[50]. In the diabetic retina and under hypoxic conditions in vitro, cyclooxygenase-2 is induced in retinal astrocytes, and inhibition of this enzyme was shown to prevent neovascularization via upregulation of thrombospondin-1 [51].

It has been suggested that astrocytes play a role in the induction of apoptosis in retinal ganglion cells [52]. Whereas ganglion cells in the human diabetic retina express proapoptotic molecules such as caspase-3, Fas, and Bax, the astrocytes express antiapoptotic molecules such as Bcl-2, in addition to the cytotoxic effector molecule Fas ligand [52].

Astrocytes of diabetic retinas upregulate the expression of the water channel protein, aqua- porin-1 (fig. 1b, c). In control retinas, the aqua- porin-1 immunoreactivity is predominantly expressed by photoreceptor cells in the outer retina [53]. After few months of experimental diabetes, immunoreactivity for aquaporin-1 is also expressed by astrocytes which are located within the nerve fiber layer and around the vessels [own unpubl. data]. Furthermore, the contact of astrocytes with the vessels of the superficial vessel plexus changes in the course of experimental diabetes. Whereas astrocytic processes have contact only to the innermost aspects of the vessel walls in control retinas (while the other parts are surrounded by Müller cell membranes), they fully surround these vessels in the diabetic retina of the rat [54]. The functional consequences of the elevated aquaporin-1 expression, and of the altered contact of astrocytes with the superficial vessels, remain to be determined. In the brain, an elevation of aquaporin-1 expression by astrocytes has been suggested to be implicated in the development or resolution of edema [55].

Müller Cells

Müller cells become abnormal early during diabetes, and it has been suggested that the functional loss and the death of neurons can be attributed to

the alterations of Müller cells [56]. Because Müller cells constitute the functional link between vasculature and neurons, changes in Müller cell function could be a key event in the development of diabetic retinopathy. Furthermore, an impairment in the supportive functions of Müller cells may increase the susceptibility of retinal neurons to additional stressful stimuli present in the diabetic retina, such as oxidative stress and inflammatory conditions.

One hallmark of reactive gliosis of Müller cells is the upregulation of the intermediate filament GFAP; it has been observed both in retinas of diabetic patients [20] and in experimental diabetes (fig. 1a). GFAP expression in Müller cells is elevated at 6 weeks and is prominent at 3 months of experimental diabetes in rats, i.e. well before overt vascular changes become demonstrable [7–9]. The upregulation of GFAP in Müller cells in the retina of diabetic rats is inhibited by the aldose reductase inhibitor, sorbinil, as well as by melatonin, suggesting that both the polyol pathway and oxidative stress contribute to the progression of Müller cell gliosis [24, 57].

Müller cells in the diabetic retina display both proliferation and apoptosis. The density of Müller cells is increased at 4 weeks of experimental diabetes [9]. During early hyperglycemia in rats, apoptosis occurs primarily in ganglion and Müller cells [58]. This is associated with the upregulation of the GFAP content in Müller cells and with an increased expression of the p75 receptor on both cell types [20, 58]. Treatment of diabetic rats with nerve growth factor prevented apoptosis in ganglion and Müller cells as well as pericyte loss and the development of acellular occluded capillaries [58]. In the rat retina, nerve growth factor is expressed by ganglion, Müller, and pigment epithelial cells, while only pigment epithelial and Müller cells express receptors for this factor [59]. Hyperglycemia induces Müller cell apoptosis in vitro, by inactivation of the Akt survival pathway [60]. A similar inactivation of Akt may contribute to the Müller cell apoptosis in the diabetic retina [60].

strong predictor of progression of diabetic retinopathy [86].

After creation of poorly perfused acellular capillaries and large areas of hypoxia within the retina, the expression of angiogenic growth factors that initiate vascular growth determine further progression of the disease toward a proliferative retinopathy. In addition to the basic fibroblast growth factor, VEGF is the major angiogenic factor in the retina promoting pathological neovascularization [87, 88]. In the healthy retina, Müller cells provide a permanent antiproliferative condition for vascular endothelial cells, by the release of antiangiogenic factors such as PEDF and thrombospondin-1 [89]. The elevated expression of VEGF by Müller cells has been related to the induction of retinal neovascularization during diabetic retinopathy [50]. The balance between proand antiangiogenic factors, such as VEGF and PEDF, is thought to be essential for angiogenic homeostasis in the retina. However, in spite of the observation that hypoxia enhances the expression of VEGF and downregulates the expression of PEDF in cultured Müller cells, conditioned media of Müller cells failed to stimulate additional proliferation of retinal endothelial cells in vitro [74, 90]. This may suggest that hypoxia-stimulated Müller cells release (in addition to VEGF) other angiogenesis-inhibiting factor(s), and points to the importance of additional signals for the triggering of neovascularization in the hypoxic retina; the nature of these signals remains to be identified.

VEGF and other angiogenic cytokines released by Müller cells, e.g. transforming growth factor-β, basic fibroblast growth factor, and TNF, increase the release of matrix metalloproteinases by endothelial cells [91, 92]. Moreover, Müller cells themselves secrete matrix metalloproteinases, e.g. upon stimulation of purinergic receptors [93]. High glucose levels increase the production of matrix metalloproteinases in retinal cells [94]. Matrix metalloproteinases impair the tight junction function in retinal endothelial and pigment

epithelial cells, by proteolytic degradation of the tight junction protein, occludin [94], and thus may facilitate vascular leakage. Moreover, the secretion of metalloproteinases allows endothelial cells to penetrate their underlying basement membrane, and eliminates the contact inhibition which normally blocks endothelial cell proliferation [91]. A further way for Müller cells to stimulate vasculogenesis may involve the reninangiotensin system which has been implicated in diabetes. Müller cells express angiotensin II and renin, with the expression being most obvious in the end feet closely apposed to retinal blood vessels, while the angiotensin receptors are expressed by neurons and by the vasculature [56, 95].

The progression of proliferative retinopathy may lead to the formation of fibrovascular scar tissues. Such scar tissues form periretinal membranes which are connected with the sensory retina via hypertrophied Müller cell fibers, and which may become contractile, resulting in traction detachment of the retina. Müller cells are the principal glial cells in the fibrovascular scar tissue [96]. Müller cells de-differentiate, migrate within and out of the neural retina, and display cellular hypertrophy. The hypertrophied Müller cell fibers form intraretinal bridges between cystic spaces, and cause subretinal fibrosis [96, 97]. Müller cells in fibrovascular membranes may transdifferentiate into myofibroblasts that generate tractional forces in response to growth factors in the vitreous, thus causing traction detachment [98]. Glial cells in fibrovascular membranes express growth factors and cytokines which may drive uncontrolled cell proliferation and neovascularization. In addition, they express receptors for various growth factors. Among these factors, the hepatocyte growth factor (HGF), which has been implicated in ocular angiogenesis and has been found to be elevated in the vitreous of patients with proliferative diabetic retinopathy [99], was shown to stimulate chemotaxis and VEGF secretion by cultured Müller cells [77]. Since

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distinct growth factors and serum stimulate the secretion of HGF by Müller cells, an autocrine/ paracrine signaling pathway has been suggested that promotes glial cell responses in the diabetic retina [77]. Particularly, vessel leakage-induced serum entry into the retinal tissue may cause a strong stimulus for HGF secretion by glial cells.

Müller cells from surgically excised retinal tissue of patients with proliferative diabetic retinopathy display physiological alterations similar to those of Müller cells from patients with proliferative vitreoretinopathy [100]. Such cells

show a strong hypertrophy, an almost complete absence of currents through inwardly rectifying K+ channels (fig. 2b), and a decreased membrane potential [97]. This current pattern suggests a de-differentiation of the cells, reminiscent of the pattern characteristic for nondifferentiated glial cells early in the ontogenesis [101]. It has been proposed that the downregulation of the K+ channels is a prerequisite for the re-entry of gliotic Müller cells into the proliferation cycle [102]. The strong downregulation of the K+ channels in Müller cells may also suggest that the retinal K+

homeostasis is largely disturbed during proliferative diabetic retinopathy (see section ‘Retinal K+ Homeostasis’).

Glutamate Metabolism

‘Neurotransmitter recycling’ is a major function of Müller cells. The cells express uptake systems for various amino acid transmitters such as glutamate [103, 104] and γ-aminobutyric acid (GABA) [105, 106]. The glutamate/aspartate transporter GLAST is expressed by Müller cells, and is the predominant transporter for the removal of glutamate within the retina [107, 108] maintaining the extracellular glutamate level below neurotoxic levels. Malfunction of glutamate transport into Müller cells results in an increased extracellular level of glutamate which may contribute to neuronal dysfunction and apoptosis in the diabetic retina [109]. After experimental inhibition of glutamate uptake by Müller cells, even low concentrations of extracellular glutamate become neurotoxic, via an activation of ionotropic glutamate receptors [110, 111]. After glutamate has been taken up by Müller cells, it is intracellularly converted into glutamine, which is transported back to neurons as a precursor for neuronal synthesis of glutamate and GABA.

Retinas of diabetic animals have been found to display an impaired glutamate metabolism, manifested by an increased level of glutamate and a reduced ability to convert glutamate into glutamine [7, 21]. Both glutamate and GABA levels are elevated in the vitreous of diabetic animals and human patients [112, 113]. These alterations have been proposed to be caused by a malfunction of the glial uptake of neurotransmitter molecules. Indeed, a decrease in the currents evoked by the GLAST transporter has been observed in isolated Müller cells already at 2–4 weeks of experimental diabetes [114]. Since a reducing agent restored the activity of the glutamate transporter, it was suggested that the dysfunction of the glutamate transport is caused by oxidative stress present in the diabetic retina [114]. However, a

recent study showed no alteration, or even an enhancement, of glutamate uptake in diabetic retinas [115]. The plasma membrane expression and -localization of transporter molecules in Müller cells does not change in the course of diabetes [54, 114, 115]. Moreover, the retinal expression of the glutamine synthetase protein is not different between diabetic and control retinas [20, 22]. Thus, an involvement of Müller cells in the disturbance of the glutamate turnover in diabetic retinas – if any – remains to be clarified by future investigations. Another possible explanation for the increased vitreal and retinal glutamate content is the breakdown of the blood-retinal barrier which may cause a leakage of plasma glutamate into the tissues.

The uptake of glutamate is also important for the retinal defense against free radicals mediated by Müller cells. Müller cells synthesize the tripeptide glutathione from glutamate, cysteine, and glycine. Reduced glutathione is provided to neurons and acts as a scavenger of free radicals and reactive oxygen compounds. Müller cells from aged animals contain less glutathione than cells from young animals [116]. An age-dependent decrease in Müller cell-mediated defense against free radicals may accelerate the pathogenesis of diabetic retinopathy in elderly patients.

Finally, it should be mentioned that Müller cells are the ‘communicators’ between vessels and neurons: they take up glucose from the circulation, metabolize glucose, and transfer energy substrates such as lactate and pyruvate to neurons [117]. Generally, the uptake and metabolization of glucose in glial cells are closely linked to the release of glutamate from neurons and its uptake by glia [118, 119]. The limiting factor in glutamate and glucose uptake by glial cells is the activity of the Na,K-ATPase which decreases very rapidly in the hyperglycemic tissue [120, 121]. An impairment of the glial sodium pump causes a depolarization of the plasma membrane that lowers the efficiency of the electrogenic glutamate uptake. This has been related to enhanced

oxidative stress, e.g. via a decrease in the synthesis of glutathione.

Retinal K+ Homeostasis

Müller cells are crucially involved in retinal K+ homeostasis, by mediating transcellular ‘spatial buffering’ K+ currents which counterbalance the changes in extracellular K+ concentration associated with neuronal activity [13, 122, 123]. A dysregulation of the K+ homeostasis causes K+- evoked neuronal hyper-excitation and, therefore, glutamate toxicity. Normally, Müller cells take up neuron-derived excess K+ particularly from the plexiform (synaptic) layers, and release a similar amount of K+ into fluid-filled spaces outside the neural retina (blood, vitreous, and subretinal space). Though also the Na,K-ATPase and transporter molecules contribute to Müller cell-mediated K+ homeostasis, it is now well-es- tablished that passive K+ currents through inwardly rectifying K+ channels play a major role in counteracting extracellular K+ imbalances. Among the various subtypes of K+ channels expressed by Müller cells [124, 125], the inwardly rectifying K+ channel of the Kir4.1 subtype has been specifically implicated in mediating the K+ buffering currents [126, 127]. The Kir4.1 channel protein is expressed in a polarized fashion in the plasma membrane of Müller cells, with a strong enrichment in such membrane domains across which the cells can dispose excess K+, i.e. in perivascular membrane sheets, and at the inner and outer limiting membranes (fig. 1d, e) [125, 127, 128].

The K+ conductance of rat Müller cells is significantly reduced at 4 and 6 months of experimental diabetes (fig. 2a). This decreased K+ conductance is associated with a mislocation of the Kir4.1 protein [54]. Though the Kir4.1 protein is still expressed within the retinal tissue, the prominent expression at both limiting membranes and around the vessels is absent (fig. 1d, e). This suggests that the decrease in the K+ currents in cells from diabetic retinas (fig. 2a) is caused by

an alteration in the expression pattern of Kir4.1 channels. The downregulation of Kir4.1 channels should cause an impairment of the transglial K+ currents and, therefore, a disturbance of the retinal K+ homeostasis that may contribute to neuronal cell death in the diabetic retina. A similar mislocation of the Kir4.1 protein has been described in retinas of mice which carry a genetic inactivation of the dystrophin gene product Dp71 proposed to be involved in the plasma membrane clustering of Kir4.1 channels [129]. In these mice, the mislocation of Kir4.1 protein is associated with an enhanced vulnerability of retinal ganglion cells during ischemic stress [130]. Glial cells are crucially implicated in activity-dependent regulation of the local blood flow, and it has been suggested that the release of K+ ions from perivascular end feet plays a role in the local dilation of arterioles [16]. A decrease in the activity-depen- dent K+ efflux in the diabetic tissue may result in a decrease in the local blood flow, thus exacerbating hypoxic insults. Since the gating of Kir4.1 channels is dependent on intracellular ATP [131, 132], a functional inactivation of these channels may disturb retinal K+ homeostasis in ischemic tissue areas even already before a mislocation of the channel proteins is observable.

The reason for the diabetes-induced downregulation of the prominent Kir4.1 expression in end feet and perivascular membranes of Müller cells is unclear. As similar changes were observed during experimental retinal ischemia-reperfusion and uveoretinitis, respectively [133, 134], a causative effect of oxidative stress and/or chronic inflammation is very likely. It has been shown that glial cells surrounding ischemic brain lesions display a reduction in their K+ currents, and that a similar reduction can be observed in cultured astrocytes in the presence of TNF [135]. Activated microglial cells or blood-derived leukocytes release nitric oxide, reactive oxygen species, and proinflammatory cytokines such as TNF, which may be implicated in the altered expression of glial K+ channels during diabetes.

It has been shown that Müller cells of patients with proliferative diabetic retinopathy display a strong reduction in their K+ conductance (fig. 2b) [97], suggesting that the downregulation of K+ channels may represent a characteristic feature also of glial cells of the diabetic human retina. Müller cells of the human retina display an age-dependent decrease in their K+ conductance [136]; this age-related downregulation should contribute to retinal complications of diabetes in elderly patients.

Cytotoxic Cell Swelling and Retinal Edema

Diabetic retinopathy is the leading cause of reduced visual acuity and acquired blindness in working-age adults. Most frequently, the presence of a macular edema is responsible for the impaired vision [137]. The macular edema may be diffuse or cystic. In cystic edema, the fluidfilled cysts are predominantly located in two retinal layers, the inner nuclear and the Henle fiber layers [138, 139]. The fluid accumulation causes cell displacement and splitting of the perifoveal neuroretina within these two layers, and the fluid-filled compartments are spanned by the trunks of Müller’s fibers. Additional fluid accumulation can occur in the subretinal space. By compression of retinal neurons, nerve fibers, and capillaries, edema contributes to photoreceptor degeneration and neuronal cell death, and exacerbates the ischemic conditions. Generally, an edema may develop by fluid accumulation within the interstitial spaces (extracellular edema, characterized by cell compression) caused by vascular leakage (vascular edema), or by fluid accumulation within the cells (intracellular or cytotoxic edema, characterized by cell swelling). In the preclinical stage of diabetic retinopathy, there are two types of increased retinal thickness that may be associated or not with angiographic vascular leakage [140]. Also the presence of cysts is associated or not with vascular leakage, suggesting that both vasogenic edema and cell swelling may contribute to cystoid macular edema. It has been

suggested that swelling of glial cells is involved in the development of cystoid edema, with the cysts being formed by swollen and dying Müller cells [141, 142]. Both hypoxic-ischemic conditions and/or inflammatory alterations of the microvasculature have been suggested as causative factors for the development of macular edema [79, 137, 143–146]. In an animal model of retinal hypoxia, the breakdown of the blood-retinal barrier was associated with an intracellular edema of Müller cells [147].

Generally, the development of chronic edema depends on two parameters: the rate of fluid entry into the retinal parenchyma through (leaky) vessel walls and the rate of fluid reabsorption from the retinal tissue back into the blood. The development of macular edema is thought to be primarily caused by a breakdown of the bloodretinal barrier, resulting in retinal vascular leakage [139, 144, 148]. However, it has been shown that clinically significant diabetic macular edema occurs only when (in addition to vascular leakage) also the active transport mechanisms of the blood-retinal barriers are dysfunctional [149], suggesting that a disturbance of the fluid (re-)absorption from the retinal tissue is a necessary step in edema formation. In the retina, the fluid absorption is carried out by pigment epithelial cells that dehydrate the subretinal space, and by Müller cells that dehydrate the inner retina by transcellular water transport [15]. It is suggested that the water transport through Müller cells is coupled to the extrusion of osmolytes from the perivascular glial cell processes into the blood. In particular, the spatial buffering K+ currents which flow through Müller cells into the blood (see the previous section) are associated with a water flow that dehydrates the retina in dependence on the neuronal activity [15, 128]. The co-localization of the Kir4.1 channel protein and of the glial water channel, aquaporin-4, in perivascular (fig. 1e) and vitreous-abutting membrane domains of Müller cells is suggested to facilitate this process of activ- ity-dependent water transport from Müller cells

into the blood and vitreous [128]. However, in the course of experimental diabetes in rats, Müller cells downregulate the expression of Kir4.1 protein in these membrane domains (fig. 1d, e) [54]; the disruption of the spatial buffering K+ currents through Müller cells should cause an impairment of the water transport through the cells. This may cause the swelling of Müller cells [134], as well as an impairment of the resolution of vasogenic and extracellular edema. Moreover, an increase in the osmotic pressure of the Müller cell interior – which must result from accumulation of K+ ions and other osmolytes within the cells – may drive water from the blood and vitreous into the cells [15].

Experimental diabetes in rats causes a significant alteration of the swelling characteristics of

Müller cells in situ. Müller cells in diabetic retinas show a swelling of their cell bodies during hypotonic stress, while Müller cells in control retinas do not alter their cell volume in response to changes in osmotic conditions (fig. 3). A similar alteration of the swelling characteristics has been observed in rat retinas during ischemiareperfusion and uveoretinitis, respectively [133, 134], implicating oxidative stress and inflammation as causative factors for osmotic Müller cell swelling. This assumption is supported by the observations that H2O2 or inflammatory mediators such as arachidonic acid and prostaglandin E2 cause osmotic Müller cell swelling in control retinas, and that inhibition of the phospholipase A2 or of the cyclooxygenase, or the presence of a reducing agent, prevents the swelling in diabetic