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.

Upregulation of P2X(7) receptor currents in Müller glial cells during proliferative vitreoretinopathy.

PURPOSE: Müller glial cells from the human retina express purinergic P2X(7) receptors. Because extracellular adenosine triphosphate (ATP) is assumed to be a mediator of the induction or maintenance of gliosis, this study was undertaken to determine whether the expression of these receptors is different in human Müller cells obtained from retinas of healthy donors and of patients with choroidal melanoma and proliferative vitreoretinopathy (PVR). METHODS: Human Müller cells were enzymatically isolated from donor retinas, and whole-cell patch-clamp recordings were made to characterize the density of the P2X(7) currents and the activation of currents through Ca2+-activated K+ channels of big conductance (I:(BK)) that reflects the increase of the intracellular Ca2+ concentration. RESULTS: Stimulation by external ATP or by benzoylbenzoyl ATP (BzATP) evoked both release of Ca2+ from thapsigargin-sensitive intracellular stores and opening of Ca2+ -permeable P2X(7) channels. These responses caused transient and sustained increases in I:(BK). In Müller cells from patients with PVR, the mean density of the BzATP-evoked cation currents was significantly greater compared with cells from healthy donors. As a consequence, such cells displayed an enlarged I:(BK) during application of purinergic agonists. ATP and BzATP increased the DNA synthesis rate of cultured cells. This effect could be reversed by blocking the I:(BK). CONCLUSIONS: The increased density of P2X(7) receptor channels may permit a higher level of entry of extracellular Ca2+ into cells from patients with PVR. Enhanced Ca2+ entry and the subsequent stronger activation of I:(BK) may contribute to the induction or maintenance of proliferative activity in gliotic Müller cells during PVR.

Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR.

PURPOSE: To determine the electrophysiological properties of Müller (glial) cells from experimentally detached rabbit retinas. METHODS: A stable local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Müller cells were acutely dissociated and studied by the whole-cell voltage-clamp technique. RESULTS: The cell membranes of Müller cells from normal retinas were dominated by a large inwardly rectifying potassium ion (K+) conductance that caused a low-input resistance (<100 M(Omega)) and a high resting membrane potential (-82 +/- 6 mV). During the first week after detachment, the Müller cells became reactive as shown by glial fibrillary acidic protein (GFAP) immunoreactivity, and their inward currents were markedly reduced, accompanied by an increased input resistance (>200 M(Omega)). After 3 weeks of detachment, the input resistance increased further (>300 M(Omega)), and some cells displayed significantly depolarized membrane potentials (mean -69 +/- 18 mV). When PVR developed (in 20% of the cases) the inward K+ currents were virtually completely eliminated. The input resistance increased dramatically (>1000 MOmega), and almost all cells displayed strongly depolarized membrane potentials (-44 +/- 16 mV). CONCLUSIONS: Reactive Müller cells are characterized by a severe reduction of their K+ inward conductance, accompanied by depolarized membrane potentials. These changes must impair physiological glial functions, such as neurotransmitter recycling and K+ ion clearance. Furthermore, the open probability of certain types of voltage-dependent ion channels (e.g., Ca2+-dependent K+ maxi channels) increases that may be a precondition for Müller cell proliferation, particularly in PVR when a dramatic downregulation of both inward current density and resting membrane potential occurs.

Age- and disease-related changes of calcium channel-mediated currents in human Müller glial cells.

PURPOSE: To determine whether the expression of voltage-gated Ca2+ channels in human Müller glial cells changes during normal aging and in cells from patients with proliferative vitreoretinopathy (PVR). METHODS: Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients with PVR, and the whole-cell, voltage-clamp technique was used to characterize the current densities of transient, low-voltage-activated calcium channels and of sustained. high-voltage-activated calcium channels, respectively. To obtain maximal currents through both channel types, Na+ ions were used as the charge carrier. RESULTS: During normal aging, Müller cells developed a hypertrophy, as indicated by an increase of the cell membrane capacitance. The mean membrane capacitance of cells from aged donors (> or = 60 years old) was elevated by 25% compared with cells from younger donors. The hypertrophy was not accompanied by a changed density of low-voltage-activated currents, whereas the density of the high-voltage-activated currents was enhanced by 76%. The density of the high-voltage-activated currents increased in correlation with the increase of the cell membrane capacitance and with the age of the donors. In the case of PVR, Müller cells displayed a strong hypertrophy accompanied by a downregulation of both current types by approximately 65%. CONCLUSIONS: Both normal aging and PVR cause a gliotic reactivity of human Müller cells, as indicated by their hypertrophy. The type of reactivity, however, differs between the two conditions. Normal aging is accompanied by an increased expression of voltage-gated Ca2+ channels, whereas in PVR Ca2+ channel expression is decreased.

Facilitation of artificial retinal detachment for macular translocation surgery tested in rabbit.

PURPOSE: For macular translocation surgery, the native attached retina has to be detached either locally or completely. Although different surgical techniques are used, there is a general search for supporting procedures that facilitate and accelerate the retinal detachment. METHODS: Pars plana vitrectomies were performed in pigmented rabbits. In all experimental groups, a local retinal detachment was created by infusing the test solution with a thin glass micropipette attached to a glass syringe. In control animals a standard balanced salt solution was used at room temperature, in combination with a standard vitrectomy light source. In two test groups, a calcium- and magnesium-free solution was used for the vitrectomy, under illumination by a standard light source in group I (solution at room temperature) and group II (solution heated up to body temperature). In group III the rabbits were dark-adapted for half an hour, and then, during surgery, a red filter was used in front of the light source (standard balanced salt solution at room temperature). After the rabbits were killed at the end of surgery, the adherence of the retinal pigment epithelium (RPE) to the neural retina in the detachment area was quantified microscopically, and the morphologic integrity of the detached retinal tissue was examined by light and electron microscopy. No electrophysiology was performed. RESULTS: In all four groups, it was possible to detach the retina. The maximum adherence of the RPE cells to the neural retina was observed in the control group. Virtually no decrease in adherence was found in test group II (36 degrees C solution without calcium and magnesium), whereas a significant decrease was seen in both group I (calcium- and magnesium-free solution at room temperature) and group III (dark adaptation-red light technique; standard balanced salt solution at room temperature). In none of the experimental groups was any obvious damage of the retinal structure observed, even after exposure to the test solutions for 60 minutes. CONCLUSIONS: Both dark adaptation (red illumination) and the use of a calcium chloride- and magnesium chloride-free solution (at room temperature) can facilitate retinal detachment in macular translocation surgery. Both techniques are proposed as a gentle support for the operation, because they protect an intact RPE cell layer and do not cause retinal damage at the ultrastructural level.

Human Müller glial cells: altered potassium channel activity in proliferative vitreoretinopathy.

PURPOSE: To determine differences of K+ channel activity between Müller glial cells obtained from retinas of healthy human donors and of patients with retinal detachment and proliferative vitreoretinopathy. METHODS: Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients. The whole-cell and the cell-attached configurations of the patch-clamp technique were used to characterize the current densities of different K+ channel types and the activity of single Ca2+ -activated K+ channels of big conductance (BK). RESULTS: Cells from patients displayed a less negative mean membrane potential (-52.8 mV) than cells from healthy donors (-80.6 mV). However, the membrane potentials in cells from patients scattered largely between -6 and -99 mV. The inwardly rectifying K+ permeability in cells from patients was strongly reduced (0.3 pA/pF) when compared with cells from healthy donors (6.0 pA/pF). At the resting membrane potential, single BK channels displayed a higher mean activity (open probability, Po, and channel current amplitude) in cells from patients (Po, 0.30) than in cells from healthy donors (Po: 0.03). The variations of BK current amplitudes were correlated with the variations of the membrane potential. CONCLUSIONS: The dominant expression of inwardly rectifying channels in cells from healthy donors is thought to support important glial cell functions such as the spatial buffering of extracellular K+. The downregulation of these channels and the less negative mean membrane potential in cells from patients should impair spatial buffering currents and neurotransmitter clearance. The increased activity of BK channels may support the proliferative activity of gliotic cells via feedback regulation of Ca2+ entry and membrane potential.

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

Enzymatically isolated, noncultured Müller glial cells from human organ donor and patient eyes were studied using the whole-cell-voltage-clamp and the patch-clamp technique. The patients suffered mainly from choroidal melanomas, retinal detachment due to proliferative vitreorentinopathy, glaucomas, and perforating eye injuries. The organ donor eyes were used as a source of corneas for corneal transplantation. Use of the human retinal tissue was approved by the Ethics Committee of the School of Medicine, University of Leipzig. Most of the patient Müller cells showed a marked or complete loss of inwardly rectifying K+ currents, causing a dramatic increase in the input resistance. The zero current potential of the patient Müller cells, which is equivalent to the membrane potential, was significantly reduced (depolarized) as compared with the donor Müller cells. In contrast to the K+ current loss, the Na+ current density was significantly higher in patient Müller cells than in donor Müller cells; the number of Müller cells depicting Na+ currents increased from 33% (3 pA/pF) in donor Müller cells to 85% (about 12pA/pF) in patient Müller cells. Application of glutamate to the Müller cells generated a glutamate-transporter-mediated current, such as that seen in other species. A highly significant increase was noted for the high-affinity Na+-dependent glutamate-transporter-current density in patient Müller cells compared with donor cells. The application of gamma-aminobutyric acid (GABA) evoked, in addition to the GABA transporter currents already known from Müller cells of other mammalian species, GABA(A)-receptor mediated currents in human Müller cells. We found that GABA(A) receptors are expressed in human Müller cells, but not in other nonprimate mammals. Whether a difference exists between the GABA(A) current density in donor and patient Müller cells remains to be seen. The results concerning the disappearance of K+ currents and diminution of the membrane potential may demonstrate early glial changes that may possibly precede pathological neuronal changes, at least in retinas from eyes with choroidal melanomas. In later stages of the diseases, the glial changes may be deleterious for the neurons, because they could diminish glutamate uptake due to the depolarized membrane potential. However, increased extracellular glutamate concentration is toxic for most neurons.

A function of delayed rectifier potassium channels in glial cells: maintenance of an auxiliary membrane potential under pathological conditions.

Müller glial cells from human and guinea-pig retinae were investigated using the whole-cell patch-clamp technique. Human Müller cells from eyes with different diseases were characterized by diminished inwardly-rectifying K(+) currents. A comparable reduction of these currents was achieved in guinea pig Müller cells by treatment with iodoacetate to generate ischemia-like conditions. Consequently, the membrane potentials were reduced significantly in both diseased human and iodoacetate-treated guinea-pig Müller cells as compared to normal controls. However, the potentials were still clearly negative. Delayed rectifier currents could still be recorded under these conditions. Application of quinine blocked the delayed rectifier K(+) channels, and resulted in a total breakdown of the membrane potentials. Thus, it becomes apparent that the glial delayed rectifier K(+) channels are necessary to maintain an 'auxiliary' membrane potential under certain pathological conditions that are characterized by an almost total loss of inward rectifier conductance. Therefore, the delayed rectifier K(+) channels of glial cells may become crucial for the support of basic glial functions.

The local and systemic secretion of the pro-inflammatory cytokine interleukin-6 after transplantation of retinal pigment epithelium cells in a rabbit model.

The rejection of retinal pigment epithelium (RPE) allografts is one of the major problems for long-term success after retinal transplantation. However, the details of the immunological interactions in the subretinal space after transplantation are still unknown. The aim of our study was to investigate the role of IL-6 in the rejection process in the subretinal space and to use IL-6 monitoring for a possible early sign of rejection after transplantation of allogeneic RPE cells. For this we used a model of transplanting pigmented RPE cells, either activated in vitro with 1000 U/ml interferon-gamma (IFN-gamma) for 8 days or non-activated, into 30 albino rabbits. The IL-6 was investigated 3, 5, 7, 9 and 14 days after transplantation. Additionally, sham operated animals and the untreated eyes served as controls. At these time-points the animals were killed, the liquid in the vitreous cavity and serum was collected and the IL-6 present in these samples was quantified with an enzyme-linked immunosorbent assay. Under these conditions, IL-6 was detected in the liquid of the vitreous cavity and in the serum of all RPE-transplanted rabbits. In the group receiving activated RPE two cytokine peaks were measured, 3 and 7 days after transplantation in the vitreous cavity. In non-activated grafts, a maximum was detected on the 5th day after transplantation. Generally, the detected quantity of IL-6 depended on the host status and on the phase of rejection. No significant changes were seen in the sera from either group. Possibly, the host RPE cells are the main source of this interleukin in the transplantation area. The measuring of IL-6 in the rejection model suggests that it plays a role in the immune cascade in the subretinal space.

Na(+) currents through Ca(2+) channels in human retinal glial (Müller) cells.

PURPOSE: To detect the presence of voltage-gated Ca(2+) channels in the plasma membranes of freshly isolated Müller glial cells from the human retina and their modulation by GABA(B) receptor agonists. METHODS: Whole cell voltage-clamp recordings were made to study Ca( 2+), Ba(2+), and Na(+) currents through voltage-gated Ca(2+) channels. RESULTS: The vast majority of the investigated cells displayed no resolvable currents through Ca(2+) channels when Ca(2+) ions (2 mM) were present in the extracellular solution. Small-amplitude inwardly directed currents ( approximately 0.6 pA/pF) were detected when Ba(2+) ions (20 mM) were used as charge carrier. However, when Na(+) ions were used as charge carrier in divalent cation-free external solution, currents of large amplitudes ( approximately 7.5 pA/pF) through voltage-gated Ca(2+) channels were detected. Human Müller cells displayed currents through both transient, low voltage-activated Ca(2+) channels and long-lasting, high voltage-activated channels. The Na(+) fluxes through low voltage-activated Ca( 2+) channels were inhibited in a voltage-independent manner in the presence of GABA(B) receptor agonists. CONCLUSIONS: Human Müller glial cells express different kinds of voltage-gated Ca(2+) channels in their plasma membranes that can be activated only under certain physiological or pathophysiological conditions. The record of Na(+) fluxes in divalent cation-free solutions may be a technique to detect the presence of "hidden" voltage-gated Ca(2+) channels in Müller glial cells.

[Blue-yellow perimetry in rhegmatogenous retinal detachment. Studies before and after therapy in comparison with white-white perimetry]. / Blau-Gelb-Perimetrie bei rhegmatogener Amotio retinae. Untersuchungen vor und nach Therapie im Vergleich zur Weiss-Weiss-Perimetrie.

INTRODUCTION: Disturbance of the blue light perceptance in rhegmatogenous retinal detachment was demonstrated by Köllner 1907 with the help of colour-perimetries. We examined the blue- and white-function after retinal reattachment by blue-on-yellow-perimetry compared with white-on-white-perimetry. PATIENTS AND METHODS: 10 Patients with rhegmatogenous retinal detachment and a visual acuity > or = 0.1 were examined preoperatively, 4 days and 6 weeks after successful buckling procedure. We performed a white-on-white-perimetry as also perimetries with blue stimuli on a yellow luminance background (program 30-2 of the Humphrey Field Analyzer 750). RESULTS: Four days after surgery the increase in sensitivity was significantly less for the blue-function compared with the white-function. Preoperatively and 6 weeks postoperatively the sensitivities were not significantly different. Between these time-points function recovered in average for both methods from MD -15 dB to MD -5 dB. CONCLUSION: After successful retinal detachment surgery the blue- and white-function increased. Blue-on-yellow-perimetry showed more retinal sensitivity loss than white-on-white-perimetry immediately after surgery. Blue-on-yellow-perimetry measured the same retinal sensitivity loss as white-on-white-perimetry preoperatively and 6 weeks postoperatively.

[Activated protein C resistance in patients with central retinal vein occlusion in comparison to patients with a history of deep-vein thrombosis and a healthy control group]. / APC-Resistenz bei Patienten mit Zentralvenenverschluss. Vergleich mit Patienten mit tiefer Becken- un Veinvenenthrombose und gesunder Kontrollgruppe.

BACKGROUND: The recently described "APC resistance" caused by a mutant form of factor V (factor V Leiden) is the most frequent cause of hereditary thrombosis. This study was carried out to investigate the association between activated protein C resistance and central retinal vein occlusion (CRVO). We evaluated the prevalence of APC resistance in patients with CRVO, patients with a history of deep-vein thrombosis, and a healthy control group. PATIENTS AND METHODS: We examined 107 patients with CRVO, 112 patients with deep-vein thrombosis and 70 healthy individuals. The test performed was a modified APC-resistance assay using factor V-deficient plasma. RESULTS: We identified APC resistance in 5.6 % of patients with CRVO and in 5.7 % of the control group. All carriers were heterozygous. In the deep-vein thrombosis group 23.2 % tested positive for APC resistance. Four patients were homozygous and 22 were heterozygous carriers. CONCLUSION: These results indicate that APC resistance has no major role in the pathogenesis of CRVO. Routine testing for the presence of factor V Leiden mutant in CRVO is not necessary.

[Autologous thrombocyte administration in treatment of idiopathic macular foramen]. / Autologe Thrombozytenapplikation bei der Behandlung des idiopathischen Makulaforamens.

BACKGROUND: Recent studies have shown the usefulness of pars plana vitrectomy with the use of growth factors in the treatment of macular holes. Autologous platelet concentrates contain many growth factors to stimulate glial wound healing. PATIENTS: Nineteen patients with idiopathic macular hole underwent vitrectomy, membrane peeling, air injection and installation of autologous platelet concentrate (0.1 ml). The platelet concentrate contained a mean of 1.8 x 10(9) platelets/ml. RESULTS: The anatomic success rate in stage 2 macular hole was 100%, in stage 3, 82% and in stage 4, 50%. Visual acuity improved in all patients with stage 2 (two lines) and in 73% of stage 3 at least (one line). CONCLUSION: Platelets are effective in the treatment of macular holes due to the high amount of different growth factors (PDGF, EGF, bFGF, IGF-1) which have a high affinity binding to Müller cells helping to seal the hole by photoreceptor adaption.