Thrombospondin-1 is produced by retinal glial cells and inhibits the growth of vascular endothelial cells.

BACKGROUND/AIMS: By the release of antiangiogenic factors, Müller glial cells provide an angiostatic environment in the normal and ischemic retina. We determined whether Müller cells produce thrombospondin-1 (TSP-1), a known inhibitor of angiogenesis. METHODS: Secretion of TSP-1 by cultured Müller cells was determined with ELISA. Slices of rat retinas and surgically excised retinal membranes of human subjects were immunostained against TSP-1 and the glial marker vimentin. The effects of TSP-1 on the growth of bovine retinal endothelial cells (BRECs) and activation of ERK1/2 were determined with DNA synthesis and migration assays, and Western blotting, respectively. RESULTS: Cultured Müller cells secrete TSP-1 under normoxic and hypoxic (0.2% O2) conditions. Secretion of TSP-1 was increased in hypoxia compared to normoxia. In rat retinal slices, glial, retinal ganglion, and possibly horizontal cells were stained for TSP-1. Retinal glial cells in preretinal membranes from human subjects with nonhypoxic epiretinal gliosis (macular pucker) and proliferative diabetic retinopathy, respectively, were immunopositive for TSP-1. Exogenous TSP-1 reduced the VEGF-induced proliferation and migration of BRECs and decreased the phosphorylation level of ERK1/2 in BRECs. CONCLUSION: The data suggest that Müller cells are one major source of TSP-1 in the normal and ischemic retina. Glia-derived TSP1 may inhibit angiogenic responses in the ischemic retina.

Müller glial cells inhibit proliferation of retinal endothelial cells via TGF-ß2 and Smad signaling.

Neovascularization is a sight-threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)-ß, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Müller) cells produce TGF-ß2 under physiological and post-ischemic conditions. To characterize glial cell-derived mediators of angiogenesis regulation in glial-endothelial interactions in the retina, we co-cultured primary Müller cells and bovine microvascular retinal endothelial cells (BRECs). Müller cell-derived TGF-ß2 was bound by the BRECs, which were found to express serine/threonine kinase TGF-ß receptors, and stimulated TGF-ß-dependent anti-proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF-ß2 as well as by the presence of Müller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti-angiogenic action of Müller cell-derived TGF-ß2: (i) binding of TGF-ß2 to BRECs is mediated by the type-II TGF-ß receptor, leading to (ii) activation and phosphorylation of receptor-activated Smads; (iii) Müller cell-derived TGF-ß2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal-regulated kinase (ERK)-1/-2. Neutralizing TGF-ß or TGF-ß type-II receptor or blocking the activation of Smads partially abrogated the effect of Müller cell-conditioned media on BRECs. Together, our data suggest that Müller cells release TGF-ß2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context-dependent action of TGF-ß2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti-angiogenic role of TGF-ß therein.

Effects of arteriolar constriction on retinal gene expression and Müller cell responses in a rat model of branch retinal vein occlusion.

BACKGROUND: To investigate the effect of induced arteriolar constriction (AC) on alterations in gene expression of factors implicated in the development of edema in branch retinal vein occlusion (BRVO). METHODS: In Brown-Norway rats, BRVO was induced by laser photocoagulation of the veins in one half of the retina. AC of the afferent arterioles was performed 30 min later. We then determined the expression of Vegfa, Vegfb, Pedf, Kir4.1, Aqp4, Aqp1, Il1ß, and Il6 with real-time polymerase chain reaction (RT-PCR) in the neuroretina and retinal pigment epithelium (RPE) after 1, 3, and 7 days. Immunostaining against GFAP, aquaporin (AQP)-4, and Kir4.1 was performed on days 1 and 3. RESULTS: BRVO resulted in transient upregulation of Vegfa in the neuroretina on day 1. The expressions of Kir4.1, AQP4, and AQP1 were downregulated, and Il1ß and Il6 were strongly upregulated, on days 1 and 3. The retinal distribution of GFAP and AQP4 proteins remained unaltered, while the Kir4.1 protein displayed redistribution from polarized to uniform retinal distribution. AC accelerated the restoration of downregulated Kir4.1, Aqp4, and Aqp1 in the RPE, of Kir4.1 in the neuroretina, and of upregulated Il6 in the neuroretina. AC did not influence the gliotic alterations of Müller cells and the redistribution of the Kir4.1 protein. CONCLUSION: Constriction of the afferent artery in the BRVO region accelerated the restoration of potassium channels and Il6. These alterations may contribute to faster resorption of retinal edema, and may decrease the level of inflammation.

Basic fibroblast growth factor contributes to a shift in the angioregulatory activity of retinal glial (Müller) cells.

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK-1/-2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under physiological conditions or transient hypoxia seem to provide an anti-angiogenic environment, but long-lasting hypoxia causes the release of bFGF, which might significantly co-stimulate neovascularization in the retina.

Three-dimensional spheroidal culture visualization of membranogenesis of Bruch's membrane and basolateral functions of the retinal pigment epithelium.

PURPOSE: Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruch's membrane, which may be related to AMD. Conventional in vitro cell culture is limited in its ability to observe the epithelial functions on the basal side. The purpose of this study was to develop a three-dimensional culture system to observe basolateral functions of the RPE. METHODS: Isolated human RPE cells were cultured in a viscous medium on a rounded-bottom culture dish, resulting in spheroid formation. The appearance and size of the spheroids were assessed by light microscopy. Spheroids were fixed in 4% paraformaldehyde for immunohistochemistry or sampled for Western blotting. For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), spheroids were postfixed in 1% osmium tetroxide. RESULTS: The spheroids had a differentiated RPE monolayer with a thin elastic layer, a main layer of Bruch's membrane, on their surface and showed outward deposition of lipoproteins with apoB-100. TEM revealed widely spaced collagen, which was identified as condensation of collagen fibrils by SEM. SEM showed deposition of membranous debris and lipid particles, which have been observed in human Bruch's membrane. Western blotting showed expression of RPE differentiation markers and components of Bruch's membrane and RPE lipoproteins. CONCLUSIONS: This model provides direct views of epithelialization processes involving elastogenesis and functions at the basolateral side such as lipoprotein deposition and may elucidate not only unknown epithelial behaviors but also the pathogenesis of RPE-related diseases.

The axon guidance molecule Netrin-4 is expressed by Müller cells and contributes to angiogenesis in the retina.

Retinal glial (Müller) cells are involved in a wide range of developmental mechanisms, including axon guidance and angiogenesis. This study was undertaken to explore whether Netrin-4, an axonal guidance molecule, is expressed by Müller cells and promotes angiogenesis-related activities. Netrin-4 was found through all retinal layers, and its expression was demonstrated in Müller cells, retinal pigment epithelium cells and bovine retinal endothelial cells (BRECs). Co-localization of Netrin-4 with Müller cell-specific molecules [cellular retinaldehyde-binding protein (cRALBP), vimentin] was observed in the ganglion cell layer, nerve fiber layer, and at the outer limiting membrane. Under hypoxic conditions, the release of Netrin-4 from Müller cells was increased, with mRNA levels upregulated in a hypoxia-inducible factor-1-dependent manner and dependent on the concomitantly induced release of vascular endothelial growth factor. These findings were consistent with an intensified immunofluorescence of Netrin-4 labeling in the postischemic retinas after ischemia-reperfusion. Netrin-4 stimulated BRECs to increase phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK)-1/-2, and p38, in a dose-dependent manner. Synthetic inhibitors of the MAP kinases were able to suppress Netrin-4-induced migration and proliferation of BRECs suggesting that both MAP kinases are differentially involved in Netrin-4-induced angiogenesis. Two receptors for Netrins, i.e., deleted in colorectal cancer (DCC) and uncoordinated-5-homolog 1 (Unc5H1), were detected in BRECs. DCC is at least partially required for Netrin-4-induced activation of ERK-1/-2. These data suggest that Müller glial cells contribute to, and may modulate, retinal Netrin-4 levels. This may be a novel pathway of Müller cell-mediated control of retinal angiogenesis, particularly under hypoxic/ischemic conditions when the cells upregulate Netrin-4 expression.

Physiologic properties of Müller cells from human eyes affected with uveal melanoma.

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.

Transcriptional regulation of aquaporins in the ischemic rat retina: upregulation of aquaporin-9.

PURPOSE/AIM: To determine the transcriptional regulation of retinal aquaporins (AQPs) in rat models of transient and permanent retinal ischemia, and to prove the effects of chemical hypoxia, oxidative stress, glucose, and osmotic alterations on the expression of AQP9 in cultured human retinal pigment epithelium (RPE) cells. MATERIALS AND METHODS: Transient retinal ischemia-reperfusion in rats was induced by elevation of the intraocular pressure for 1 hour. Permanent retinal ischemia was induced by argon laser-induced retinal vein occlusion. The mRNA levels were determined one day after ischemia. RESULTS: Transient and permanent ischemia of the rat retina resulted in downregulation of AQPs 1, 3, 4, 5, 6, 8, and 11 in the RPE and/or neural retina. Pressure-induced transient retinal ischemia-induced upregulation of AQP9 in the neuroretina and RPE, and of AQ12 in the neuroretina. Retinal vein occlusion induced upregulation of AQP0 in the neuroretina and RPE, and of AQP9 and AQP12 in the neuroretina. In cultured human RPE cells, transcriptional expression of AQP9 was stimulated by chemical hypoxia, oxidative stress, VEGF, and high glucose. CONCLUSIONS: The data may suggest that the expression of retinal AQP9 is regulated by metabolic and oxidative stress. Upregulation of AQP9 in RPE cells may prevent lactic acidosis and subretinal edema under ischemic and oxidative stress conditions.

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.

Immunolocalization of aquaporin-6 in the rat retina.

Previous RT-PCR experiments revealed that the neural retina of the rat contains gene transcripts of numerous aquaporins (AQPs), including AQP6 (Tenckhoff et al., Neuroreport 16 (2005) 53-56). In the present study, we investigated the localization of AQP6 immunoreactivity in slices of the rat neural retina, and determined whether blue light injury of the retina affects the tissue distribution of this channel. AQP6 immunoreactivity was found to be selectively localized to the outer plexiform layer. Around the ribbon synapses in this layer, AQP6 labeling was co-localized with the glial water channel AQP4. AQP6 labeling was not colocalized with the marker of horizontal cells, calbindin, nor with the marker of rod bipolar cells, protein kinase Cα. Along with the degeneration of photoreceptor cells after blue light treatment of the retina, AQP6-labeled ribbon synapses disappeared, and a punctate AQP6 staining redistributed into the inner nuclear layer. The co-localization of AQP6 and the glial water channel AQP4 suggests a preferential localization of AQP6 in glial membranes that surround the ribbon synapses in the outer plexiform layer. AQP6 might be involved in the glia-mediated osmo and ion regulation of the extracellular space in this layer.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression.

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.

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.

Alterations in protein expression and membrane properties during Müller cell gliosis in a murine model of transient retinal ischemia.

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.

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.

Role of retinal glial cells in neurotransmitter uptake and metabolism.

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.

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.

Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina.

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.