Frank Faude

Role of glial K(+) channels in ontogeny and gliosis: a hypothesis based upon studies on Müller cells.

The electrophysiological properties of Müller cells, the principal glial cells of the retina, are determined by several types of K+ conductances. Both the absolute and the relative activities of the individual types of K+ channels undergo important changes in the course of ontogenetic development and during gliosis. Although immature Müller cells express inwardly rectifying K+ (KIR) currents at a very low density, the membrane of normal mature Müller cells is predominated by the KIR conductance. The KIR channels mediate spatial buffering K+ currents and maintain a stable hyperpolarized membrane potential necessary for various glial‐neuronal interactions. During “conservative” (i.e., non‐proliferative) reactive gliosis, the KIR conductance of Müller cells is moderately reduced and the cell membrane is slightly depolarized; however, when gliotic Müller cells become proliferative, their KIR conductances are dramatically down‐regulated; this is accompanied by an increased activity of Ca2+‐activated K+ channels and by a conspicuous unstability of their membrane potential. The resultant variations of the membrane potential may increase the activity of depolarization‐activated K+, Na+ and Ca2+ channels. It is concluded that in respect to their K+ current pattern, mature Müller cells pass through a process of dedifferentiation before proliferative activity is initiated. GLIA 29:35–44, 2000.

Loss of inwardly rectifying potassium currents by human retinal glial cells in diseases of the eye

We compared the inward K+ currents of Müller glial cells from healthy and pathologically changed human retinas. To this purpose, the whole‐cell voltage‐clamp technique was performed on noncultured Müller cells acutely isolated from human retinas. Cells originated from retinas of four healthy organ donors and of 24 patients suffering from different vitreoretinal and chorioretinal diseases. Müller cells from organ donors displayed inward K+ currents in the whole‐cell mode similar to those found in other species. In contrast, this pattern was clearly changed in the Müller cells from patient retinas. In whole‐cell recordings many Müller cells had strongly decreased inward K+ current amplitudes or lost these currents completely. Thus, the mean input resistance of Müller cells from patients was significantly increased to 1,129 ± 812 MΩ, compared to 279 ± 174 MΩ in Müller cells from healthy organ donor retinas. Accordingly, since the membrane potential is mainly determined by the K+ inward conductance in healthy Müller cells, a large amount of Müller cells from patient retinas had a membrane potential which was significantly lower than that of Müller cells from control eyes. The mean membrane potentials were −37 ± 24 mV and −63 ± 25 mV for patient and donor Müller cells, respectively. The newly described membrane characteristic changes of Müller cells from patient eyes are assumed to interfere severely with normal retinal function: (1) the retinal K+ homeostasis, which is partly regulated by the Müller cell‐mediated spatial buffering, should be disturbed, and (2) the diminished membrane potential should influence voltage‐dependent transporter systems of the Müller cells, e.g., the Na+‐dependent glutamate uptake. GLIA 20:210–218, 1997.

Comparative studies on mammalian Muller (retinal glial) cells

Muller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the ‘mid-periphery’ of retinae immunocytochemically labeled for vimentin, estimates of Muller cell densities per unit retinal surface area, and of neuron-to-(Muller) glia indices were performed. Muller cell densities were strikingly similar among the species studied (around 8000–11000 mm−2) with the extremes of the horse (≤5000 mm−2) and the tree shrew (≥20000 mm−2). By contrast, the number of neurons per Muller cell varied widely, being clustered at 6–8 (in retinae with many cones), at about 16, and at up to more than 30 (in strongly rod-dominated retinae). Isolated Muller cell volumes were estimated morphometrically, and cell surface areas were calculated from membrane capacities. Muller cells isolated from thick vascularized retinae (carnivores,rats, mice, ungulates) were longer and thinner, and had smaller volumes but higher surface-to-volume ratios than cells from thin paurangiotic (i.e. with blood vessels only near the optic disc) or avascular retinae (rabbits, guinea pigs, horses, zebras). In whole-cell voltage-clamp studies, Muller cells from all mammals studied displayed two dominant K+ conductances, inwardly rectifying currents and delayed rectifier currents. TTX-sensitive Na+ currents were recorded only in some species. Based on these data, the following hypotheses are presented, (a) neuron-to-(Muller) glia indices are determined by precursor cell proliferation rather than by metabolic demands; (b) Muller cell volumes depend on available space rather than on the number of supported neurons; and (c) it follows that, the specific metabolic activities of Muller cells must differ greatly between species, a difference that may contribute to distinct patterns of retinal vascularization.

The prevalence of activated protein C (APC) resistance and factor V Leiden is significantly higher in patients with retinal vein occlusion without general risk factors - Case-control study and meta-analysis

Several small case-control studies have investigated whether factor V Leiden (FVL) is a risk factor for retinal vein occlusion (RVO) and generated conflicting data. To clarify this question we performed a large two-centre case-control study and a meta-analysis of published studies. Two hundred seven consecutive patients with RVO and a control group of 150 subjects were screened between 1996 and 2006. A systematic meta-analysis was done combining our study with further 17 published European case-control studies. APC resistance was detected in 16 out of 207 (7.7%) patients and eight out of 150 (5.3%) controls. The odds ratio (OR) estimated was 1.49 with a (non-significant) 95% confidence interval (CI) of 0.62-3.57. The meta-analysis including 18 studies with a total of 1,748 patients and 2,716 controls showed a significantly higher prevalence of FVL in patients with RVO compared to healthy controls (combined OR 1.66; 95% CI 1.19-2.32). All single studies combined in the meta-analysis were too small to reliably detect the effect individually. This explains the seemingly contradictory data in the literature. In conclusion, the prevalence of APC resistance (and FVL) is increased in patients with RVO compared to controls, but the effect is only moderate. Therefore, there is no indication for general screening of factor V mutation in all patients with RVO. We recommend this test to be performed in patients older than 50 years with an additional history of thromboembolic event and in younger patients without general risk factors like hypertension.

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.

Glial cell-mediated spread of retinal degeneration during detachment: A hypothesis based upon studies in rabbits

In human subjects with peripheral retinal detachments, visual deficits are not restricted to the detached retina but are also present in the non-detached tissue. Based upon studies on a rabbit model of rhegmatogenous retinal detachment, we propose a glial cell-mediated mechanism of spread of retinal degeneration into non-detached retinal areas which may also have importance for the understanding of alterations in the human retina. Both detached and attached portions of the rabbit retina display photoreceptor cell degeneration and cystic degeneration of the innermost layers. An inverse mode of photoreceptor cell degeneration in the attached tissue suggests a disturbed support of the photoreceptor cells by Müller cells which show various indications of gliosis (increased expression of intermediate filaments, cell hypertrophy, decreased plasma membrane K(+) conductance, increased Ca(2+) responsiveness to purinergic stimulation) in both detached and attached tissues. We propose that gliotic alterations of Müller cells contribute to the degeneration of the attached retina, via disturbance of glial homeostasis mechanisms. A down-regulation of the K(+) conductance of Müller cells may prevent effective retinal K(+) and water clearance, and may favor photoreceptor cell degeneration and edema development.

Alterations of potassium channel activity in retinal Müller glial cells induced by arachidonic acid.

Arachidonic acid, which is thought to be involved in pathogenetic mechanisms of the central nervous system, has been shown previously to modulate neuronal ion channels and the glutamate uptake carrier of retinal glial (Müller) cells. We have used various configurations of the patch-clamp technique to determine the effects of arachidonic acid on the K+ currents of freshly isolated Müller glial cells from rabbit and human. Arachidonic acid reduced the peak amplitude of the transient (A-type) outward K+ currents in a dose-dependent and reversible manner, with a 50% reduction achieved by 4.1 microM arachidonic acid. The inward rectifier-mediated currents remained unchanged after arachidonic acid application. The amplitude of the Ca(2+)-activated K+ outward currents (KCa), which were blocked by 1 mM tetraethylammonium chloride and 40 nM iberiotoxin, respectively, was dose-dependently elevated by bath application of arachidonic acid. The activation curve of the KCa currents shifted towards more negative membrane potentials. Furthermore, arachidonic acid was found to suppress inwardly directed Na+ currents. In cell-attached recordings with 3 mM K+ in the bath and 130 mM K+ in the pipette, the KCa channels of rabbit Müller cells displayed a linear current-voltage relation, with a mean slope conductance of 102 pS. In excised patches, the slope conductance was 220 pS (150 mM K+i/130 mM K+o). The opening probability of the KCa channels increased during membrane depolarization and during elevation of the free Ca2+ concentration at the intracellular face of the membrane patches. Bath application of arachidonic acid caused a reversible increase of the single-channel opening probability, as well as an increase of the number of open channels. Arachidonic acid did not affect the single-channel conductance. Since arachidonic acid also stimulates the KCa channel activity in excised patches, the action of arachidonic acid is assumed to be independent of changes of the intracellular calcium concentration. Our results demonstrate that arachidonic acid exerts specific effects on distinct types of K+ channels in retinal glial, cells. In pathological cases, elevated arachidonic acid levels may contribute to prolonged Müller cell depolarizations, and to the initiation of reactive glial cell proliferation.

Sodium current amplitude increases dramatically in human retinal glial cells during diseases of the eye.

Müller cells, the main macroglial cells of the retina, express several types of voltage and ligand‐activated ion channels, including Na‐ channels. Using the whole‐cell voltage‐clamp technique, we studied the expression of Na‐ currents in acutely isolated, non‐cultivated human Muller cells from retinas of healthy organ donors and patients suffering from different eye diseases. In both types of retinas transient Na+ currents could be recorded from Muller cells. The tetrodotoxin‐resistant Na+ currents, which were not completely blocked even at a concentration of 10

Mechanisms of Graft Rejection in the Transplantation of Retinal Pigment Epithelial Cells

muM tetrodotoxin, had a mean current density of 3.0± 3.0 pA/pF (mean ± SD, n = 10) in Muller cells from donor retinas and of 12.2± 2 9.6 pA/pF (n= 74) in Muller cells from patient retinas. Only 33.3% of healthy but 88.4% of pathological Muller cells depicted such currents. The GNa+/GK+ ratio was very high in several Muller cells from patient retinas, such that action potential‐like activity could be generated after prehyperpolarizing current injection in some of these cells. Apparently, the Na’channels, due to their negative steady‐state inactivation curve (Vh= ‐84.5 mV), do not influence the lowered membrane potential of the pathological cells, since they are inactivated at these voltages. Currently, we do not have an explanation for the increase in amplitude and frequency of Na+ currents in human Muller cells under pathological conditions. However, the up‐regulation of Na channels may mirror a basic glial response to pathological conditions, since it has also been found previously in human hippocampal astrocytes from epileptic foci and in rat cortex stab wounds lined by an astrocytic scar.

Comparison between functional characteristics of healthy and pathological human retinal Müller glial cells.

PURPOSE The role of activated retinal pigment epithelium (RPE) cells was investigated in the rejection after subretinal transplantation. METHODS RPE cells from 7 pigmented rabbits were separated and evaluated regarding their MHC class II expression as the sign of activation. The activation of the RPE cells was augmented with a treatment of 1,000 U/ml interferon gamma (IFN-gamma) for 8 days. These cells were then transplanted into 7 albino rabbits. As control, RPE transplantations without a pretreatment were performed in 7 albino rabbits. Six weeks after the transplantation, the transplanted eyes were enucleated and histology was performed. RESULTS In culture, without IFN-gamma addition, 11.38 +/- 0.94% of the RPE cells presented MHC class II. After IFN-gamma treatment, this quantity increased to 78.26 +/- 1.46% of the RPE cells. These cells transplanted into the rabbits caused an obvious rejection in the transplantation area which was verified histologically. The control group presented a transplantation area without signs of rejection or inflammation. CONCLUSION In culture, some of the adult RPE cells are activated. These cells may accelerate the rejection cascade after transplantation. An elimination of activated RPE cells from the transplant should be recommended before transplantation.