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

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

Effect of Aβ protein on inhibiting proliferation and promoting apoptosis of retinal pigment epithelial cells

AIM: To identify the effect and regulatory mechanism of amyloid β (Aβ) protein on retinal pigment epithelial (RPE) cells in cell proliferation and apoptosis, and clarify Aβ role in the pathogenesis of age-related macular degeneration (AMD).METHODS: The model of Aβ25-35 protein cytotoxicity in RPE cell was successfully established to investigate the effect of Aβ protein on RPE cells in vitro. Based on Aβ protein, the specific inhibitors (HY-50682 or BAY11-7082) or activating agent (lipopolysaccharide) was used to analyze the regulatory mechanism of Aβ protein to RPE cells on cell proliferation and apoptosis by flow cytometry, real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay and dual-luciferase reporter gene assay.RESULTS: The number of RPE cells, treated with Aβ25-35 from 0.3 to 60 μmol/L, significantly reduce (P<0.01), and had the dose-dependent effect. Aβ protein 60 μmol/L inhibits the G1/S phase transition (P<0.01) and down-regulated cyclin E mRNA level (P<0.01). Similarly, Aβ25-35 induced a significant increase of cell apoptosis, accompanied by the significantly higher level of activated caspase 3 protein. Furthermore, nuclear factor-kappaB (NF-κB) activity and phosphorylated Iκ-Ba level would significantly lower in treated RPE cells. Using specific inhibitors or activating agent based on the Aβ, the cell numbers, NF-κB activity, phosphorylated Iκ-Ba level, receptor for advanced glycation endproducts (RAGE) gene expression levels, cyclin E mRNA level and activated caspase 3 level had accordingly changed by different methods, confirming that RAGE/NF-κB signaling pathway involved in the regulation of Aβ protein on RPE cell apoptosis and proliferation.CONCLUSION: Aβ protein inhibits cell proliferation and activates apoptosis via inactivation of the RAGE/NF-κB signaling pathway in RPE cell.     

Effect of carnosine on steroid-induced modification of lens α-crystallin

To explore whether carnosine can protect α-crystallin modification and decrease chaperone by a steroid, and whether carnosine could directly react with a steroid. · METHODS: Bovine lens α-crystallin was separated by size- exclusion chromatography on a Sephacyl S-300 HR column. α-Crystallin was incubated with different concentrations of prednisolone-21-hemisuccinate (P-21-H) with or without carnosine for different times. The chaperone activity of α-crystallin was monitored using the prevention thermal aggregation of α-crystallin. The modified α-crystallin was examined by SDS-PAGE and fluorescence measurements. The absorbance spectra of solutions of carnosine and P-21-H were investigated. · RESULTS: P-21-H decreased the chaperone activity of α-crystallin in a concentration- and time-dependent fashion. Carnosine only worsened this effect. The tryptophan fluorescence intensity of α-crystallin modified by P-21-H was significantly decreased compared with unmodified crystallin, whereas its non-tryptophan fluorescence was increased with a shift to longer wavelengths in a time- and dose-dependent manner, suggesting that new fluorophores were possibly formed. Carnosine readily reacted with P-21-H thereby inhibiting steroid-mediated protein modification as revealed electrophoretically. The increased absorbance was time-dependent, suggesting adducts may be formed between carnosine and P-21-H. · CONCLUSION: Carnosine reacts with P-21-H, which suggests carnosine's potential as a possible anti-steroid agent.     

Protective effects of curcumin on retinal Müller cell in early diabetic rats

AIM: To explore the effects and potential mechanisms of curcumin on retinal Müller cell in early diabetic rats.METHODS: Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ). Male Sprague-Dawley (SD) rats were randomly assigned into 4 groups:control group (naïve SD rats administered with a single intraperitoneal injection of citric buffer), diabetic group (STZ-diabetic rats), dimethyl sulfoxide (DMSO) group (diabetic rats intraperitoneally administered with mixture of DMSO and normal saline, once a day) and curcumin group (diabetic rats intraperitoneally administered with curcumin, 80mg/kg, once a day). Three months after diabetes onset, malondialdehyde (MDA, indication of oxidative stress level) and reduced glutathione (GSH) in retina were detected with kits, glial fibrillary acidic protein (GFAP) in retina was revealed by immunohistochemistry and Western blot, and retinal glutamine synthetase (GS) were observed by Western blot.RESULTS: Compared with control group, retinal MDA was increased, and GSH was decreased in diabetic and DMSO groups (P<0.05, respectively). While, retinal MDA and GSH in curcumin group showed no difference compared with control group (P>0.05). Furthermore, up-regulation of retinal GFAP and down-regulation of retinal GS were detected in diabetic and DMSO groups, and no alteration could be observed in curcumin group revealed with Western blot. Compared with control group, retinal Müller cells showed significant increase in GFAP immunochemistry staining in diabetic and DMSO groups. Moreover, GFAP-positive staining was decreased in curcumin group compared with diabetic group.CONCLUSION: Curcumin inhibits diabetic retinal oxidative stress, protects Müller cell, and prevents the down-regulation of GS in diabetic retina. Therefore, curcumin has a therapeutic potential in the treatment of diabetic retinopathy (DR).     

Effect of bevacizumab (Avastin?) on mitochondrial function of in vitro retinal pigment epithelial,neurosensory retinal and microvascular endothelial cells

Purpose:

To evaluate the effect of bevacizumab on the mitochondrial function of human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28) and human microvascular endothelial (HMVEC) cells in culture.

Materials and Methods:

ARPE-19 and R28 cells were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab. The HMVEC cultures were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab or 1 mg/ml of immunoglobulin G (control). Mitochondrial function assessed by mitochondrial dehydrogenase activity (MDA) was determined using the WST-1 assay.

Results:

Bevacizumab doses of 0.125 to 1 mg/ml for 5 days did not significantly affect the MDA of ARPE-19 cells. Bevacizumab treatment at 0.125 and 0.25 mg/ml (clinical dose) did not significantly affect the MDA of R28 cells; however, 0.50 and 1 mg/ml doses significantly reduced the R28 cell mitochondrial function. All doses of bevacizumab significantly reduced the MDA of proliferating and non-proliferating HMVEC.

Conclusion:

Bevacizumab exposure for 5 days was safe at clinical doses in both ARPE-19 and R28 retinal neurosensory cells in culture. By contrast, bevacizumab exposure at all doses show a significant dose-dependent decrease in mitochondrial activity in both the proliferating and non-proliferating HMVEC in vitro. This suggests a selective action of bevacizumab on endothelial cells at clinical doses.     

Effects of arteriolar constriction on retinal gene expression and Müller cell responses in a rat model of branch retinal vein occlusion

Background

To investigate the effect of induced arteriolar constriction (AC) on alterations in gene expression of factors implicated in the development of edema in branch retinal vein occlusion (BRVO).

Methods

In Brown-Norway rats, BRVO was induced by laser photocoagulation of the veins in one half of the retina. AC of the afferent arterioles was performed 30 min later. We then determined the expression of Vegfa, Vegfb, Pedf, Kir4.1, Aqp4, Aqp1, Il1ß, and Il6 with real-time polymerase chain reaction (RT-PCR) in the neuroretina and retinal pigment epithelium (RPE) after 1, 3, and 7 days. Immunostaining against GFAP, aquaporin (AQP)-4, and Kir4.1 was performed on days 1 and 3.

Results

BRVO resulted in transient upregulation of Vegfa in the neuroretina on day 1. The expressions of Kir4.1, AQP4, and AQP1 were downregulated, and Il1ß and Il6 were strongly upregulated, on days 1 and 3. The retinal distribution of GFAP and AQP4 proteins remained unaltered, while the Kir4.1 protein displayed redistribution from polarized to uniform retinal distribution. AC accelerated the restoration of downregulated Kir4.1, Aqp4, and Aqp1 in the RPE, of Kir4.1 in the neuroretina, and of upregulated Il6 in the neuroretina. AC did not influence the gliotic alterations of Müller cells and the redistribution of the Kir4.1 protein.

Conclusion

Constriction of the afferent artery in the BRVO region accelerated the restoration of potassium channels and Il6. These alterations may contribute to faster resorption of retinal edema, and may decrease the level of inflammation.     

Arachidonic acid-induced inhibition of Ca2+ channel currents in retinal glial (Müller) cells

BACKGROUND: Arachidonic acid is a second messenger that has been implicated in several pathological conditions in nervous tissues. The present study was carried out to determine whether the second messenger arachidonic acid modulates currents through voltage-gated Ca2+ channels in freshly isolated Müller glial cells. METHODS: Whole-cell voltage-clamp recordings were made in human Muller cells to investigate Ba2+ and Na+ currents through high-voltage-activated (HVA) channels, and in rabbit Muller cells to study Na+ currents through low-voltage-activated (LVA) channels. RESULTS: Extracellular application of arachidonic acid reversibly and dose-dependently depressed the amplitude of both LVA (rabbit cells) and HVA currents (human cells). 10 microM arachidonic acid reduced the peak LVA and HVA currents by approximately 70%. A 50% reduction of LVA currents was achieved at 4.7 microM. The block of HVA and LVA currents was not accompanied by alterations in the voltage dependences of current activation and inactivation. A similar reduction of the currents was achieved by 20 microM eicosatetraynoic acid. CONCLUSION: Since eicosatetraynoic acid mimics the effects of arachidonic acid, it is assumed that arachidonic acid itself rather than its degradation products modulates glial Ca2+ channel activity. This Ca2+ channel inhibition may stabilize Muller cell function during pathological conditions in which arachidonic acid levels are elevated and may participate in the cellular action of neurotransmitters.     

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

PURPOSE: To determine whether experimental retinal detachment causes an alteration in Ca2 +-activated, big conductance K+ (BK) currents of Müller glial cells. METHODS: Rhegmatogenous retinal detachment was induced in porcine eyes. Müller cells were acutely isolated from control retinas and from retinas that were detached for 7 days. BK currents were detected by using the BK channel opener and the blocker phloretin and tetraethylammonium, respectively. RESULTS: In addition to cellular hypertrophy and a decrease in inward rectifier K+ currents, Müller cells from detached retinas showed an increase in the amplitude of currents mediated by BK channels (850 +/- 105 pA) when compared with cells from control retinas (228 +/- 60 pA; p < 0.001). Similarly, the density of the BK channel-mediated currents was greater in cells from detached retinas (12.32 +/- 1.52 pA/pF) compared with control cells (4.07 +/- 1.07 pA/pF; p < 0.001). The increase in BK currents was correlated with the decrease of the inward rectifier K+ currents. CONCLUSIONS: It is suggested that an increase in the expression of functional BK channels may be involved in gliotic responses of Müller cells after retinal detachment (e.g., in mitogen-induced Ca2+ responses and cellular proliferation).     

P2Y receptor-mediated stimulation of Müller glial cell DNA synthesis: dependence on EGF and PDGF receptor transactivation

PURPOSE: To determine whether P2Y receptor-evoked proliferation of Müller glial cells depends on transactivation of receptor tyrosine kinases. METHODS: Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine (BrdU) immunoassay, and the phosphorylation states of the extracellular signal-regulated kinase (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) were determined by Western blot analysis. RESULTS: In Müller cells, the mitogenic effect of P2Y receptor activation by extracellular adenosine triphosphate (ATP) depended on transactivation of both the platelet-derived growth factor (PDGF) and the epidermal growth factor (EGF) receptor tyrosine kinases, as suggested by the blocking effects of the tyrphostins AG1296 and AG1478 on the ATP-induced proliferation and phosphorylation of ERK1/2. Moreover, the PDGF-induced proliferation may depend on transactivation of the EGF receptor kinase. Antibodies against heparin-binding EGF (HB-EGF) or PDGF, as well as inhibition of matrix metalloproteinases (MMPs) blocked ATP-evoked proliferation. At least one metalloproteinase (MMP-9), was implicated in the signal transfer from P2Y to EGF receptors. In contrast, the mitogenic effect of fetal calf serum was independent of growth factor receptor activity. P2Y receptor activation stimulated Müller cell proliferation by activating the ERK1/2 and the phosphatidylinositol 3 (PI3) kinase signaling pathways, whereas the p38 MAPK pathway was not involved in mitogenic signaling. CONCLUSIONS: The present data suggest that P2Y-receptor-induced mitogenic signaling in Müller cells is mediated by transactivation of the PDGF and EGF receptor tyrosine kinases. The transactivation may be mediated by release of PDGF and MMP-dependent shedding of HB-EGF from the Müller cell matrix, respectively. The transactivation of the receptor tyrosine kinases may result in activation of ERK1/2 and PI3 kinase and an increase in the proliferation rate.     

Effect of geniposide on retinal microangiogenesis in rats with diabetic retinopathy and its mechanism北大核心

Objective To investigate the effect of geniposide (Gen) on retinal microangiogenesis in rats with diabetic retinopathy (DR) and its mechanism. Methods Fifty 6-week-old SPF male Wistar rats with normal eyes were randomly divided into the normal group, model group, low-dose Gen group, high-dose Gen group, and calcium dobesilate group, with 10 rats in each group. Except for the normal group, rats in the other groups were fed high-sugar and high-fat diets and induced by streptozotocin to establish the DR rat models. After modeling, drug intervention was carried out immediately. Rats in the low- and high-dose Gen groups were given 25 mg·kg-1 and 100 mg·kg-1 Gen intragastrically, rats in the calcium dobesilate group were given 5.8 mg·kg-1 calcium dobesilate intragastrically, while rats in the normal and model groups were given the same amount of solvent intragastrically, once a day for 4 weeks. During the drug administration period, rats in the normal group continued to be fed normal diets, and rats in the other groups continued to be fed high-sugar and high-fat diets. Levels of vascular endothelial growth factor (VEGF) and soluble VEGF receptor 1 (sFlt-1) in serum were measured by the enzyme-linked immunosorbent assay. Retinal pathological changes were exhibited by hematoxylin-eosin staining. Retinal microangiogenesis was revealed by periodic acid-Schiff staining. The expression levels of VEGF, hypoxia-inducible factor-1α (HIF-1α), intercellular adhesion molecule-1 (ICAM-1), and sFlt-1 in the retina were measured by Western blot. Results At the end of drug intervention (week 4), serum VEGF level in the model group was higher than that in the normal group, while sFlt-1 level was lower than that in the normal group (both P<0.001); serum VEGF level in the high-dose Gen and calcium dobesilate groups was lower than that in the model group, while sFlt-1 level was higher than that in the model group (all P<0.001); there were no significant differences in the above indexes between the low-dose Gen group and the model group (all P>0.05). At the end of drug intervention (week 4), the retinal morphology of rats in the normal group was normal, and the cells in the inner and outer nuclear layers were arranged neatly; cells in the inner and outer nuclear layers were arranged loosely, ganglion cells were reduced, and capillary congestion was observed in the model and low-dose Gen groups; the arrangement of cells in the inner and outer nuclear layers in the high-dose Gen and calcium dobesilate groups tended to be normal, and ganglion cells increased compared with the model group. At the end of drug intervention (week 4), the retinal vascular diameter in the model group was uneven, with segmental enlargement, and retinal microangiogenesis was more significant than that in the normal group (P<0.001); retinal microangiogenesis in the high-dose Gen and calcium dobesilate groups was less significant than that in the model group (both P<0.001); there was no significant difference in retinal microangiogenesis between the low-dose Gen group and the model group (P>0.05). At the end of drug intervention (week 4), the relative expression levels of VEGF, HIF-1α and ICAM-1 proteins in the model group were higher than those in the normal group, while the relative expression level of sFlt-1 protein was lower than that in the normal group (all P<0.001); the relative expression levels of VEGF, HIF-1α and ICAM-1 proteins in the high-dose Gen and calcium dobesilate groups were lower than those in the model group, while the relative expression level of sFlt-1 protein was higher than that in the model group (all P<0.001); there were no significant differences in the above indexes between the low-dose Gen group and the model group (all P>0.05). Conclusion Gen can inhibit the expression of VEGF and promote the expression of sFlt-1, which in turn reduces retinal microangiogenesis in DR rats to treat DR. © The Author(s) 2023.     

Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment: the value of oxygen supplementation.

PURPOSE: To assess the role of hypoxia in inducing the proliferation, hypertrophy, and dysfunction of Muller cells in detached retina and the effectiveness of supplemental oxygen in limiting these reactions. METHODS: Retinal detachments were produced in the right eye of each of 13 cats; the cats survived surgery for 3 days, during which six were kept in normoxia (room air, 21%) and seven in hyperoxia (70% oxygen). Retinas were labeled for proliferation with an antibody (MIB-1) to a cell cycle protein (Ki-67), for evidence of hypertrophy employing antibodies to the intermediate filament protein glial fibrillary acidic protein (GFAP) and to beta-tubulin and for disturbance of glutamate neurochemistry employing antibodies to glutamate to a glutamate receptor (GluR-2) and to glutamine synthetase. RESULTS: Results from the two animals kept in normoxia after retinal detachment confirmed previous reports that detachment caused the proliferation of Muller cells, the hypertrophy of Muller cell processes, and the disruption of glutamate recycling by Muller cells. Oxygen supplementation during detachment reduced Muller cell proliferation and hypertrophy and reduced the abnormalities in the distributions of glutamate, GluR-2, and glutamine synthetase. CONCLUSIONS: Oxygen supplementation reduced the reaction of retinal Muller cells to retinal detachment, limiting their proliferation and helping to maintain their normal structure and function. In the clinical setting, oxygen supplementation between diagnosis and reattachment surgery may reduce the incidence and severity of glial-based complications, such as proliferative vitreoretinopathy.     

Effect of polydatin on lens epithelial cell injury in rats with cataract北大核心

Objective To investigate the effect of polydatin on lens epithelial cell (LEC) injury in rats with cataract. Methods A total of 50 newborn rats were randomly divided into the control group, model group, low-dose polydatin group, high-dose polydatin group, and activator group, with 10 rats in each group. Except for the control group, rats in the rest groups were injected with sodium selenite subcutaneously to establish the cataract rat models. Since the second day, rats in the low- and high-dose polydatin groups were given eye drops containing 1.3 g·L-1 and 5.0 g·L-1 polydatin, respectively; in addition to 5.0 g·L-1 polydatin eye drops, rats in the activator group were also given the SRI-011381 hydrochloride (10 mg·kg-1) intragastrically; rats in the control and model groups were given normal saline in eyes. The lens opacity was examined by a slit lamp. Hematoxylin-eosin (HE) staining was performed to monitor the pathological changes in the lens. The total superoxide dismutase (SOD) and malondialdehyde (MDA) levels in LECs were measured. The LEC apoptosis was detected by flow cytometry. The expression levels of transforming growth factor-β (TGF-β), Smad2, Smad3, α smooth muscle actin (α-SMA), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) in LECs were measured by Western blot. Results The anterior capsule of rats in the control group was structurally clear, and LECs and lens fiber cells were closely arranged. Compared with the control group, the model group had structural disorder in the anterior capsule, degeneration and swelling in lens fiber cells, loose LEC arrangement, and vacuolization near the posterior capsule; besides, the lens opacity grade, the LEC apoptosis, and the expression levels of MDA, TGF-β, Smad2, Smad3, α-SMA, and Bax in the model group increased greatly (all P<0.05), and the expression levels of SOD and Bcl-2 decreased greatly (both P<0.05). Compared with the model group, the lens structure of rats in the low- and high-dose polydatin groups improved significantly, the lens opacity grade, the LEC apoptosis, and the expression levels of MDA, TGF-β, Smad2, Smad3, α-SMA, and Bax decreased greatly (all P<0.05), and the expression levels of SOD and Bcl-2 increased greatly (both P<0.05). Compared with the high-dose polydatin group, the lens structure damage in the activator group was aggravated, the lens opacity grade, the LEC apoptosis, and the expression levels of MDA, TGF-β, Smad2, Smad3, α-SMA, and Bax increased greatly (all P<0.05), and the expression levels of SOD and Bcl-2 decreased greatly (both P<0.05). Conclusion Polydatin can inhibit the apoptosis and oxidative stress of LECs in rats with cataract by regulating the TGF-β/Smads signal pathway, which in turn alleviates the LEC injury. © The Author(s) 2023.     

Gold nanoparticles downregulate VEGF-and IL-1β-induced cell proliferation through Src kinase in retinal pigment epithelial cells

Proliferative vitreo retinopathy (PVR) is one of the ocular complications, marked by the enhanced proliferation of various cells including retinal pigment epithelial cells (RPE). The aim of the present study is to analyze the effect of gold nanoparticles (Au-NP) on vascular endothelial growth factor (VEGF) and interleukin-1 beta (IL-1β)-induced cell spreading, migration and proliferation in RPE cells. Au-NP (300 nM) significantly blocked the VEGF-and IL-1β-induced cell spreading, migration and proliferation in bovine RPE cells (BRPEs). To elucidate the signaling mechanism of VEGF- and IL-1β-induced cell proliferation, BRPEs were treated with PP2, a Src inhibitor. Further, to clarify the possible involvement of the Src pathway on the inhibitory effect of Au-NPs, transient transfection assay was performed using dominant negative (DN) and constitutively active (CA) mutant plasmid of Src kinase. The results showed that VEGF and IL-1β exert their proliferative effects through the activation of Src kinase whereas CA Src rescued the inhibitory effect of Au-NP in presence or absence of VEGF and IL-1β in BRPEs. Further, an in vitro kinase assay was performed to identify the status of Src phosphorylation at Y419. We found that VEGF and IL-1β increased Src phosphorylation in BRPEs and Au-NP blocked the VEGF- and IL-1β-induced Src phosphorylation at Y419. Taken together, our result suggests that Au-NP could effectively inhibit the VEGF- and IL-1β-induced proliferation and migration by suppressing the Src kinase pathway in BRPEs and Au-NP might act as an effective therapeutic agent for the treatment of ocular diseases such as proliferative vitreo retinopathy.     

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.     

Mitochondria of retinal Müller (glial) cells: the effects of aging and of application of free radical scavengers

Age-related changes of mitochondria were studied in Müller (retinal glial) cells from guinea pigs fed with or without externally applied Ginkgo biloba extract EGb 761, an established radical scavenger. When Müller cell mitochondria from aged animals were compared with those from young adults, they displayed (1) a diminished number of well-defined cristae at the ultrastructural level, (2) a reduced membrane potential, as revealed by fluorimetry using the voltage-sensitive dye tetramethyl rhodamine methylester, and (3) a slightly reduced index of vitality assayed by tetrazolium salt colorimetry. Müller cell mitochondria were also studied in aged guinea pigs which had been fed daily by EGb 761 during the last 2 months before they were sacrificed. Such mitochondria displayed (1) many well-defined cristae at the ultrastructural level, and, compared with mitochondria from untreated aged animals, (2) a significantly enhanced membrane potential and (3) a significantly enhanced index of vitality. No age- or drug-related changes were observed in the mitochondrial content of GABA transaminase, as revealed by immunocytochemistry/densitometry. These results suggest that many but not all structural and functional parameters of aging Müller cell mitochondria are impaired by accumulating oxidative damage, and that externally applied radical scavengers may protect the organelles from the damaging actions of free radicals. As it has been shown earlier that EGb 761 treatment enhances the intrinsic glutathione content of aged guinea pig Müller cells, the protective radical-scavenging effect of the drug may be mediated both directly and indirectly.     

Intravitreal ranibizumab (Lucentis®) in the treatment of retinal angiomatous proliferation (RAP)

Background  Retinal angiomatous proliferation (RAP) is a distinct variant of neovascular age-related macular degeneration (AMD). The aim of this study is to evaluate the functional and anatomic outcome after intravitreal ranibizumab (Lucentis) treatment in patients with RAP. Methods  Prospective study of consecutive patients with newly diagnosed or recurrent RAP treated with intravitreal ranibizumab at the Jules Gonin Eye Hospital between March 2006 and December 2007. Baseline and monthly follow-up visits included best-corrected visual acuity (BCVA), fundus exam and optical coherence tomography. Fluorescein and indocyanine green angiography were performed at baseline and repeated at least every 3 months. Results  Thirty-one eyes of 31 patients were treated with 0.5 mg of intravitreal ranibizumab for RAP between March 2006 and December 2007. The mean age of the patients was 82.6 years (SD:4.9). The mean number of intravitreal injections administered for each patient was 5 (SD: 2.4, range 3 to 12). The mean follow up was 13.4 months (SD: 3, range 10 to 22). The baseline mean logMAR BCVA was 0.72 (SD: 0.45) (decimal equivalent of 0.2). The mean logMAR BCVA was improved significantly (P < 0.0001) at the last follow-up to 0.45, SD: 0.3 (decimal equivalent 0.35). The visual acuity (VA) improved by a mean of 2.7 lines (SD 2.5). Mean baseline central macular thickness (CMT) was 376 μm, and decreased significantly to a mean of 224 μm (P < 0.001) at the last follow-up. Mean reduction of CMT was 152 μm (SD: 58). An average of 81.5% of the total visual improvement and 85% of the total CMT reduction occurred during the first post-operative month after one intravitreal injection of ranibizumab. During follow-up, an RPE tear occurred in one eye (3.2%) of the study group. No injection complications or systemic drug-related side-effects were noted during the follow-up period. Conclusions  Intravitreal ranibizumab injections appeared to be an effective and safe treatment for RAP, resulting in visual gain and reduction in macular thickness. Further long-term studies to evaluate the efficacy of intravitreal ranibizumab in RAP are warranted.