Klinik und Poliklinik für Augenheilkunde Universitätsklinikum Regensburg

Seeing eye to eye: Plasma-protein binding is effective in improving the pharmacokinetic properties of otherwise short-lived molecules. One compound in a class of small portable albumin binders can be used to improve the in vivo circulatory half-life of two widely used contrast agents. It improves the imaging performance of fluorescein in angiographic analysis of the retina of mice (see picture). Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z704936_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

AIM: To evaluate intravitreal VEGF Trap-Eye (VTE) in patients with macular oedema secondary to central retinal vein occlusion (CRVO). METHODS: In this double-masked study, 177 patients were randomised (3:2 ratio) to intravitreal injections of VTE 2 mg or sham procedure every 4 weeks for 24 weeks. Best-corrected visual acuity was evaluated using the Early Treatment Diabetic Retinopathy Study chart. Central retinal thickness (CRT) was measured with optical coherence tomography. RESULTS: From baseline until week 24, more patients receiving VTE (60.2%) gained ≥ 15 letters compared with those receiving sham injections (22.1%) (p<0.0001). VTE patients gained a mean of 18.0 letters compared with 3.3 letters with sham injections (p<0.0001). Mean CRT decreased by 448.6 and 169.3 µm in the VTE and sham groups (p<0.0001). The most frequent ocular adverse events in the VTE arm were typically associated with the injection procedure or the underlying disease, and included eye pain (11.5%), increased intraocular pressure (9.6%) and conjunctival haemorrhage (8.7%). CONCLUSIONS: VTE 2 mg every 4 weeks was efficacious in CRVO with an acceptable safety profile. Vision gains with VTE were significantly higher than with observation/panretinal photocoagulation if needed. Based on these data, VTE may provide a new treatment option for CRVO.

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca(2+)-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca(2+)-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca(2+)-dependent chloride channel in the photoreceptor synapse.

PURPOSE: To evaluate the efficacy and safety of a silicone oil-RMN3 mixture ("heavy silicone oil") as heavier as water internal retinal tamponade after vitrectomy for complicated retinal detachment. The relative density of the heavier-than-water silicone oil was 1.03 g/cm3, and the viscosity was 3,800 cSt. Heavy silicone oil is designed to tamponade the inferior retina in complicated retinal detachment. METHODS: Patients with a complicated retinal detachment involving the inferior part of the retina requiring internal tamponade with silicone oil were recruited for this prospective study. Inclusion criteria were retinal detachment secondary to proliferative vitreoretinopathy (stage > or = C2), inferior or posterior tears, or penetrating trauma. The heavy silicone oil was injected at the end of surgery after peeling of retinal membranes or retinotomy. Follow-up examinations were scheduled at 1, 3, 6 months, and 1 year after the initial surgery. RESULTS: A total of 33 eyes of 33 patients aged from 20 to 84 years (mean, 56 +/- 18 years) were treated with heavy silicone oil. Follow-up ranged from 12 to 16 months. Rhegmatogenous retinal detachment with significant proliferative vitreoretinopathy accounted for 17 cases, inferior holes for three, and trauma with retinal detachment for three. Initial visual acuity ranged from 20/50 to hand motions. Initial retinal reattachment was achieved in all cases. Complications included increased intraocular pressure in six eyes (18%), intraocular inflammation and synechia formation in one eye (3%), a central retinal artery occlusion after heavy oil removal in one eye, and scattered retinal hemorrhages during follow-up in two eyes (6%). Significant emulsification was not observed during intraocular tamponade with heavy silicone oil. At the last follow-up, all eyes had macular attachment, and 24 eyes had a visual acuity better than or equal to 20/400. CONCLUSIONS: The results of this prospective study show the good intraocular tolerance of heavy silicone oil as tamponade in complicated retinal detachment. Its specific gravity allows for sufficient tamponade of inferior retinal tears for at least 3 months without significant side effects.

Mutations in the peripherin/RDS gene, which encodes a photoreceptor-specific membrane glycoprotein, have been identified in a variety of retinal phenotypes. However, the mechanisms by which specific mutations in this gene can cause typical features of retinal dystrophies clinically as distinct as retinitis pigmentosa or macular degeneration are still unknown. Recently, a single case of adult vitelliform macular dystrophy (AVMD) has been associated with a Y258Stop mutation. To assess the frequency of peripherin/RDS mutations in the clinically heterogeneous group of AVMD, we analyzed the entire coding region of the gene in 28 unrelated patients. We identified five novel mutations including two presumed null allele mutations. Thus, our results demonstrate that a significant portion of AVMD patients (18%) carry point mutations in peripherin/RDS, suggesting that this gene is frequently involved in the pathogenesis of this macular disorder. In addition, this study shows that the variable phenotypes in AVMD are due, at least in part, to genetic heterogeneity and are likely to be caused by mutations in disease genes thus far unknown.

BACKGROUND: Epidemic keratoconjunctivitis (EKC) is a highly contagious infection of the ocular surface. 316 cases were diagnosed in Germany in the first 8 months of 2010, corresponding to a 300% increase above the typical figures for recent years. This outbreak motivates us to present the current recommendations concerning EKC. METHODS: Selective literature review. RESULTS: EKC is an adenoviral infection that typically starts with a unilateral foreign body sensation and then develops, within a few hours or days, into bilateral keratoconjunctivitis with marked chemosis, epiphora, and photophobia. Visual impairment can persist for months because of subepithelial corneal infiltrates (nummuli) and irregular astigmatism. Randomized clinical trials have not shown any clear benefit in the acute phase from any of a variety of treatments, including steroids, calcineurin inhibitors, virostatic drugs and disinfecting agents. In the chronic phase, cyclosporin A eye drops can accelerate the regression of subepithelial infiltrates. Hygienic measures, including conscientious hand and surface disinfection, can lessen the spread of the disease. CONCLUSION: The first priority in the treatment of patients with definite or suspected EKC is the rigorous application of hygienic measures in medical facilities, particularly because there is still no effective drug treatment for this disease. No virostatic agent has yet been demonstrated to influence its course, either subjectively or objectively.

The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a-Myrip-MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.

PURPOSE: Intravitreal injection of VEGF inhibitors has become the standard of care for different macular diseases within the last years resulting in improved visual outcomes. Under real-life conditions, however, the necessity for frequent retreatments and reexaminations poses a burden for patients and treatment centers. Non-adherence and non-persistence to intravitreal treatment may lead to inferior clinical outcomes, and knowledge of contributing factors is crucial to improve adherence. This systematic review analyzes current literature for potential factors involved in non-adherence and non-persistence. METHODS: A systematic search was conducted in PubMed and Embase including three different aspects of intravitreal injection therapy: (1) diseases with intravitreal injections as treatment, (2) intravitreal injection, and (3) aspects of therapy adherence or therapy persistence. Data from identified quantitative studies were further extracted and grouped according to WHO criteria (condition, socio-economy, therapy, patient, and health system). The methodological quality of identified studies was graded. Identified qualitative studies (i.e., interviews) were descriptively analyzed and their findings narratively reported. RESULTS: Twenty-four publications were included. In 16 of those publications, a quantitative data analysis was conducted, analyzing factors associated with non-adherence. Worse visual acuity at baseline and unfavorable development of visual acuity, higher age, and greater distance to the treatment center were associated with non-adherence, while there was inconsistent evidence for an association of comorbidity. In qualitative studies, high follow-up/treatment burden, fear and anxiety, disappointed patient expectations, and lack of motivation to continue treatment were reported as reasons for non-persistence. CONCLUSIONS: Knowledge of potential barriers in IVT treatment may improve adherence and potentially clinical results. Improvements can be achieved particularly in the healthcare complex (organizational improvements) and the "patient" complex by establishing realistic expectations. Recurrent education of the patient may be necessary.

<b>Aims:</b> This study describes a prospective consecutive case series of the initial six eyes of five patients undergoing implantation of the trabecular bypass tube shunt. <b>Methods:</b> A prospective consecutive case series. The initial six eyes of five patients with uncontrolled open angle glaucoma who had never previously undergone ocular surgery. Implantation of the trabecular bypass tube shunt measuring 150 μm outer diameter and 50 μm inner diameter was performed with the distal end placed in Schlemm’s canal and the proximal end in the anterior chamber. The main outcome measures were visual acuity, intraocular pressure, glaucoma medication use. <b>Results:</b> The tube was successfully implanted in five of six eyes. In four eyes longer term follow up of 5–9 months showed no loss of visual acuity with decreased intraocular pressure from preoperative levels (mean 23.4–16.5 mm Hg) and reduced requirement of glaucoma medications (mean 3–0.5). In a subset of two eyes, there was no measured increase in aqueous flare or reduction of endothelial cell count. In one eye the tube was explanted because of presumed misplacement by excessive bleeding during surgery. Two eyes showed a diffuse bleb. <b>Conclusions:</b> This study reports the initial experience with a novel approach to surgical glaucoma therapy. This very small tube allows a direct communication to be established between the anterior chamber and Schlemm’s canal, effecting a trabecular bypass. In this small number of eyes this procedure reduced intraocular pressure and the need for glaucoma medications without appreciable side effects.

AIM: To investigate effects of photodynamic therapy (PDT) on human choroidal neovascularisation (CNV). METHODS: Two patients with recurrences after PDT with verteporfin underwent surgical extraction of the CNV. Immediately after surgical excision the subfoveal neovascular membranes were divided for light microscopic and for electron microscopic processing. For light microscopy tissues were embedded in paraffin. Sections were stained with haematoxylin and eosin, and the periodic acid Schiff (PAS) reaction was performed to determine histological diagnosis and to ensure tissue quality. For electron microscopy the specimens were fixed in glutaraldehyde and embedded in epoxy resin. Semithin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope. RESULTS: Light microscopy showed thick fibrovascular membranes in both cases. On the outer surface remnants of retinal pigment epithelial cells resting on thickened inner aspect of Bruch's membrane were found. On the retinal side some outer segments were found. The membrane showed areas with irregularly shaped vessels. Electron photomicrographs showed occluded vessels within the CNV containing thrombotic masses and/or ultrastructural damage of the neovascular endothelium. Most of the vessels presented regressive changes with vacuolisation and fragmentation of the neovascular endothelium accompanied by disintegration of the endothelial cell layer. Extravasation of red blood cells was observed. Occasionally, vessels with normal endothelium containing intact red blood cells were observed. Some vessels contained immature endothelial cells. At some locations the retinal pigment epithelium cells (RPE) were metaplastic showing highly vacuolised cytoplasm. CONCLUSIONS: These findings suggest that the evidence of fluorescein leakage from the CNV and enlargement of the neovascular complex following PDT could be related to new vessel growth and recanalisation of occluded vessels. Additionally, RPE disturbances were observed in the specimens. This finding may be related to the original pathology or could indicate that PDT treatment may result in RPE atrophy.

PURPOSE: Choroidal neovascularization in age-related macular degeneration is caused, to a large extent, by increased secretion of vascular endothelial growth factor (VEGF)-A by the retinal pigment epithelium (RPE). The purpose of the study was to identify pathways that lead to increased VEGF secretion by the RPE. METHODS: Ca(2+) signaling was studied in ARPE-19 and human RPE cells in primary culture by means of Ca(2+) imaging. Membrane conductance was measured in the whole-cell configuration of the patch-clamp technique. VEGF-A secretion was measured by using ELISA. RESULTS: Freshly isolated RPE cells or ARPE-19 cells were shown to express TRPV1, -2, -3, and -4 channels. Increasing the temperature or stimulation by IGF-1 increased the VEGF-A secretion rate in both cell types. These effects were both sensitive to the TRPV channel blocker ruthenium red (20 μM). The heat-inducible Ca(2+) signals were blocked by the TRPV channel blockers La(3+) and ruthenium red by 68% and 52%, respectively. In contrast, high concentrations of 2-APB (3 mM) increased [Ca(2+)](i), whereas the TRPV1 channel opener capsaicin and the TRPV3 channel opener camphor had no effect. Reduction of TRPV2 expression by siRNA attenuated the heat-evoked Ca(2+) response. In addition, a heat-activated inwardly rectifying current was measured that was completely blocked by ruthenium red. IGF-1 also increased whole-cell current with a corresponding increase in [Ca(2+)](i), which was blocked by the PI3-kinase blocker LY294002. CONCLUSIONS: The data strongly suggest that TRPV2 channels expressed by the RPE are involved in the Ca(2+) signaling that mediates both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion.

The OPA1 gene, encoding a dynamin-related GTPase that plays a role in mitochondrial biogenesis, is implicated in most cases of autosomal dominant optic atrophy (ADOA). Sixty-nine pathogenic OPA1 mutations have been reported so far. Most of these are truncating mutations located in the GTPase domain coding region (exons 8-16) and at the 3'-end (exons 27-28). We screened 44 patients with typical ADOA using PCR-sequencing. We also tested 20 sporadic cases of bilateral optic atrophy compatible with ADOA. Of the 18 OPA1 mutations found, 14 have never been previously reported. The novel mutations include one nonsense mutation, 3 missense mutations, 6 deletions, one insertion and 3 exon-skipping mutations. Two of these are de novo mutations, which were found in 2 patients with sporadic optic atrophy. The recurrent c.2708_2711delTTAG mutation was found in 2 patients with a severe congenital presentation of the disease. These results suggest that screening for OPA1 gene mutations may be useful for patients with optic atrophy who have no affected relatives, or when the presentation of the disease is atypical as in the case of early onset optic atrophy.

BACKGROUND: Reduced choroidal perfusion is hypothesized to play a role in the pathogenesis of normal tension glaucoma. Thus the impact of antiglaucomatous eye drops on ocular perfusion has been the focus of recent research and the subject of intensive investigations. The present study investigates whether topically applied latanoprost or bimatoprost influence ocular perfusion in patients with normal tension glaucoma and compares these effects with that changes detected after the treatment with dorzolamide. METHODS: Ocular hemodynamics were assessed by color Doppler imaging (CDI) shortly before and after a one-month treatment with either latanoprost, bimatoprost or dorzolamide. Primary end-points of the study were peak systolic and end-diastolic blood flow velocities in the short posterior ciliary artery (SPCA) under the new therapy. Intraocular pressure (IOP) and additional perfusion parameters in the SPCA and other retrobulbar vessels were tracked as observational parameters. n = 42 patients with normal tension glaucoma were enrolled in the study. RESULTS: Systolic and diastolic blood flow velocities in the SPCA showed no significant alteration after the treatment with latanoprost or bimatoprost. Dorzolamide lead to increase of peak systolic velocity. IOP was reduced by all three agents in a range reported in the literature. CONCLUSION: Topically applied latanoprost and bimatoprost act in a hemodynamically neutral manner and have the capability to lower IOP even in patients with normal tension glaucoma and low initial IOP level. Dorzolamide accelerates blood flow in systole. None of the tested compounds has a negative impact on hemodynamics in the short posterior ciliary arteries.

Inwardly rectifying K+ (Kir) channels have been implicated in the mediation of retinal K+ homeostasis by Muller glial cells. To assess possible involvement of altered glial K+ channel expression in ischemia-reperfusion injury, transient retinal ischemia was induced in rat eyes. Acutely isolated Muller cells from postischemic retinae displayed a fast downregulation of their Kir currents, which began within 1 day and reached a maximum at 3 days of reperfusion, with a peak decrease to 20% as compared with control. This strong decrease of Kir currents was accompanied by an increase of the incidence of cells which displayed depolarization-evoked fast transient (A-type) K+ currents. While no cell from untreated control rats expressed A-type K+ currents, all cells investigated from 3- and 7-day postischemic retinae displayed such currents. An increased incidence of cells displaying fast transient Na+ currents was observed at 7 days after ischemia. These results suggest a role of altered glial Kir channel expression in postischemic neuronal degeneration via disturbance of retinal K+ siphoning.

PURPOSE: To evaluate the impact of intravitreal aflibercept (EYLEA, Regeneron Pharmaceuticals, Tarrytown, NY) versus laser on progression of diabetic retinopathy (DR) severity in Intravitreal Aflibercept Injection in Vision Impairment due to DME (VIVID-DME) and Study of Intravitreal Aflibercept Injection in Patients with Diabetic Macular Edema (VISTA-DME). DESIGN: Secondary and exploratory analyses of 2 phase 3, randomized, controlled studies. PARTICIPANTS: All patients with a baseline Diabetic Retinopathy Severity Scale (DRSS) score based on fundus photograph (full analysis), patients who progressed to proliferative DR (PDR) (safety analysis) in VIVID-DME (n = 403) and VISTA-DME (n = 459), or both. METHODS: We randomized patients with diabetic macular edema (DME) to intravitreal aflibercept 2 mg every 4 weeks (2q4), intravitreal aflibercept 2 mg every 8 weeks after 5 initial monthly doses (2q8), or macular laser photocoagulation at baseline and sham injections at every visit. MAIN OUTCOME MEASURES: Proportions of patients with 2-step or more and 3-step or more improvements from baseline in DRSS score, who progressed to PDR, and who underwent panretinal photocoagulation (PRP). RESULTS: Among patients with an assessable baseline DRSS score, most showed moderately severe or severe nonproliferative DR. The proportions of patients treated with 2q4, 2q8, and laser with a 2-step or more improvement in DRSS score at week 100 were 29.3%, 32.6%, and 8.2%, respectively, in VIVID-DME and 37.0%, 37.1%, and 15.6%, respectively, in VISTA-DME; the proportions with a 3-step or more improvement in DRSS score were 7.3%, 2.3%, and 0%, respectively, and 22.7%, 19.9%, and 5.2%, respectively. Fewer patients in the 2q4 and 2q8 groups versus the laser group progressed to PDR at week 100 in VISTA-DME (1.5% and 2.2% vs. 5.3%) and VIVID-DME (3.2% and 2.0% vs. 12.3%). The proportions of patients who underwent PRP were 2.9%, 0.7%, and 4.5%, respectively, in VIVID-DME and 1.9%, 0.7%, and 5.2%, respectively, in VISTA-DME. The most frequent serious ocular adverse event at week 100 was cataract (pooled intravitreal aflibercept, 1.7% of patients; laser, 3.5% of patients). CONCLUSIONS: These analyses demonstrate the benefit of intravitreal aflibercept over laser with respect to DR progression, suggesting a benefit on DME, and on underlying DR.

Membrane differential filtration is able to optimize rheologic parameters by eliminating high molecular weight proteins and lipoproteins from the blood. Following the hypothesis that these changes result in an improvement of the microcirculation, the authors tested the efficacy of membrane differential filtration in improving visual function in patients with age-related macular degeneration (ARMD).Forty patients (40 eyes) were randomized into two groups. The treatment group was treated five times over a period of 21 weeks. In both groups, 9/20 of the eyes showed subfoveolar subretinal neovascularization. The main parameter of the study was visual acuity (VA). Electroretinogram (ERG), electrooculogram, and macular visual evoked potentials were also recorded. Plasma and whole blood viscosity and erythrocyte aggregation were measured.The 20 patients treated repeatedly over a period of 21 weeks showed a mean improvement of 0.63 lines (SD 1.8) of VA on Early Treatment Diabetic Retinopathy Study charts. The control group showed a deterioration of 0.94 lines (SD 1.7) compared to VA at baseline examination. The amplitude of the ERG photopic a-wave and the flicker ERG was significantly increased. The rheologic parameters were lowered in all treated patients.Repetitive treatment with membrane differential filtration is able to improve visual acuity of patients with ARMD and the natural course of this disease. Several questions arise from the results of this study. Further research will show if it is possible to optimize the selection of patients for subgroups with predictive responses through morphologic and functional tests and how to create an optimized and individual treatment strategy determined by the quality, intensity, and frequency of treatment sessions.

PURPOSE: This study evaluated the outcome of severely injured eyes treated with early primary vitrectomy with silicone oil filling. METHODS: A total of 435 eye injuries, which required surgical intervention, were reviewed retrospectively. In 13 eyes (3%) pars plana vitrectomy and silicone oil filling were performed as primary surgical repair. Silicone oil filling was restricted to cases with laceration of the retina larger than 4 disc diameters (nine eyes), primary retinal detachment larger than two quadrants (two eyes) and/or persistent intrasurgical hemorrhage (12 eyes). All patients underwent surgery within 24 hours. RESULTS: After a mean follow-up period of 28.7 months (range, 9-70 months), 11 eyes achieved a visual acuity ranging from 20/25 to 20/200. Silicone oil was removed in 11 of 13 eyes after 5.8 +/- 4.6 months. Recurrent proliferative vitreoretinopathy developed in two eyes. CONCLUSIONS: Silicone oil tamponade after early primary pars plana vitrectomy may be an alternative for primary repair after trauma, especially in severely injured eyes with retinal lacerations larger than 4 disc diameters, persistent intrasurgical bleeding, and/or primary retinal detachments.

PURPOSE: Retinal Müller glial cells are known to express metabotropic P2Y receptors. The present study was conducted to identify certain subtypes of P2Y receptors in human Müller cells. METHODS: The patch-clamp technique was used to measure increases of Ca(2+)-dependent K+ currents mediated by the activation of P2Y receptors in freshly isolated human Müller cells. Several P2 agonists were used. Subsequently, the cells were harvested into the patch pipette and a single cell RT-PCR was performed. Moreover, retinal tissue from organ donors was used for immunohistochemistry. RESULTS: The electrophysiological data were consistent with the expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes. RT-PCR revealed that mRNA for all these subtypes was present in Müller cells. However, the incidence of P2Y2 receptor mRNA was significantly lower than that of the other subtypes. Immunoreactivity for all four subtypes was found in retinal tissue, partly colocalized with immunoreactivity for vimentin. CONCLUSIONS: The presented data obtained by different techniques revealed that human Müller cells express P2Y1, P2Y2, P2Y4, and P2Y6 receptors. The specific roles of these receptor subtypes in retinal physiology and/or pathophysiology remain to be investigated in future studies.

PURPOSE: Transient retinal ischemia-reperfusion is associated with neuronal degeneration and activation of Müller glial cells. Reactive gliosis may impede the homeostatic functions of Müller cells. A viable animal model for human ischemic events should display similarities in eye size and retinal blood supply. Therefore, pigs were used in this investigation of physiological alterations in Müller cells after ischemia-reperfusion. METHODS: Transient retinal ischemia was induced in young adult pigs by high intraocular pressure in one eye for 1 hour. After 3 days of reperfusion, the retinal tissue and isolated Müller cells were used for osmotic swelling recordings, whole-cell patch-clamp experiments, Ca(2+) microfluorimetry, and immunohistochemistry. RESULTS: Müller cells in retinal slices from postischemic eyes but not control cells displayed a significant swelling of the somata when osmotic stress was applied by hypotonic extracellular solution. The amplitude of K(+) inward currents was significantly reduced (∼60% of the control value). This decrease was accompanied by a depolarization of the cell membrane. The number of Müller cell end feet displaying a Ca(2+) increase after application of adenosine 5'-triphosphate was increased in the ischemic retina. Moreover, reactive Müller cell gliosis was characterized by an (increased) expression of vimentin, glial fibrillary acidic protein, the phosphorylated mitogen-activated protein kinases extracellular signal-related kinase (ERK) 1 and 2, and the transcription factor c-fos. CONCLUSIONS: The alterations of reactive Müller cells after transient ischemia of the pig eye were similar to those found in rat and rabbit models, demonstrating that the porcine retina is a suitable model for the investigation of ischemic injury.

PURPOSE: Ca(2+) is a major regulator of cell function. In the retinal pigment epithelium (RPE), intracellular free Ca(2+) concentration ([Ca(2+)](i)) is essential for the maintenance of normal retinal function. Therefore, accurate control of [Ca(2+)](i) is vital in these cells. Because Ca(2+) is permanently extruded from the cytosol, RPE cells need a basal Ca(2+) entry pathway that counteracts this Ca(2+) efflux. The purpose of this study was to identify the molecular basis of basal Ca(2+) entry into the RPE. METHODS: [Ca(2+)](i) was measured using Fura-2-loaded ARPE-19 cells. The expression pattern of TRPC channels was investigated by RT-PCR with RNA extracted from ARPE-19 cells and freshly isolated RPE cells from human donor eyes. RESULTS: In most cells, basal [Ca(2+)](i) is highly controlled by cell membranes that are only slightly permeable to Ca(2+) and by the activity of Ca(2+) pumps and transporters. The authors show here that RPE cells have a basal Ca(2+) conductance that is dose dependently blocked by La(3+). Basal [Ca(2+)](i) was also strongly reduced by the TRP channel blockers Gd(3+), Ni(2+), 2-APB, and SKF96365 and was insensitive to blockers of other Ca(2+) channels. In confirmation of this pharmacologic profile, RPE cells expressed TRPC1 and TRPC4 channels, as shown by RT-PCR experiments. CONCLUSIONS: Ca(2+) is needed for several permanently occurring regulatory processes in RPE cells. The Ca(2+) influx pathway identified in this study is essential to define a resting basal [Ca(2+)](i). This resting [Ca(2+)](i) may contribute, for example, to basal cytokine secretion essential for the maintenance of normal retinal function.