Müller glial cells in retinal disease

Virtually all pathogenic stimuli activate Müller cells. Reactive Müller cells exert protective and toxic effects on photoreceptors and neurons. They contribute to oxidative stress and glutamate toxicity due to malfunctions of glutamate uptake and glutathione synthesis. Downregulation of potassium conductance disrupts transcellular potassium and water transport, resulting in neuronal hyperexcitability and edema. Protective effects of reactive Müller cells include upregulation of adenosine 5'-triphosphate (ATP)-degrading ectoenzymes, which enhances the extracellular availability of the neuroprotectant adenosine, abrogation of the osmotic release of ATP, which might protect retinal ganglion cells from apoptosis, and the release of antioxidants and neurotrophic factors. The dedifferentiation of reactive Müller cells to progenitor-like cells might have an impact on future therapeutic approaches. A better understanding of the gliotic mechanisms will be helpful in developing efficient therapeutic strategies aiming at increased protective and regenerative properties and decreased toxicity of reactive Müller cells.     

Energy metabolism in human retinal Müller cells

PURPOSE: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells. METHODS: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase. RESULTS: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. CONCLUSIONS: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.     

Role of interleukin-1β in hypoxia-induced depression of glutamate uptake in retinal Müller cells

Background

It is suggested that hypoxic–ischemic retinal diseases induce loss of retinal ganglion cells. Excess glutamate release is involved in these conditions. A predominant function of Müller cells is to regulate glutamate levels, but in these diseases the function is compromised. The present study was performed to investigate the role of interleukin-1β(IL-1β)on the glutamate uptake in retinal Müller cells under hypoxia and to study the possible mechanism.

Methods

The levels of IL-1β,Kir4.1, and GLAST in retinal Müller cells under hypoxia were analyzed by Western blotting and realtime-RT-PCR, and glutamate uptake assay was undertaken to investigate the activity of GLAST. After being treated with IL-1βunder normoxia, these proteins (Kir4.1 and GLAST) and their mRNAs, and glutamate uptake activity in Müller cells were investigated. To confirm the effect of IL-1βon glutamate uptake activity in Müller cells, addition of IL-1ra was used.

Results

Under hypoxia, Müller cells glutamate uptake, Kir4.1 and GLAST expressions were decreased significantly; however, IL-1βexpression was increased. IL-1βtreatment induced depression of glutamate uptake, decrease of Kir4.1 and GLAST expressions in retinal Müller cells under normoxia. Moreover, addition of IL-1ra significantly ameliorated decreases in Kir4.1 and GLAST expressions, and compromise of glutamate uptake activity in retinal Müller cells under hypoxia.

Conclusions

These findings indicated that decreases in Kir4.1 and GLAST expressions and depression of glutamate uptake in retinal Müller cells under hypoxia may be induced by the inflammatory cytokine IL-1β.     

Regulation of gamma-glutamylcysteine synthetase subunit gene expression in retinal Müller cells by oxidative stress.

PURPOSE: To study regulation of gamma-glutamylcysteine synthetase (GCS) heavy and light subunit gene expression in Müller cells under conditions of oxidative stress. METHODS: Experiments were carried out with an SV40 transformed cell line (rMC-1) that exhibits the phenotype of rat retinal Müller cells. Endogenous glutathione levels were modified by treating cells with diethyl maleate (DEM), D,L-buthionine sulfoximine (BSO), or tert-butylhydroquinone (TBH). In other experiments, cells were grown in either high (28 mM) or normal (5.5 mM) glucose medium for 1 week to examine the effects of hyperglycemia. Cells were processed for reduced glutathione (GSH) measurement, RNA extraction, cell count, and, in some cases, lactate dehydrogenase activity. The steady state mRNA levels of GCS heavy and light subunits were measured by northern blot analysis using specific cDNA probes. Changes in mRNA levels were normalized to beta-actin or 18S rRNA. RESULTS: Treatment with DEM for 30 minutes depleted cell GSH to 20% to 30% of the normal value. GSH content recovered completely 6 hours after returning to normal medium. BSO treatment for 12 hours followed by a medium change for 6 hours resulted in a cell GSH level that was 26% that of untreated cells. If cells were left in BSO for 18 hours, however, GSH levels were reduced to < 1%. Treatment with TBH for 12 hours led to a 77% increase in cellular GSH level. Treatment with DEM, TBH, or BSO for 18 hours led to a significant induction of the mRNA level of the GCS subunits, regardless of glucose concentration in the medium. Shorter BSO treatment exerted no effect. Prolonged hyperglycemia resulted in 30% lower GSH level, 55% lower GCS heavy subunit, and 30% lower GCS light subunit mRNA levels. CONCLUSIONS: Oxidative stress induced the gene expression of GCS heavy and light subunits in Müller cells. The effect of BSO on mRNA levels correlated with the degree of GSH depletion. Prolonged hyperglycemia lowered GCS subunit mRNA and GSH levels.     

Müller cells as players in retinal degeneration and edema

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.     

The role of Müller cells in fibrocontractive retinal disorders

Despite advances in surgical management of fibrocontractive retinal disorders, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) remain major causes of blindness and there is still considerable uncertainty about the origins and roles of the cell types involved. Muller cells and cells identified as retinal glia are consistently identified in epiretinal tissues from both types of disorders. However, their abundance relative to total cell populations is generally low, leaving their role in these disorders uncertain. Studies of Müller cell biology using tissue culture and animal models provide evidence of the remarkable capacity of this cell type for graded responses to environmental insult, the capacity to proliferate, translocate from the retina and alter phenotype and thus, functional characteristics. This review considers the potential roles of Müller cells in fibrocontractive retinal disorders and, in particular, evidence that Müller cells function as an effector cell type in traction retinal detachment associated with PVR and PDR.     

Responses of Müller glia to retinal injury in adult zebrafish

In an effort to identify the cellular events that enable neuronal regeneration in the vertebrate retina, the identity and characteristics of mitotic and apoptotic cells were examined in lesioned retinas of adult zebrafish. Following lesion a complex spatiotemporal pattern of mitosis was observed, including a delayed entry of Müller glia into the cell cycle. Characteristics of these proliferative Müller glia indicated they might serve as a stem/precursor cell of regenerated retina. The results suggested a model of retinal regeneration in which lesions are filled, in part, by a localized en place cytogenesis within intact retina surrounding the lesion site.     

Diabetes-induced dysfunction of the glutamate transporter in retinal Müller cells

PURPOSE: A decrease in the ability of Müller cells to remove glutamate from the extracellular space may play a critical role in the disruption of glutamate homeostasis that occurs in the diabetic retina. Because this amino acid is toxic to retinal neurons and is likely to exacerbate oxidative stress, elucidation of the mechanisms by which glutamate levels are elevated in diabetes may help in the understanding of the pathogenesis of diabetic retinopathy. This study tested the hypothesis that the function of the glutamate transporter in Müller cells of the diabetic retina is compromised by a mechanism involving oxidation. METHODS: Müller cells were freshly isolated from normal rats and those made diabetic by streptozotocin injection. The activity of the Müller cell glutamate transporter, which is electrogenic, was monitored by the perforated-patch configuration of the patch-clamp technique. RESULTS: Four weeks after the onset of hyperglycemia, a significant dysfunction of the Müller cell glutamate transporter was detected. After 13 weeks of streptozotocin-induced diabetes, the activity of this transporter was decreased by 67%. Consistent with oxidation's causing this dysfunction, exposure to a disulfide-reducing agent rapidly restored the activity of the glutamate transporter in Müller cells of diabetic retinas. CONCLUSIONS: Early in the course of diabetic retinopathy, the function of the glutamate transporter in Müller cells is decreased by a mechanism that is likely to involve oxidation.     

Functional and molecular analysis of D-serine transport in retinal Müller cells

D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.     

Rabbit retinal Müller cells undergo antigenic changes in response to experimentally induced proliferative vitreoretinopathy.

Experimental proliferative vitreoretinopathy (PVR) was induced in the rabbit eye by injecting mitotically active Müller cells into the vitreal chamber. Two weeks after the initiation of PVR, the retina and the epiretinal membrane that formed were examined to ascertain the antigenic expression of Müller cells in the retina and in the epiretinal membrane. Examination of various regions of the retina from the experimental PVR eye demonstrated that vimentin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde binding protein (CRALBP), and beta-amyloid precursor protein (beta-APP), which were present in the Müller cells of the retina from the control eye, increased their expression, while the antigenicity of glutamine synthetase (GS), did not change; these proteins were also present in the cells contained within the experimentally induced epiretinal membrane. Alpha smooth muscle actin (alpha-SMA), a cytoskeletal protein that is associated with migration and tractional forces in many cell types, was not only present in the cells embedded within the epiretinal membrane, but was also present in the Müller cells underlying the epiretinal membrane. However, Müller cells that were in the inferior portion of the retina, where epiretinal membrane pathology was absent, did not express alpha-SMA. Although this protein is not normally found in Müller cells, they do express it de novo when they are maintained in culture. This suggests that a localized mechanism associated with epiretinal membrane formation induces the expression of alpha-SMA in Müller cells while the increased expression of GFAP, beta-APP, vimentin, and CRALBP are probably regulated via a more general mechanism.     

Porcine Müller glial cells increase expression of BKCa channels in retinal detachment

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).     

Conditioned medium from mixed retinal pigmented epithelium and Müller cell cultures reduces in vitro permeability of retinal vascular endothelial cells

AIM: To investigate the in vitro effect of laser photocoagulation on blood-retinal barrier permeability. METHODS: Retinal capillary endothelial cells were exposed to supernatants from long term co-cultured cells that were argon laser treated. Endothelial cell permeability was analysed by (1) measurement of transendothelial electrical resistance and (2) equilibration of [(3)H] inulin and [(14)C] albumin across the cell monolayer. RESULTS: Laser photocoagulation of various retinal cells and control ECV304 cells in the lower chamber did not appreciably improve permeability of the endothelial cell monolayer compared with that of unlasered cells. However, medium that was conditioned by mixed retinal pigmented epithelium and Müller cells significantly reduced both inulin (43.2% (SD 6.5%) equilibration in mixed cultures v 59.8% (SD 7.0%) control cells, p<0.05) and albumin (15.1% (SD 3.8%) v 31.1% (SD 6.7%), p<0.05) permeability of the endothelial cell monolayers. A fourfold increase in transendothelial electrical resistance was also seen. CONCLUSIONS: These results are consistent with the hypothesis that interaction of Müller cells with retinal pigmented epithelium induced by laser treatment results in secretion of soluble factor(s), which reduces permeability of retinal vascular endothelium. Identification of these factor(s) may have implications for the clinical treatment of macular oedema secondary to diabetic retinopathy and other diseases.     

Neuropeptide Y-evoked proliferation of retinal glial (Müller) cells

Background Glial cells in human retinas and in fibrocellular membranes from patients with proliferative vitreoretinopathy (PVR) have been described to upregulate their expression of Y₁ receptors for neuropeptide Y (NPY) (Soler et al.: Glia 39:320, 2002). However, it is unknown whether Y₁ receptor activation causes proliferation of retinal glial cells. We investigated whether NPY exerts a proliferation-stimulating effect on retinal glial cells, and compared the NPY-evoked signaling with the signaling of purinergic P2Y receptors.Methods Proliferation assays using bromodeoxyuridine were carried out on primarily cultured Müller glial cells of the guinea pig, in the absence and presence of blockers of Y₁ receptors, of receptor tyrosine kinases (RTKs), of mitogen-activated protein kinases (MAPKs) and of phosphatidylinositol-3 kinase (PI3K).Results NPY exerted a biphasic effect on Müller cell proliferation. At low concentrations (0.1 ng/ml and 1 ng/ml) it decreased the proliferation rate of the cells, while at higher concentration (100 ng/ml) it increased Müller cell proliferation. The NPY-evoked proliferation was mediated by Y₁ receptor stimulation and by activation of the p44/p42 MAPKs and partially of the p38 MAPK. Moreover, Y₁ receptor-induced activation of PI3K as well as transactivations of the platelet-derived and the epidermal growth factor RTKs were necessary for full mitogenic effect of NPY. Y₁ and P2Y receptors share partially common signal transduction pathways in Müller cells.Conclusion It is suggested that NPY may be involved in stimulation of retinal glial cell proliferation during PVR when it is released at higher amounts into the injured retina.