Changes in Membrane Conductance Play a Pathogenic Role in Osmotic Glial Cell Swelling in Detached Retinas
2006; Elsevier BV; Volume: 169; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.060628
ISSN1525-2191
AutoresAntje Wurm, Thomas Pannicke, Ianors Iandiev, Eva Bühner, Uta-Carolin Pietsch, Andreas Reichenbach, Peter Wiedemann, Susann Uhlmann, Andreas Bringmann,
Tópico(s)Retinal Diseases and Treatments
ResumoDetachment of the neural retina from the pigment epithelium may be associated with tissue edema; however, the mechanisms of fluid accumulation are not understood. Because retinal detachment is usually not accompanied by vascular leakage, we investigated whether the osmotic swelling characteristics of retinal glial (Müller) cells are changed after experimental detachment of the porcine retina. Osmotic stress, induced by application of a hypotonic bath solution to retinal slices, caused swelling of Müller cell bodies in 7-day-detached retinas, but no swelling was inducible in slices of control retinas. Müller cell somata in slices of retinal areas that surround local detachment in situ also showed osmotic swelling, albeit at a smaller amplitude. The amplitude of osmotic Müller cell swelling correlated with the decrease in the K+ conductance, suggesting a causal relationship between both gliotic alterations. Further factors implicated in Müller cell swelling were inflammatory mediators and oxidative stress. We propose that a dysregulation of the ion and water transport through Müller cells may impair the fluid absorption from the retinal tissue, resulting in chronic fluid accumulation after detachment. This knowledge may lead to a better understanding of the mechanisms involved in retinal degeneration after detachment. Detachment of the neural retina from the pigment epithelium may be associated with tissue edema; however, the mechanisms of fluid accumulation are not understood. Because retinal detachment is usually not accompanied by vascular leakage, we investigated whether the osmotic swelling characteristics of retinal glial (Müller) cells are changed after experimental detachment of the porcine retina. Osmotic stress, induced by application of a hypotonic bath solution to retinal slices, caused swelling of Müller cell bodies in 7-day-detached retinas, but no swelling was inducible in slices of control retinas. Müller cell somata in slices of retinal areas that surround local detachment in situ also showed osmotic swelling, albeit at a smaller amplitude. The amplitude of osmotic Müller cell swelling correlated with the decrease in the K+ conductance, suggesting a causal relationship between both gliotic alterations. Further factors implicated in Müller cell swelling were inflammatory mediators and oxidative stress. We propose that a dysregulation of the ion and water transport through Müller cells may impair the fluid absorption from the retinal tissue, resulting in chronic fluid accumulation after detachment. This knowledge may lead to a better understanding of the mechanisms involved in retinal degeneration after detachment. Detachment of the neural retina from the pigment epithelium is a major cause of vision loss, with approximately 15,000 new cases of nontraumatic retinal detachment every year in the United States.1Regillo CD Bensen WE Retinal Detachment: Diagnosis and Management. JB Lippincott, Philadelphia1988Google Scholar In recent years, retinal detachment has become part of a surgical procedure for treating age-related macular degeneration by macular translocation. Likewise, a number of proposed retinal therapies, such as transplantation of pigment epithelium or photoreceptors, electronic retinal implants, or injection of trophic factors or vectors into the subretinal space, may include short- and long-term detachments. Though reattachment surgery has a success rate of over 90% in producing morphological recovery, often there are visual deficits that persist for long time periods after retinal reattachment, suggesting that functional impairments of retinal cells are not recovered. Experimental detachment causes complex cellular responses in the neural retina.2Fisher SK Lewis GP Linberg KA Verardo MR Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment.Prog Retin Eye Res. 2005; 24: 395-431Crossref PubMed Scopus (256) Google Scholar The damage to the outer segments and the apoptotic death of photoreceptor cells,3Cook B Lewis GP Fisher SK Adler R Apoptotic photoreceptor degeneration in experimental retinal detachment.Invest Ophthalmol Vis Sci. 1995; 36: 990-996PubMed Google Scholar, 4Mervin K Valter K Maslim J Lewis GP Fisher SK Stone J Limiting photoreceptor death and deconstruction during experimental retinal detachment: the value of oxygen supplementation.Am J Ophthalmol. 1999; 128: 155-164Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar as well as the morphological and biochemical alterations of inner retinal neurons,5Lewis GP Linberg KA Fisher SK Neurite outgrowth from bipolar and horizontal cells after experimental retinal detachment.Invest Ophthalmol Vis Sci. 1998; 39: 424-434PubMed Google Scholar, 6Marc RE Murry RF Fisher SK Linberg KA Lewis GP Amino acid signatures in the detached cat retina.Invest Ophthalmol Vis Sci. 1998; 39: 1694-1702PubMed Google Scholar, 7Faude F Francke M Makarov F Schuck J Gartner U Reichelt W Wiedemann P Wolburg H Reichenbach A Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina.J Neurocytol. 2001; 30: 379-390Crossref PubMed Scopus (55) Google Scholar, 8Coblentz FE Radeke MJ Lewis GP Fisher SK Evidence that ganglion cells react to retinal detachment.Exp Eye Res. 2003; 76: 333-342Crossref PubMed Scopus (59) Google Scholar are associated with activation of macro- and microglial cells.9Anderson DH Guerin CJ Erickson PA Stern WH Fisher SK Morphological recovery in the reattached retina.Invest Ophthalmol Vis Sci. 1986; 27: 168-183PubMed Google Scholar, 10Fisher SK Erickson PA Lewis GP Anderson DH Intraretinal proliferation induced by retinal detachment.Invest Ophthalmol Vis Sci. 1991; 32: 1739-1748PubMed Google Scholar, 11Lewis GP Guerin CJ Anderson DH Matsumoto B Fisher SK Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment.Am J Ophthalmol. 1994; 118: 368-376Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 12Lewis GP Mervin K Valter K Maslim J Kappel PJ Stone J Fisher S Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment: the value of oxygen supplementation.Am J Ophthalmol. 1999; 128: 165-172Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Geller SF Lewis GP Fisher SK FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Müller and RPE cells.Invest Ophthalmol Vis Sci. 2001; 42: 1363-1369PubMed Google Scholar Among the various gliotic responses being characteristic for the detached retina, Müller glial cells display a decrease in their K+ conductance.14Francke M Faude F Pannicke T Bringmann A Eckstein P Reichelt W Wiedemann P Reichenbach A Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR.Invest Ophthalmol Vis Sci. 2001; 42: 1072-1079PubMed Google Scholar, 15Uhlmann S Bringmann A Uckermann O Pannicke T Weick M Ulbricht E Goczalik I Reichenbach A Wiedemann P Francke M Early glial cell reactivity in experimental retinal detachment: effect of suramin.Invest Ophthalmol Vis Sci. 2003; 44: 4114-4122Crossref PubMed Scopus (40) Google Scholar, 16Iandiev I Uckermann O Pannicke T Wurm A Tenckhoff S Pietsch UC Reichenbach A Wiedemann P Bringmann A Uhlmann S Glial cell reactivity in a porcine model of retinal detachment.Invest Ophthalmol Vis Sci. 2006; 47: 2161-2171Crossref PubMed Scopus (111) Google Scholar The decrease in K+ conductance is associated with a decrease in the gene and protein expression of the major K+ channel subtype of the cells, inwardly rectifying Kir4.1 channel.16Iandiev I Uckermann O Pannicke T Wurm A Tenckhoff S Pietsch UC Reichenbach A Wiedemann P Bringmann A Uhlmann S Glial cell reactivity in a porcine model of retinal detachment.Invest Ophthalmol Vis Sci. 2006; 47: 2161-2171Crossref PubMed Scopus (111) Google Scholar It has been suggested that impaired retinal K+ clearance, normally performed by Müller cells by means of their K+ channels,17Newman EA Reichenbach A The Müller cell: a functional element of the retina.Trends Neurosci. 1996; 19: 307-312Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar may contribute to neuronal hyperexcitation and glutamate toxicity and, therefore, to neuronal degeneration in the detached retina.14Francke M Faude F Pannicke T Bringmann A Eckstein P Reichelt W Wiedemann P Reichenbach A Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR.Invest Ophthalmol Vis Sci. 2001; 42: 1072-1079PubMed Google Scholar, 16Iandiev I Uckermann O Pannicke T Wurm A Tenckhoff S Pietsch UC Reichenbach A Wiedemann P Bringmann A Uhlmann S Glial cell reactivity in a porcine model of retinal detachment.Invest Ophthalmol Vis Sci. 2006; 47: 2161-2171Crossref PubMed Scopus (111) Google Scholar Various studies suggest that retinal detachment may be associated with fluid accumulation in the retinal tissue. Intra- and extracellular edema in and around Müller cells is an early alteration in the detached retina of the pig.18Jackson TL Hillenkamp J Williamson TH Clarke KW Almubarak AI Marshall J An experimental model of rhegmatogenous retinal detachment: surgical results and glial cell response.Invest Ophthalmol Vis Sci. 2003; 44: 4026-4034Crossref PubMed Scopus (45) Google Scholar Experimental detachment of the primate retina causes edematous swelling and cystoid degeneration of the inner retinal layers.19Machemer R Experimental retinal detachment in the owl monkey. II. Histology of retina and pigment epithelium.Am J Ophthalmol. 1968; 66: 396-410Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 20Machemer R Norton EW Experimental retinal detachment and reattachment: I. Methods, clinical picture and histology.Bibl Ophthalmol. 1969; 79: 80-90PubMed Google Scholar Cystoid fluid-filled spaces have been described to develop in the detached human retina.21Arruga H Die Netzhautablösung. Barcelona, 1936Google Scholar Optical coherence tomography performed before reattachment surgery often demonstrated edema in the macular tissue,22Wolfensberger TJ Gonvers M Optical coherence tomography in the evaluation of incomplete visual acuity recovery after macula-off retinal detachments.Graefes Arch Clin Exp Ophthalmol. 2002; 240: 85-89Crossref PubMed Scopus (150) Google Scholar, 23Hagimura N Suto K Iida T Kishi S Optical coherence tomography of the neurosensory retina in rhegmatogenous retinal detachment.Am J Ophthalmol. 2000; 129: 186-190Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar even in cases when the macula remained attached.24Siwiec-Proœcińska J Rakowicz P Pecold K The evaluation of macular thickness in patients with retinal detachment treated with conventional surgery—preliminary report.Klin Oczna. 2004; 106: 581-584PubMed Google Scholar Fluid accumulation within the retinal tissue may contribute to neuronal degeneration and to the decrease in visual acuity after detachment. The fluid accumulation in the retina suggests that the water homeostasis in the detached retina is altered. Generally, two cell types are implicated in the water homeostasis of the neural retina. The subretinal space, which encloses the photoreceptor segments, is dehydrated by the pigment epithelium,25Marmor MF Mechanisms of fluid accumulation in retinal edema.Doc Ophthalmol. 1999; 97: 239-249Crossref PubMed Google Scholar whereas the inner retina is dehydrated by transcellular water transport through Müller cells.26Nagelhus EA Horio Y Inanobe A Fujita A Haug FM Nielsen S Kurachi Y Ottersen OP Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Müller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains.Glia. 1999; 26: 47-54Crossref PubMed Scopus (411) Google Scholar, 27Bringmann A Reichenbach A Wiedemann P Pathomechanisms of cystoid macular edema.Ophthalmic Res. 2004; 36: 241-249Crossref PubMed Scopus (246) Google Scholar The water transport through pigment epithelial and Müller cells is tightly coupled to a transcellular transport of ions, especially of K+ and Cl−. In distinct membrane domains of Müller cells, eg, around blood vessels, Kir4.1 and aquaporin-4 water channels are co-localized, suggesting that the transglial water transport is predominantly coupled to K+ currents that flow through the cells into the blood.26Nagelhus EA Horio Y Inanobe A Fujita A Haug FM Nielsen S Kurachi Y Ottersen OP Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Müller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains.Glia. 1999; 26: 47-54Crossref PubMed Scopus (411) Google Scholar It has been suggested that the down-regulation of Kir4.1 channels in Müller cells under pathological conditions will disrupt this water transport—in addition to the transglial K+ clearance currents—through the cells.27Bringmann A Reichenbach A Wiedemann P Pathomechanisms of cystoid macular edema.Ophthalmic Res. 2004; 36: 241-249Crossref PubMed Scopus (246) Google Scholar, 28Pannicke T Iandiev I Uckermann O Biedermann B Kutzera F Wiedemann P Wolburg H Reichenbach A Bringmann A A potassium channel-linked mechanism of glial cell swelling in the postischemic retina.Mol Cell Neurosci. 2004; 26: 493-502Crossref PubMed Scopus (196) Google Scholar The formation of chronic edema is caused by an imbalance between the fluid inflow into the tissue and the fluid absorption from the tissue. Normally, retinal detachment is not associated with vascular leakage, suggesting that the fluid accumulation in the detached retina is caused by other mechanisms. We hypothesize that an impairment of the fluid absorption function of Müller cells may contribute to the fluid accumulation in the detached retina. To investigate whether Müller cells alter the water transport across their plasma membranes after detachment, we determined the volume changes of the cells in response to hypotonic stress (a situation resembling hypoxia-induced intracellular edema in the brain). We found that osmotic stress causes swelling of Müller cells in detached retinas but not in control retinas. Furthermore, we found that the decrease in K+ conductance characteristically for Müller cells of detached retinas14Francke M Faude F Pannicke T Bringmann A Eckstein P Reichelt W Wiedemann P Reichenbach A Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR.Invest Ophthalmol Vis Sci. 2001; 42: 1072-1079PubMed Google Scholar, 15Uhlmann S Bringmann A Uckermann O Pannicke T Weick M Ulbricht E Goczalik I Reichenbach A Wiedemann P Francke M Early glial cell reactivity in experimental retinal detachment: effect of suramin.Invest Ophthalmol Vis Sci. 2003; 44: 4114-4122Crossref PubMed Scopus (40) Google Scholar, 16Iandiev I Uckermann O Pannicke T Wurm A Tenckhoff S Pietsch UC Reichenbach A Wiedemann P Bringmann A Uhlmann S Glial cell reactivity in a porcine model of retinal detachment.Invest Ophthalmol Vis Sci. 2006; 47: 2161-2171Crossref PubMed Scopus (111) Google Scholar is significantly correlated with the alteration in the osmotic swelling characteristics of the cells. Mitotracker Orange (chloromethyltetramethylrosamine) was purchased from Molecular Probes (Eugene, OR). Adenosine 5′-diphosphate (ADP), adenosine 5′-triphosphate (ATP), uridine triphosphate (UTP), triamcinolone acetonide (9α-fluoro-16α-hydroxyprednisolone), prostaglandin E2 (PGE2), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), N-nitrobenzylthioinosine (NBTI), N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and all other substances used were purchased from SigmaAldrich (Taufkirchen, Germany). The following antibodies were used: mouse anti-glutamine synthetase (1:250; Chemicon), mouse anti-vimentin (1:400, V9 clone; Immunotech, Marseille, France), rabbit anti-cyclooxygenase-2 (1:100; Cayman Chemical, Ann Arbor, MI), rabbit anti-Kir4.1 (1:200; Alomone Labs, Jerusalem, Israel), Cy3-conjugated goat anti-rabbit IgG (1:400; Dianova, Hamburg, Germany), and Cy2-coupled goat anti-mouse IgG (1:200; Dianova). All experiments were performed in accordance with applicable German laws and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Twelve young adult domestic white pigs (17–22 kg; both sexes) were used. Twenty-four hours before and after surgery, the food intake of the animals was restricted, with free access to water. Intramuscular azaperone (15 mg/kg; Cilag-Janssen, Neuss, Germany), atropine (0.2 mg/kg; Braun, Melsungen, Germany), and ketamine (3 mg/kg; Ratiopharm, Ulm, Germany) were administered for premedication. By stepwise application of ketamine (5 mg/kg) and propofol (5 mg/kg; Ratiopharm), a totally intravenous anesthesia was performed. Deltajonin (Delta Select, Pfullingen, Germany) was continuously infused via a vein of the ear. Pulse rate and pO2 were monitored during anesthesia, and O2 (3 l/min) was applied. Rhegmatogenous detachment was created in one eye per animal; the other eye served as nonoperated control. The pupils of the eyes were dilated by topical tropicamide (1%; Ursapharm, Saarbrücken, Germany) and phenylephrine hydrochloride (5%; Ankerpharm, Rudolstadt, Germany), and a lateral canthotomy was created. Hemostasis was achieved with wet-field cautery. After pars plana sclerotomy, a circumscript vitrectomy was performed in the area of the future detachment, and balanced salt solution (Delta Select) was infused into the eye to replace the vitreous. Thin glass micropipettes attached to 250-μl glass syringes (Hamilton, Reno, NV) were used to create a retinal detachment by subretinal injection of saline, followed by 0.25% sodium hyaluronate in saline (Healon; Pharmacia & Upjohn, Dübendorf, Switzerland). The retina ventral of the optic nerve head was detached, whereas the dorsal retina remained attached. After surgery, gentamicin (5 mg) and dexamethasone (0.5 mg) were injected subconjunctivally. The lateral canthotomy was closed with 5-0 silk sutures, and atropine (1%) eye drops were instilled into the conjunctival sac. After a survival time of 7 days, the animals were anesthetized as described, the eyes were excised, and the animals were sacrificed by intravenous T61 (embutramid mebezonium iodide; 0.65 ml/kg body weight; Hoechst, Unterschleißheim, Germany). To investigate whether operation procedure per se alters osmotic swelling of glial cells, vitrectomy (without retinal detachment) was performed in one further animal, and the retinas and cells were investigated at 7 days after surgery. Whole-cell patch-clamp recordings were performed using Müller cells acutely isolated in papain and DNase I-containing solutions, as described previously.14Francke M Faude F Pannicke T Bringmann A Eckstein P Reichelt W Wiedemann P Reichenbach A Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR.Invest Ophthalmol Vis Sci. 2001; 42: 1072-1079PubMed Google Scholar The cell suspensions were stored in serum-free modified Eagle's medium at 4°C (up to 6 hours) before use. Voltage-clamp records were performed at room temperature using the Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and the ISO-2 computer program (MFK, Niedernhausen, Germany). Patch pipettes were pulled from borosilicate glass (WPI, Sarasota, FL) and had resistances between 4 and 6 megaΩ when filled with the intracellular solution that contained 10 mmol/L NaCl, 130 mmol/L KCl, 1 mmol/L CaCl2, 2 mmol/L MgCl2, 10 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.1. The signals were low-pass filtered at 1, 2, or 6 kHz (eight-pole Bessel filter) and digitized at 5, 10, or 30 kHz, respectively, using a 12-bit analog/dialog converter. The recording chamber was continuously perfused with extracellular solution consisting of 135 mmol/L NaCl, 3 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 1 mmol/L Na2HPO4, 10 mmol/L HEPES-Tris, and 11 mmol/L glucose, pH 7.4. To evoke K+ currents, depolarizing and hyperpolarizing voltage steps of 250-ms duration, with increments of 10 mV, were applied from a holding potential of −80 mV. The membrane capacitance of the cells was measured by the integral of the uncompensated capacitive artifact (filtered at 6 kHz) evoked by a hyperpolarizing voltage step from −80 to −90 mV when Ba2+ (1 mmol/L) was present in the bath solution. Membrane potentials were measured in the current-clamp mode. Isolated retinas were fixed in 4% paraformaldehyde for 2 hours. After several washing steps in buffered saline, the tissue was embedded in saline containing 3% agarose (w/v), and 70-μm-thick slices were cut by using a vibratome. The slices were incubated in 5% normal goat serum plus 0.3% Triton X-100 in saline for 2 hours at room temperature and, subsequently, in primary antibodies overnight at 4°C. After washing in 1% bovine serum albumin in saline, the secondary antibodies were applied for 2 hours at room temperature. The lack of unspecific staining was demonstrated by negative controls omitting the primary antibodies (not shown). Images were recorded with the confocal laser scanning microscope LSM 510 Meta (Carl Zeiss GmbH, Jena, Germany) at single planes; excitation and emission settings were held constant for all images acquired. The experiments were performed at room temperature. To determine volume changes of Müller glial cells in situ evoked by hypotonic stress, the cross-sectional area of Müller cell somata in the inner nuclear layer of retinal slices was measured. Acutely isolated retinal slices (thickness, 1 mm) were placed in a perfusion chamber and loaded with the vital dye Mitotracker Orange (10 μmol/L). It has been shown that Mitotracker Orange is taken up selectively by Müller glial cells in the retina, whereas neurons remain unstained.29Uckermann O Iandiev I Francke M Franze K Grosche J Wolf S Kohen L Wiedemann P Reichenbach A Bringmann A Selective staining by vital dyes of Müller glial cells in retinal wholemounts.Glia. 2004; 45: 59-66Crossref PubMed Scopus (70) Google Scholar The dye was dissolved in extracellular solution that contained 136 mmol/L NaCl, 3 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L HEPES-Tris, and 11 mmol/L glucose, pH 7.4. A gravity-fed system with multiple reservoirs was used to perfuse the recording chamber continuously with extracellular solutions; the hypotonic solution was added by fast changing of the perfusate. The hypotonic solution contained 60% of control osmolarity and was made by adding distilled water to the extracellular solution. Ba2+ (1 mmol/L) was preincubated for 10 minutes in extracellular solution before it was applied within hypotonic solution, and blocking substances were preincubated for 15 minutes before hypotonic challenge. The slices were examined by using the LSM. Mitotracker Orange was excited at 543 nm, and emission was recorded with a 560-nm long-pass filter. During the experiments, the Mitotracker Orange-stained cell somata in the inner nuclear layer were recorded at the plane of their largest extension. To assure that the maximum soma areas were precisely recorded, the focal plane was continuously adjusted during the course of the experiments. To determine the extent of soma swelling, the cross-sectional area of Mitotracker Orange-stained cell bodies in the inner nuclear layer of retinal slices was measured using the image analysis software of the LSM. Bar diagrams display the mean cross-sectional areas of Müller cell somata that were measured after a 4-minute perfusion of the hypotonic solution, in percentage of the soma area measured before osmotic challenge (100%). The amplitude of the steady-state inward K+ conductance was measured at the end of the 250-ms voltage step from −80 to −140 mV. Statistical analysis was made using SigmaPlot (SPSS Inc., Chicago, IL) and the Prism program (Graphpad Software, San Diego, CA); significance was determined by Mann-Whitney U-test for two groups and by analysis of variance followed by comparisons for multiple groups. Data are expressed as means ± SEM. As shown recently,16Iandiev I Uckermann O Pannicke T Wurm A Tenckhoff S Pietsch UC Reichenbach A Wiedemann P Bringmann A Uhlmann S Glial cell reactivity in a porcine model of retinal detachment.Invest Ophthalmol Vis Sci. 2006; 47: 2161-2171Crossref PubMed Scopus (111) Google Scholar Müller cells isolated 7 days after surgery from detached porcine retinas displayed a strong decrease in the amplitude of their inward K+ currents (Figure 1A), in the mean by 85% compared with control (Figure 1B). Moreover, Müller cells isolated from peri-detached retinal areas (ie, attached areas that surrounded the local detachment in situ) displayed a decrease of their K+ currents, in the mean by 63%. Cells from vitrectomized control eyes (without retinal detachment) displayed current amplitudes similar to the native controls (Figure 1B). The decrease of the K+ conductance was associated with a decrease in the membrane potential of Müller cells (Figure 1C). The cell membrane capacitance, which is a marker of the cell membrane area, was significantly enlarged in Müller cells isolated from detached retinas compared with cells from nonoperated control eyes (Figure 1D). Cells isolated from peri-detached retinal areas displayed a weaker, but still significant, increase in their membrane capacitance, whereas cells from vitrectomized eyes (without retinal detachment) showed a normal membrane capacitance (Figure 1D). The data suggest that gliotic alterations occur also in attached retinal areas that surround local detachments in operated eyes, albeit at a lower degree. The swelling of Müller cell somata was investigated in acutely isolated retinal slices (Figure 2A) by perfusing the slices with a hypotonic solution that contained 60% of control osmolarity. Exposure to hypotonic solution did not alter the size of Müller cell bodies in retinal slices from nonoperated and vitrectomized control eyes (Figure 2, B–D). However, perfusion of slices from detached retinas caused a time-dependent increase in the size of Müller cell bodies (Figure 2B). After a 4-minute perfusion with hypotonic solution, the cross-sectional area of Müller cell bodies in detached retinas increased by 12.3 ± 0.7% (P < 0.001) (Figure 2C). Interestingly, Müller cell somata in slices from peri-detached retinal areas showed a similar swelling on hypotonic stress (Figure 2B), albeit with a significantly (P < 0.001) lower amplitude than cells from detached retinas; as a mean, cells in the peri-detached retina swelled by 6.5 ± 1.0% (P < 0.001) (Figure 2C). Müller cell bodies in slices from control, detached, and peri-detached retinas swelled on hypotonic stress when K+ channel-blocking Ba2+ ions were present in the bath solution (Figure 2, B–D). The data indicate that Müller cells in detached and, to a lower extent, peri-detached retinal areas are more sensitive to osmotic stress than cells in control retinas and lack the ability of rapid volume regulation under hypotonic conditions. We show here that Müller cells in detached retinas decrease their K+ conductance (Figure 1, A and B) and are more sensitive to osmotic stress conditions. The observation that K+ channel-blocking Ba2+ ions induce osmotic swelling in cells from control retinas (Figure 2, B–D) suggests a causal relationship between the alterations of both physiological parameters during detachment. To support this assumption, we determined whether there is a correlation between the decrease of the K+ conductance and the extent of cellular swelling in Müller cells of 11 operated animals. Figure 3 displays a scatter plot of both parameters obtained in cells from operated and control eyes for each animal. As shown, there was a negative correlation between both parameters; the smaller the mean K+ currents of Müller cells, the larger was the amplitude of cellular swelling under hypotonic conditions (r = −0.805; P < 0.001). The data suggest that changes in the expression or functional state of K+ channels play a pathogenic role in osmotic swelling of Müller cells in detached retinas. However, because cells from different retinal areas (eg, from control retinas and peri-detached retinal areas) may show different swelling amplitudes despite similar K+ currents (Figure 3), other causative factors—in addition to the decrease of functional K+ channels—may contribute to the alteration of glial swelling characteristics after retinal detachment. Such factors may be inflammatory mediators and oxidative stress.30Pannicke T Iandiev I Wurm A Uckermann O vom Hagen F Reichenbach A Wiedemann P Hammes H-P Bringmann A Diabetes alters osmotic-swelling characteristics and membrane conductance of glial cells in rat retina.Diabetes. 2006; 55: 633-639Crossref PubMed Scopus (165) Google Scholar, 31Uckermann O Wolf A Kutzera F Kalisch F Beck-Sickinger AG Wiedemann P Reichenbach A Bringmann A Glutamate release by neurons evokes a purinergic inhibitory mechanism of osmotic glial cell swelling in the rat retina: activation by neuropeptide Y.J Neurosci Res. 2006; 83: 538-550Crossref PubMed Scopus (86) Google Scholar The anti-inflammatory glucocorticoid triamcinolone acetonide is used clinically for the rapid resolution of retinal edema.32Martidis A Duker JS Greenberg PB Rogers AH Puliafito CA Reichel E Baumal C Intravitreal triamcinolone for refractory diabetic macular edema.Ophthalmology. 2002; 109: 920-927Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 33Ip M Kahana A Altaweel M Treatment of central retinal vein occlusion with triamcinolone acetonide: an optical coherence tomography study.Semin Ophthalmol. 2003; 18: 67-73Crossref PubMed Scopus (62) Go
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