Ca2+ microfluorimetry in retinal Müller glial cells.

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.

The human Müller cell line MIO-M1 expresses opsins.

PURPOSE: To determine whether the human Müller cell line Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) expresses opsins. METHODS: The gene expression of opsins was determined by reverse-transcription PCR (RT-PCR). The presence of opsin proteins was determined by western blotting and immunocytochemistry. The light sensitivity of the cells was examined with imaging experiments using the calcium-sensitive dye Fluo-4. RESULTS: MIO-M1 cells express glial (glutamine synthase [GLUL], vimentin [VIM], glial fibrillary acidic protein [GFAP], cellular retinaldehyde-binding protein [RLBP1], glial high-affinity glutamate transporter [SLCA1], aquaporin-4 [AQP4], inwardly rectifying potassium channel Kir4.1 [Kir4.1]), neuronal (Thy-1 cell surface antigen [THY1], heavy neurofilament polypeptide [NEFH], microtubule-associated protein 2 [MAP2], neurogenic differentiation 1 [NEUROD1], neuronal nuclei [NEUN]), and neural progenitor markers (Nestin [NES], paired-type homeobox transcription factor [PAX6], neurogenic locus notch homolog 1 [NOTCH1]). The cells contain mRNA for the following opsins: blue opsin (OPN1SW), rhodopsin (OPN2), panopsin (OPN3), melanopsin (OPN4), neuropsin (OPN5), and peropsin (RRH), as well as for the transducins (guanine nucleotide binding protein [GNAZ], alpha transducing activity polypeptide 1 [GNAT1], alpha transducing activity polypeptide 2 [GNAT2]). The presence of blue opsin and melanopsin was confirmed with immunocytochemistry and western blotting. The immunoreactivity and mRNA of red-green opsin were found in some but not all cultures, while the immunoreactivity for rhodopsin was absent in all cultures investigated. Repetitive stimulation with 480 nm light evoked slow and fast transient calcium responses in the majority of cells investigated, while irradiation with 600 nm light was ineffective. CONCLUSIONS: The human Müller cell line MIO-M1 expresses opsins. This suggests immortalized Müller cells could be used as a cellular source to produce human opsins for their potential application as therapeutic agents in patients with retinitis pigmentosa.

Effects of intravitreal bevacizumab (Avastin) on the porcine retina.

BACKGROUND: To evaluate the effects of intravitreal bevacizumab (Avastin) on the porcine retina, with respect to structural alterations, expression of proteins involved in apoptosis (bax, caspase-3, caspase-9) and gliosis (vimentin, GFAP), expression of factors which influence the development of vascular edema (VEGF, PEDF), and of membrane channels implicated in retinal osmohomeostasis (Kir4.1, aquaporin-1, aquaporin-4). METHODS: One eye of seven adult pigs received a single intravitreal injection of bevacizumab (1.25 mg). Control eyes received buffered saline. For light and electron microscopy, the eyes were prepared 3 (one animal) and 7 days (two animals) after injection. Retinal slices were immunostained against gliosis- and apoptosis-related proteins. The gene expression was determined in the neuroretina and the retinal pigment epithelium of the remaining four animals with real-time RT-PCR 2 days after injection of bevacizumab. RESULTS: Intravitreal bevacizumab did not induce alterations in the retinal structure, neither at light microscopic nor at electron microscopic level. The photoreceptors were well-preserved; no signs of photoreceptor damage or mitochondrial swelling were observed. Bevacizumab did also not induce reactive gliosis (as indicated by the unaltered immunolocalization of the glial proteins vimentin, GFAP, and glutamine synthetase) or apoptosis (as indicated by the unaltered immunolocalization of bax, caspase-3, and caspase-9). Intravitreal bevacizumab decreased the transcriptional expression of VEGF-A, and increased the expression of Kir4.1 in the neuroretina and pigment epithelium, and of PEDF in the pigment epithelium. Bevacizumab did not alter the transcriptional expression of GFAP, bax, caspase-3, VEGF receptor-1 and -2, and aquaporin-1 and -4. CONCLUSIONS: A single intravitreal injection of bevacizumab does not result in structural changes of the porcine retina, nor in induction of gliosis or apoptosis. The bevacizumab-induced transcriptional downregulation of VEGF and upregulation of Kir4.1 might protect the retina from the development of vascular and cytotoxic edema.

Purinergic signaling involved in Müller cell function in the mammalian retina.

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.

Effects of ischemia-reperfusion on physiological properties of Müller glial cells in the porcine retina.

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.

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina.

In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal/noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones-responsible for acute vision-is roughly 1. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its individual Müller cell-light guide.

Synergistic action of hypoosmolarity and glutamine in inducing acute swelling of retinal glial (Müller) cells.

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.

Involvement of oxidative stress and mitochondrial dysfunction in the osmotic swelling of retinal glial cells from diabetic rats.

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.

Altered immune response in mice deficient for the G protein-coupled receptor GPR34.

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.

Deletion of aquaporin-4 renders retinal glial cells more susceptible to osmotic stress.

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.

Sex steroids inhibit osmotic swelling of retinal glial cells.

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.

Serum albumin induces osmotic swelling of rat retinal glial cells.

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.

Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells.

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.

Functional implication of Dp71 in osmoregulation and vascular permeability of the retina.

Functional alterations of Müller cells, the principal glia of the retina, are an early hallmark of most retina diseases and contribute to their further progression. The molecular mechanisms of these reactive Müller cell alterations, resulting in disturbed retinal homeostasis, remain largely unknown. Here we show that experimental detachment of mouse retina induces mislocation of the inwardly rectifying potassium channels (Kir4.1) and a downregulation of the water channel protein (AQP4) in Müller cells. These alterations are associated with a strong decrease of Dp71, a cytoskeleton protein responsible for the localization and the clustering of Kir4.1 and AQP4. Partial (in detached retinas) or total depletion of Dp71 in Müller cells (in Dp71-null mice) impairs the capability of volume regulation of Müller cells under osmotic stress. The abnormal swelling of Müller cells In Dp71-null mice involves the action of inflammatory mediators. Moreover, we investigated whether the alterations in Müller cells of Dp71-null mice may interfere with their regulatory effect on the blood-retina barrier. In the absence of Dp71, the retinal vascular permeability was increased as compared to the controls. Our results reveal that Dp71 is crucially implicated in the maintenance of potassium homeostasis, in transmembraneous water transport, and in the Müller cell-mediated regulation of retinal vascular permeability. Furthermore, our data provide novel insights into the mechanisms of retinal homeostasis provided by Müller cells under normal and pathological conditions.

Involvement of A(1) adenosine receptors in osmotic volume regulation of retinal glial cells in mice.

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.

Cellular signaling and factors involved in Müller cell gliosis: neuroprotective and detrimental effects.

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.

Calcium responses mediated by type 2 IP3-receptors are required for osmotic volume regulation of retinal glial cells in mice.

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.

Expression and function of P2Y receptors on Müller cells of the postnatal rat retina.

In the postnatal and mature retina, many processes are controlled by the action of nucleotides. Their effects are partly mediated via activation of metabotropic P2Y receptors. However, little is known about the developmental regulation and cellular localization of P2Y receptor subtypes. Combining immunohistochemical and neurophysiological methods, we investigated the developmental expression of P2Y receptors on 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.

Retinal gene expression and Müller cell responses after branch retinal vein occlusion in the rat.

PURPOSE: In a rat model of branch retinal vein occlusion (BRVO), changes in gene expression of factors implicated in the development of retinal edema and alterations in the properties of Müller cells were determined. METHODS: In adult Long-Evans rats, BRVO was induced by laser photocoagulation of retinal veins; untreated eyes served as controls. The mRNA levels of after factors were determined with real-time RT-PCR in the neural retina and retinal pigment epithelium after 1 and 3 days of BRVO: VEGF-A, pigment epithelium-derived factor (PEDF), tissue factor, prothrombin, the potassium channel Kir4.1, and aquaporins 1 and 4. Potassium currents were recorded in isolated Müller cells, and cellular swelling was assessed in retinal slices. RESULTS: In the neural retina, the expression of VEGF was upregulated within 1 day of BRVO and returned to the control level after 3 days. PEDF was upregulated in the neuroretina and retinal pigment epithelium after 3 days of BRVO. Prothrombin, Kir4.1, and both aquaporins were downregulated in the neuroretina. After BRVO, Müller cells displayed a decrease in their potassium currents and an altered distribution of Kir4.1 protein, an increase in the size of their somata, and cellular swelling under hypoosmotic stress that was not observed in control tissues. CONCLUSIONS: BRVO results in a rapid transient increase in the expression of VEGF and a delayed increase in the expression of PEDF. The downregulation of Kir4.1 and aquaporins, the mislocation of Kir4.1 protein, and the osmotic swelling of Müller cells may contribute to the development of edema and neuronal degeneration.

Müller cell gliosis in retinal organ culture mimics gliotic alterations after ischemia in vivo.

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.