CXCR1, and CXCR2 in human and rabbit retinas. Functionality of the receptors in cultured glial cells was tested by Ca(2+) imaging., Results: Immunohistochemical examinations of normal human and rabbit retinas revealed a distinct expression of CXCR1 and CXCR2 in several neuronal cell types. CXCL8 mRNA was demonstrated only by RT-PCR in normal retinas, and receptor expression was confirmed by Western blotting and RT-PCR. The presence of CXCR1 and CXCR2, but not CXCL8, was detected by immunostaining in glial fibrillary acidic protein-positive glial cells of cellular PVR membranes. Immunoreactivity for CXCL8, CXCR1, and CXCR2 was observed in virtually all cultured glial cells and in the human Müller cell line MIO-M1. Müller cells responded to the application of CXCL8 with increased cytosolic Ca(2+) concentrations. In PVR rabbit retinas, CXCR1 expression is increased in Müller cells, and CXCL8 and CXCR2 are strongly expressed in microglial cells., Conclusions: Expression of CXCL8 and CXCL8 receptors in glial cells of human PVR membranes and rabbit PVR retinas suggests an involvement in glial reactivity. Furthermore, the prominent expression of CXCR1 and CXCR2 in neurons of the healthy human and rabbit retina suggests additional physiological functions.

The anti-inflammatory glucocorticoid, triamcinolone acetonide, is used clinically for the rapid resolution of diabetic macular edema. Osmotic swelling of glial cells may contribute to the development of retinal edema. Triamcinolone inhibits the swelling of retinal glial cells of diabetic rats. Here, we determined whether the effect of triamcinolone is mediated by a receptor-dependent mechanism. Hyperglycemia was induced in rats with streptozotocin injection. After 6-10 months, the swelling properties of glial cells in retinal slices upon hypotonic challenge were determined. Nucleotide-degrading ecto-enzymes were immunostained in retinal slices and glial cells. Hypotonic challenge did not change the size of glial cell bodies from control retinas but induced swelling of cells from diabetic animals. Triamcinolone inhibited glial cell swelling; this effect was prevented by a selective antagonist of adenosine A1 receptors, an inhibitor of nucleoside transporters, inhibitors of adenylyl cyclase and protein kinase A activation, and inhibitors of potassium and chloride channels. In diabetic (but not control) retinas, the effect of triamcinolone apparently involves extracellular nucleotide degradation. Glial cells from diabetic retinas displayed immunolabeling against nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) which was not observed in control retinas. The mRNA expression for NTPDase1 was significantly increased in the retina of diabetic rats. It is suggested that triamcinolone induces the release and formation of endogenous adenosine that subsequently activates A1 receptors resulting in ion efflux through potassium and chloride channels and prevention of osmotic swelling. Whereas adenosine is liberated via facilitated transport in control retinas, an extracellular formation of adenosine contributes to the effect of triamcinolone in diabetic retinas.

Purpose: To investigate the membrane physiology of Müller glial cells from retinae of DBA/2J mice (which develop ocular hypertension) and of C57BL/6 control mice of different ages., Methods: Retinae were obtained at the ages of 3, 6, and 12 months from DBA/2J mice and from C57BL/6 controls. Immunohistochemistry was performed using antibodies against glial fibrillary acidic protein (GFAP). Whole-cell membrane currents, membrane potentials and capacitances were recorded from freshly isolated Müller cells., Results: Strong immunostaining for GFAP was found in Müller cells of 12-month-old DBA/2J mice, whereas only astrocytes were immunopositive in C57BL/6 retinae. No significant alterations of membrane currents or potentials of Müller cells from DBA/2J mice as compared to controls were observed at any age; however, the membrane capacitance was increased in Müller cells from DBA/2J mice at the age of 6 months., Conclusions: Although Müller cells of DBA/2J mice display some symptoms of reactive gliosis, the lack of significant alterations of the membrane physiology confirm previous data demonstrating that these cells undergo a nonproliferative gliosis.

Purpose: In addition to photoreceptor degeneration, excessive light causes degenerative alterations in the inner retina and ganglion cell death. A disturbance in osmohomeostasis may be one causative factor for the alterations in the inner retina. Because Müller cells mediate inner retinal osmohomeostasis (mainly through channel-mediated transport of potassium ions and water), the authors investigated whether these cells alter their properties in response to excessive blue light., Methods: Retinas of adult rats were exposed to blue light for 30 minutes. At various time periods after treatment, retinal slices were immunostained against glial fibrillary acidic protein and potassium and water channel proteins (Kir4.1, aquaporin-1, aquaporin-4). Patch-clamp recordings of potassium currents were made in isolated Müller cells, and the swelling of Müller cell bodies was recorded in retinal slices., Results: After blue light treatment, Müller cells displayed hypertrophy and increased glial fibrillary acidic protein. The immunostaining of the glial water channel aquaporin-4 was increased in the outer retina, whereas the immunostaining of the photoreceptor water channel aquaporin-1 disappeared. Blue light treatment resulted in a decrease and a dislocation of the Kir4.1 protein in the whole retinal tissue and a decrease in the potassium conductance of Müller cells. Hypo-osmotic stress evoked a swelling of Müller cell bodies in light-treated retinas that was not observed in control tissues., Conclusions: The decrease in functional Kir channels may result in a disturbance of retinal potassium and water homeostasis, contributing to the degenerative alterations of the inner retina after exposure to blue light.

The dystrophin-associated proteins (DAPs) complex consisting of dystroglycan, syntrophin, dystrobrevin, and sarcoglycans in muscle cells is associated either with dystrophin or its homolog utrophin. In rat retina, a similar complex was found associated with dystrophin-Dp71 that serves as an anchor for the inwardly rectifying potassium channel Kir4.1 and the aqueous pore, aquaporin-4 (AQP4). Here, using immunofluorescence imaging of isolated retinal Müller glial cells and co-immunoprecipitation experiments performed on an enriched Müller glial cells end-feet fraction, we investigated the effect of Dp71 deletion on the composition, anchoring, and membrane localization of the DAPs-Kir4.1 and/or -AQP4 complex. Two distinct complexes were identified in the end-feet fraction associated either with Dp71 or with utrophin. Upon Dp71 deletion, the corresponding DAPs complex was disrupted and a compensating utrophin upregulation was observed, accompanied by diffuse overall staining of Kir4.1 along the Müller glial cells and redistribution of the K(+) conductance. Dp71 deficiency was also associated with a marked reduction of AQP4 and beta-dystroglycan expression. Furthermore, it was observed that the Dp71-DAPs dependent complex could be, at least partially, associated with a specific membrane fraction. These results demonstrate that Dp71 has a central role in the molecular scaffold responsible for anchoring AQP4 and Kir4.1 in Müller cell end-feet membranes. They also show that despite its close relationship to the dystrophin proteins and its correlated upregulation, utrophin is only partially compensating for the absence of Dp71 in Müller glial cells., ((c) 2008 Wiley-Liss, Inc.)

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

Retinal glial (Müller) cells are proposed to mediate retinal potassium homeostasis predominantly by potassium transport through inwardly rectifying K(+) (Kir) channels. Retinal gliosis is often associated with a decrease in glial potassium conductance. To determine whether this decrease is caused by a downregulation of glial Kir channels, we investigated a rabbit model of proliferative vitreoretinopathy (PVR) which is known to be associated with proliferative gliosis. The membrane conductance of control Müller cells is characterized by large Kir currents whereas Müller cells of PVR retinas displayed an almost total absence of Kir currents. In control tissues, Kir2.1 immunoreactivity is localized in the inner stem processes and endfeet of Müller cells whereas Kir4.1 immunoreactivity is largely confined to the Müller cell endfeet. In PVR retinas, there is a mislocation of Kir channel proteins, with Kir4.1 immunoreactivity detectable in Müller cell fibers throughout the whole retina, and a decrease of immunoreactivity in the cellular endfeet. Real-time PCR analysis revealed no alteration of the Kir4.1 mRNA levels in PVR retinas as compared to the controls but a slight decrease in Kir2.1 mRNA. Western blotting showed no difference in the Kir4.1 protein content between control and PVR retinas. The data suggest that proliferative gliosis in the retina is associated with a functional inactivation of glial Kir channels that is not caused by a downregulation of the channel proteins but is associated with their mislocation in the cell membrane.

Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y(1) and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors.

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.

Background: Under normal conditions, Müller cells support neuronal activity and the integrity of the blood-retinal barrier, whereas gliotic alterations of Müller cells under pathological conditions may contribute to retinal degeneration and edema formation. A major function of Müller cells is the fluid absorption from the retinal tissue, which is mediated by transcellular water transport coupled to currents through potassium channels., Methods: Alterations of retinal Müller cells under pathological conditions were investigated by immunohistochemistry and recording their behavior under osmotic stress., Results: In animal models of various retinopathies, e.g., retinal ischemia, ocular inflammation, retinal detachment, and diabetes, it was found that Müller cells decrease the expression of their major potassium channel (Kir4.1). This alteration is associated with an impairment of the rapid water transport across Müller cell membranes, as recognizable in the induction of cellular swelling under hypoosmolar conditions. Osmotic swelling of Müller cells is also induced by oxidative stress and by inflammatory mediators such as arachidonic acid and prostaglandins., Conclusions: The data suggest that a disturbed fluid transport through Müller 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 Müller 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 Müller 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 Müller cells.

Purpose: To determine whether experimental retinal detachment causes an alteration in Ca2 +-activated, big conductance K+ (BK) currents of Müller glial cells., Methods: Rhegmatogenous retinal detachment was induced in porcine eyes. Müller cells were acutely isolated from control retinas and from retinas that were detached for 7 days. BK currents were detected by using the BK channel opener and the blocker phloretin and tetraethylammonium, respectively., Results: In addition to cellular hypertrophy and a decrease in inward rectifier K+ currents, Müller cells from detached retinas showed an increase in the amplitude of currents mediated by BK channels (850 +/- 105 pA) when compared with cells from control retinas (228 +/- 60 pA; p < 0.001). Similarly, the density of the BK channel-mediated currents was greater in cells from detached retinas (12.32 +/- 1.52 pA/pF) compared with control cells (4.07 +/- 1.07 pA/pF; p < 0.001). The increase in BK currents was correlated with the decrease of the inward rectifier K+ currents., Conclusions: It is suggested that an increase in the expression of functional BK channels may be involved in gliotic responses of Müller cells after retinal detachment (e.g., in mitogen-induced Ca2+ responses and cellular proliferation).

Detachment of the neural retina from the pigment epithelium may be associated with tissue edema; however, the mechanisms of fluid accumulation are not understood. Because retinal detachment is usually not accompanied by vascular leakage, we investigated whether the osmotic swelling characteristics of retinal glial (Müller) cells are changed after experimental detachment of the porcine retina. Osmotic stress, induced by application of a hypotonic bath solution to retinal slices, caused swelling of Müller cell bodies in 7-day-detached retinas, but no swelling was inducible in slices of control retinas. Müller cell somata in slices of retinal areas that surround local detachment in situ also showed osmotic swelling, albeit at a smaller amplitude. The amplitude of osmotic Müller cell swelling correlated with the decrease in the K+ conductance, suggesting a causal relationship between both gliotic alterations. Further factors implicated in Müller cell swelling were inflammatory mediators and oxidative stress. We propose that a dysregulation of the ion and water transport through Müller cells may impair the fluid absorption from the retinal tissue, resulting in chronic fluid accumulation after detachment. This knowledge may lead to a better understanding of the mechanisms involved in retinal degeneration after detachment.

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.

A major function of glial cells is the control of osmotic and ionic homeostasis, mediated by K+ and water movements predominantly through inwardly rectifying K+ (Kir) and aquaporin water channels. It has been suggested that K+ currents through Kir channels are implicated in the regulation of glial cell volume. Here, we investigated whether the developmental increase in Kir channel expression in Müller glial cells of the rat retina is associated with an alteration of cell volume regulation under anisoosmotic conditions. Around the time of eye opening at postnatal day (P) 15, developing retinal glial cells fully alter the profile of their membrane conductances, from a current pattern with prominent fast transient K+ and Na+ currents to a pattern of noninactivating currents through Kir and delayed rectifier K+ channels. Concomitantly, aquaporins-1 and -4 are expressed in the developing retina. This is accompanied by a conspicuous alteration of the swelling characteristics of cells; somata of immature glial cells in early postnatal retinas (P5-P15) swell under hypotonic stress but no swelling is inducible in mature cells at P18 and thereafter. However, glial cells at all developmental stages swell when their Kir channels are blocked by Ba2+. The postnatal maturation of Kir channel currents and volume regulation in retinal glial cells is delayed by visual deprivation. The data suggest that Kir channels are crucially involved in osmotic volume homeostasis of mature glial cells, and that the absence of Kir channels in immature cells is a major cause of their insufficient volume regulation., (2006 Wiley-Liss, Inc.)

Müller glial cells span the entire thickness of the tissue, and ensheath all retinal neurons, in vertebrate retinae of all species. This morphological relationship is reflected by a multitude of functional interactions between neurons and Müller cells, including a 'metabolic symbiosis' and the processing of visual information. Müller cells are also responsible for the maintenance of the homeostasis of the retinal extracellular milieu (ions, water, neurotransmitter molecules, and pH). In vascularized retinae, Müller cells may also be involved in the control of angiogenesis, and the regulation of retinal blood flow. Virtually every disease of the retina is associated with a reactive Müller cell gliosis which, on the one hand, supports the survival of retinal neurons but, on the other hand, may accelerate the progress of neuronal degeneration: Müller cells protect neurons via a release of neurotrophic factors, the uptake and degradation of the excitotoxin, glutamate, and the secretion of the antioxidant, glutathione. However, gliotic Müller cells display a dysregulation of various neuron-supportive functions. This contributes to a disturbance of retinal glutamate metabolism and ion homeostasis, and causes the development of retinal edema and neuronal cell death. Moreover, there are diseases evoking a primary Müller cell insufficiency, such as hepatic retinopathy and certain forms of glaucoma. Any impairment of supportive functions of Müller cells, primary or secondary, must cause and/or aggravate a dysfunction and loss of neurons, by increasing the susceptibility of neurons to stressful stimuli in the diseased retina. On the contrary, Müller cells may be used in the future for novel therapeutic strategies to protect neurons against apoptosis (somatic gene therapy), or to differentiate retinal neurons from Müller/stem cells. Meanwhile, a proper understanding of the gliotic responses of Müller cells in the diseased retina, and of their protective vs. detrimental effects, is essential for the development of efficient therapeutic strategies that use and stimulate the neuron-supportive/protective-and prevent the destructive-mechanisms of gliosis.

Purpose: Detachment of the neural retina from the pigment epithelium causes, in addition to photoreceptor deconstruction and neuronal cell remodeling, an activation of glial cells. It has been suggested that gliosis contributes to the impaired recovery of vision after reattachment surgery that may involve both formerly detached and nondetached retinal areas. Müller and microglial cell reactivity was monitored in a porcine model of rhegmatogenous retinal detachment, to determine whether gliosis is present in detached and nondetached retinal areas., Methods: Local detachment was created in the eyes of adult pigs by subretinal application of hyaluronate. Retinal slices were immunostained against glial intermediate filaments and K+ and water channel proteins (aquaporin-4, Kir4.1, Kir2.1), and P2Y receptor proteins. In retinal wholemounts, adenosine 5'-triphosphate (ATP)-induced intracellular Ca2+ responses of Müller cells were recorded, and microglial and immune cells were labeled with Griffonia simplicifolia agglutinin isolectin I-B4. K+ currents were recorded from isolated Müller cells., Results: At 3 and 7 days after surgery, Müller cells in detached retinas showed a pronounced gliosis, as revealed by the increased expression of the intermediate filaments glial fibrillary acidic protein and vimentin, by the decrease of Kir4.1 immunoreactivity and of the whole-cell K+ currents, and by the increased incidence of cells that showed Ca2+ responses on stimulation of purinergic (P)2 receptors by ATP. By contrast, the immunohistochemical expression of Kir2.1 and aquaporin-4 were not altered after detachment. The increase in the expression of intermediate filaments, the decrease of the whole-cell K+ currents and of the Kir4.1 immunolabeling, and the increase in the Ca2+ responsiveness of Müller cells were also observed in attached retinal areas surrounding the focal detachment. The density of microglial-immune cells at the inner surface of the retinas increased in both detached and nondetached retinal areas. The immunoreactivities for P2Y1 and P2Y2 receptor proteins apparently increased only in detached areas., Conclusions: Reactive responses of Müller and microglial cells are not restricted to detached retinal areas but are also observed in nondetached regions of the porcine retina. The gliosis in the nondetached retina may reflect, or may contribute to, neuronal degeneration that may explain the impaired recovery of vision observed in human subjects after retinal reattachment surgery.

Ischemia-reperfusion of the rat retina causes gliosis of Müller cells that is associated with a decrease of their K+ conductance. By using quantitative PCR and immunohistochemical staining of retinal slices, we investigated the effect of transient ischemia-reperfusion on retinal expression of two inward-rectifying K+ (Kir) channels, Kir4.1 and Kir2.1. In control retinas, Müller cells prominently expressed both Kir4.1 and Kir2.1 proteins. At 7 days after reperfusion, the expression of Kir4.1 protein was strongly downregulated, while the Kir2.1 protein expression remained unaltered. The expression of Kir4.1 mRNA was reduced by 55% after ischemia while the expression of Kir2.1 mRNA was not altered. The data suggest that the glial expression of distinct Kir channels is differentially regulated after retinal ischemia, with deletarious consequences for K+ ion and water homeostasis.

The development of edema in the diabetic retina may be caused by vascular leakage and glial cell swelling. To determine whether diabetic retinopathy alters the swelling characteristics of retinal glial cells and changes the properties of the glial membrane K+ conductance, isolated retinas and glial cells of rats were investigated at 4 and 6 months of chemical diabetes. After 6 months of hyperglycemia, application of a hypotonic solution to retinal slices induced swelling of glial cell bodies, a response not observed in control retinas. The osmotic glial cell swelling was blocked by inhibitors of phospholipase A2 or cyclooxygenase and by a thiol-reducing agent. Glial cells from diabetic retinas displayed a decrease of K+ currents that was associated with an altered subcellular distribution of the K+ conductance and a loss of perivascular Kir4.1 protein. The observation that swelling of cells in control retinas was inducible with K+ channel-blocking Ba2+ ions suggests a relationship between decreased K+ inward currents and osmotic cell swelling in diabetic retinas. The data show that glial cells in diabetic retinas are more sensitive to osmotic stress, which is associated with a decrease of K+ currents, than cells in control retinas. It is suggested that these alterations may be implicated in the development of diabetic retinal edema.

The glucocorticoid triamcinolone acetonide is clinically used for the treatment of macular edema. However, the edema-resolving mechanisms of triamcinolone are incompletely understood. Since cell swelling is a central cause of cytotoxic edema in the brain and retina, we determined the effects of triamcinolone acetonide on the swelling of retinal ganglion and Müller glial cells in acutely isolated retinas from rats and guinea pigs in situ. Triamcinolone acetonide (100 microM) had no effect on the swelling of ganglion cells that was evoked in isolated whole mounts of the guinea pig retina by acute application of glutamate (1 mM) or high K+ (50 mM). However, triamcinolone reversed the osmotic swelling of Müller glial cells in retinas of the rat that was observed under various experimental conditions: in retinas isolated at 3 days after transient retinal ischemia, in retinas of eyes with lipopolysaccharide-induced ocular inflammation, and in control retinas in the presence of Ba2+ (1 mM), H2O2 (200 microM), arachidonic acid (10 microM), or prostaglandin E2 (30 nM). The inhibiting effect of triamcinolone on osmotic glial cell swelling was mediated by stimulation of transporter-mediated release of endogenous adenosine and subsequent A1 receptor activation, resulting in an elevation of the intracellular cAMP level and activation of the protein kinase A, and, finally, in an opening of extrusion pathways for K+ and Cl- ions. The inhibitory effect on the cytotoxic swelling of glial cells may contribute to the fast edema-resolving effect of vitreal triamcinolone observed in human patients.