VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway.

The voltage-dependent anion channel 1 (VDAC1) was first described as a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. In the nervous system, the functional roles of VDAC1 remain poorly understood. Herein, the rat retina was employed to study VDAC1. First, it was observed that even subtle changes in VDAC1 levels affect neuronal survival, inducing severe alterations in the retinal morphology. We next examined the regulation of VDAC1 after traumatic retinal injury. After mechanical trauma, SOD1 translocates towards the nucleus, which is insufficient to contain the consequences of oxidative stress, as determined by the evaluation of protein carbonylation. Using in vitro models of oxidative stress and mechanical injury in primary retinal cell cultures, it was possible to determine that inhibition of VDAC1 oligomerization by 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) rescues cell viability, impacting microglial cell activation. We next focused on the regulation of VDAC1 after retinal mechanical injury. VDAC1 was promptly upregulated 2 h after lesion in the plasma membrane and endoplasmic reticulum rather than in the mitochondria, and multimers of VDAC1 were assembled after lesion. DIDS intraocular application decreased apoptosis and prevented microglial polarization, which confirmed in vitro observations. Considering the role of microglia in neuroinflammation, multiplex evaluation of cytokines showed that DIDS application disorganized the inflammatory response 2 h after the lesion, matching the fast regulation of VDAC1. Taken together, data disclosed that fine regulation of VDAC1 influences neuronal survival, and pharmacological inhibition after trauma injury has neuroprotective effects. This protection may be attributed to the effects on VDAC1 abnormal accumulation in the plasma membrane, thereby controlling the activation of microglial cells. We concluded that VDAC1 is a putative therapeutic target in neuronal disorders since it integrates both death and survival cellular signaling.

Damaging effects of BMAA on retina neurons and Müller glial cells.

B-N-methylamino-L-alanine (BMAA), a cyanotoxin produced by most cyanobacteria, has been proposed to cause long term damages leading to neurodegenerative diseases, including Amyotrophic Lateral Sclerosis/Parkinsonism Dementia complex (ALS/PDC) and retinal pathologies. Previous work has shown diverse mechanisms leading to BMAA-induced degeneration; however, the underlying mechanisms of toxicity affecting retina cells are not fully elucidated. We here show that BMAA treatment of rat retina neurons in vitro induced nuclear fragmentation and cell death in both photoreceptors (PHRs) and amacrine neurons, provoking mitochondrial membrane depolarization. Pretreatment with the N-Methyl-D-aspartate (NMDA) receptor antagonist MK-801 prevented BMAA-induced death of amacrine neurons, but not that of PHRs, implying activation of NMDA receptors participated only in amacrine cell death. Noteworthy, BMAA stimulated a selective axonal outgrowth in amacrine neurons, simultaneously promoting growth cone destabilization. BMAA partially decreased the viability of Müller glial cells (MGC), the main glial cell type in the retina, induced marked alterations in their actin cytoskeleton and impaired their capacity to protect retinal neurons. BMAA also induced cell death and promoted axonal outgrowth in differentiated rat pheochromocytoma (PC12) cells, implying these effects were not limited to amacrine neurons. These results suggest that BMAA is toxic for retina neurons and MGC and point to the involvement of NMDA receptors in amacrine cell death, providing new insight into the mechanisms involved in BMAA neurotoxic effects in the retina.

Neuroprotective Effect of Brazilian Green Propolis on Retinal Ganglion Cells in Ischemic Mouse Retina.

PURPOSE: The current study was undertaken to investigate whether Brazilian green propolis (BGP) can increase the viability of retinal ganglion cells (RGCs) in ischemic mouse retina, and examined the possible mechanisms underlying this neuroprotection. MATERIALS AND METHODS: C57BL/6J mice were subjected to constant elevation of intraocular pressure for 60 min to establish retinal ischemia-reperfusion injury. Mice then received saline or BGP (200 mg/kg) intraperitoneally once daily until sacrifice. The expression of hypoxia-inducing factor (HIF)-1α and glial fibrillary acidic protein (GFAP) and the level of histone acetylation were assessed at 1, 3, and 7 days after injury. The expression of Bax, Bcl-2, p53, NF-κB, Nrf2, and HO-1 were also analyzed at 3 days after injury. The neuroprotective effect of BGP treatment on RGC survival was evaluated using Brn3a immunohistochemical staining. RESULTS: The expression of HIF-1α and GFAP was increased and the level of histone acetylation decreased in saline-treated ischemic retinas within 7 days. BGP treatment effectively attenuated the elevated expression of HIF-1α, GFAP, Bax, NF-κB and p53. The expression of Bcl-2, Nrf2, HO-1 and the level of histone acetylation increased by BGP treatment, resulting in a significant difference between BGP-treated and saline-treated retinas. Immunohistochemical staining for Brn3a also revealed that BGP treatment protected against RGC loss in ischemic retina. CONCLUSIONS: Our results suggest that BGP has a neuroprotective effect on RGCs through the upregulation of histone acetylation, downregulation of apoptotic stimuli, and suppression of NF-κB mediated inflammatory pathway in ischemic retina. These findings suggest that BGP is a potential neuroprotective agent against RGC loss under oxidative stress.

Pre-ischemic enriched environment increases retinal resilience to acute ischemic damage in adult rats.

Retinal ischemia is a condition associated with several degenerative diseases leading to visual impairment and blindness worldwide. Currently, there is no highly effective therapy for ischemic retinopathies. This study was designed to determine possible benefits of pre-exposure to enriched environment against retinal damage induced by acute ischemia. For this purpose, adult male Wistar rats were randomly assigned to a pre-ischemic standard environment or a pre-ischemic enriched environment for 3 weeks, followed by unilateral ischemia induced by increasing intraocular pressure above 120 mm Hg for 40 min and reperfusion for 1 or 2 weeks in standard environment. Animals were subjected to electroretinography and histological analysis. Pre-ischemic enriched environment afforded significant functional protection in eyes exposed to ischemia/reperfusion injury. A marked reduction in retinal layer thickness, reduced synaptophysin-immunoreactivity and retinal ganglion cell (RGC) number, and increased microglia/macrophage reactivity were observed in ischemic retinas from animals submitted to pre-ischemic standard environment, which were prevented by pre-ischemic enriched environment. A deficit in anterograde transport from the retina to the superior colliculus and the lateral geniculate nucleus was observed in animals exposed to pre-ischemic standard environment, which was lower in animals previously exposed to enriched environment. The exposure to enriched environment before ischemia increased retinal brain derived neurotrophic factor (BDNF) protein levels in ischemic retinas and the administration of ANA-12 (a TrkB antagonist) abolished the protective effect of enriched environment on retinal function and retinal ganglion cell number. These results indicate that pre-ischemic enriched environment increases retinal resilience to acute ischemic damage, possibly through a BDNF/TrkB mediated pathway.

Histological, morphometric, protein and gene expression analyses of rat retinas with ischaemia-reperfusion injury model treated with sildenafil citrate.

The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.

Cannabinoid receptors and TRPA1 on neuroprotection in a model of retinal ischemia.

Retinal ischemia is a pathological event present in several retinopathies such as diabetic retinopathy and glaucoma, leading to partial or full blindness with no effective treatment available. Since synthetic and endogenous cannabinoids have been studied as modulators of ischemic events in the central nervous system (CNS), the present study aimed to investigate the involvement of cannabinoid system in the cell death induced by ischemia in an avascular (chick) retina. We observed that chick retinal treatment with a combination of WIN 55212-2 and cannabinoid receptor antagonists (either AM251/O-2050 or AM630) decreased the release of lactate dehydrogenase (LDH) induced by retinal ischemia in an oxygen and glucose deprivation (OGD) model. Further, the increased availability of endocannabinoids together with cannabinoid receptor antagonists also had a neuroprotective effect. Surprisingly, retinal exposure to any of these drugs alone did not prevent the release of LDH stimulated by OGD. Since cannabinoids may also activate transient receptor potential (TRP) channels, we investigated the involvement of TRPA1 receptors (TRPA1) in retinal cell death induced by ischemic events. We demonstrated the presence of TRPA1 in the chick retina, and observed an increase in TRPA1 content after OGD, both by western blot and immunohistochemistry. In addition, the selective activation of TRPA1 by mustard oil (MO) did not worsen retinal LDH release induced by OGD, whereas the blockage of TRPA1 completely prevented the extravasation of cellular LDH in ischemic condition. Hence, these results show that during the ischemic event there is an augment of TRPA1, and activation of this receptor is important in cell death induction. The data also indicate that metabotropic cannabinoid receptors, both type 1 and 2, are not involved with the cell death found in the early stages of ischemia. Therefore, the study points to a potential role of TRPA1 as a target for neuroprotective approaches in retinal ischemia.

PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina.

The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-D-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina.

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Global and ocular hypothermic preconditioning protect the rat retina from ischemic damage.

Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.

Staining of the internal limiting membrane with the use of heavy brilliant blue G.

BACKGROUND: Brilliant blue G (BBG) is frequently used in chromovitrectomy to facilitate internal limiting membrane (ILM) peeling. A study was initiated to evaluate if heavy BBG is safe and effective in staining the ILM. METHODS: We studied 30 eyes, 23 with idiopathic macular holes and 7 of patients with diabetic macular edema. Removal of the ILMs was assisted by heavy BBG staining. In cases with histopathological correlation the ILMs were evaluated with hematoxylin and eosin, Masson's trichrome, periodic acid-Schiff and glial fibrillary acidic protein staining. In addition, immunohistochemistry was also performed using specific antibodies for vimentin, neuron-specific enolase, factor VIII and CD68. Using the Image-Pro Plus software of Media Cybernetics Co. we found an average thickness in ILMs. RESULTS: Of the ILM specimens sent, 19/30 (63.33%) could not be processed properly because of the limited sample material, recognizing only fragments of dispersed fibrillar material. In macular hole ILMs we found an average thickness of 1.3 ± 0.65 µm, and in diabetic macular edema ILMs an average thickness of 6.2 ± 1.4 µm. CONCLUSIONS: In heavy BBG-assisted ILM peeling we observed no intraoperative or postoperative complications after a mean follow-up of 12 months. Heavy BBG could be an effective and safe vehicle for staining the ILM.

Toxicity and retinal penetration of infliximab in primates.

PURPOSE: To evaluate the retinal penetration and toxicity of two doses of intravitreal infliximab in primates. METHODS: Ten marmosets (Callithrix jacchus) were given intravitreal injection of 100 µg or 400 µg of infliximab, and balanced salt solution served as control. At baseline and after 24 hours (5 animals) and 7 days (the other 5), the eyes were examined by electroretinography. They were then killed (at 24 hours and 7 days) and assessed by light microscopy and transmission electron microscopy for toxicity and immunohistochemistry, using a biotinylated anti-human immunoglobulin G, to evaluate retinal penetration. RESULTS: There was no difference over 50% of the electroretinography b-wave between baseline and the time points studied in all animals. Light and electron microscopy, and electroretinography analysis, showed no signs of toxicity in any of the animals. Strong presence of infliximab was observed in all retinal layers 7 days after intravitreal injection at both doses (100 and 400 µg). CONCLUSION: Infliximab at doses of 100 and 400 µg seemed to cause no damage to the retina 24 hours and 7 days after its intravitreal injection, and deeply penetrated all its layers, in primates. These results encourage future perspectives for the treatment of chronic inflammatory diseases of the retina in humans.

Phoneutria spider toxins block ischemia-induced glutamate release and neuronal death of cell layers of the retina.

PURPOSE: To investigate the effect of calcium channel blockers, spider toxins, on cell viability and the glutamate content of ischemic retinal slices. METHODS: Rat retinal slices were subjected to ischemia via exposure to oxygen-deprived low-glucose medium for 45 minutes. Slices were either treated or not treated with the toxins PhTx3, Tx3-3, and Tx3-4. After oxygen-deprived low-glucose insult, glutamate content and cell viability were assessed in the slices by confocal and optical microscopy. RESULTS: In the retinal ischemic slices that were treated with PhTx3, Tx3-3, and Tx3-4, confocal imaging showed a decrease in cell death of 79.5 ± 3.1%, 75.5 ± 5.8%, and 61 ± 3.8%, respectively. Neuroprotective effects were also observed 15, 30, 60, and 90 minutes after the onset of the retinal ischemic injury. As a result of the ischemia, glutamate increased from 6.2 ± 1.0 nMol/mg protein to 13.2 ± 1.0 nMol/mg protein and was inhibited by PhTx3, Tx3-3, and Tx3-4 to 8.6 ± 0.7, 8.8 ± 0.9, and 7.4 ± 0.8 nMol/mg protein, respectively. Histologic analysis of the live cells in the outer, inner, and ganglion cell layers of the ischemic slices showed a considerable reduction in cell death by the toxin treatment. CONCLUSION: Spider toxins reduced glutamate content and cell death of retinal ischemic slices.

Hypothermia prevents nitric oxide system changes in retina induced by severe perinatal asphyxia.

One-third of asphyctic neonates develop long-term neurological injuries, including several degrees of ischemic proliferative retinopathy (IPR) such as retinopathy of prematurity (ROP). Given that the retina is altered by perinatal asphyxia, our aim was to study the effects of nitric oxide (NO) in the retina in order to analyze its impact on the retinal injury. Application of hypothermia was evaluated as preventive treatment. Sprague-Dawley rats were subjected to perinatal asphyxia [either at 37°C (PA group) or at 15°C (HYP group)]. Full-term rats were used as controls (CTL). A significantly increased activity of both constitutive NO synthase (nNOS, Ca(2+)-dependent) and inducible NO synthase (iNOS, Ca(2+)-independent) was observed in PA retinas from 21 days old up to 60 days old with respect to age-matched CTL, with a significant increase along the time course in the PA. nNOS was immunolocalized at amacrine, horizontal, and ganglion cells of the PA group, with a significant increase in relative optical density (R.O.D.), cellular area, and number of cells. iNOS immunoreactivity was observed in the inner nuclear layer and in the internal Müller cell processes of PA, with a significant increase in R.O.D. and colocalizing with GFAP in the 60-day-old PA group. Six nitrated protein species were increased in retinas from PA rats. Nitrotyrosine immunoreactivity showed a localization similar to that of iNOS, with increased R.O.D. in the PA group and colocalization with GFAP in 60-day-old animals. HYP prevented all the changes observed in PA rats. Although the NO system displays changes induced by hypoxia-ischemia, hypothermia application shows a strong protective effect.

Novel and recurrent NDP gene mutations in familial cases of Norrie disease and X-linked exudative vitreoretinopathy.

PURPOSE: To present the results of molecular analysis of the NDP gene in Mexican families with Norrie disease (ND) and X-linked familial exudative vitreoretinopathy (XL-FEVR). METHODS: Two unrelated families with ND and two with XL-FEVR were studied. Clinical diagnosis was suspected on the basis of a complete ophthalmologic examination. Molecular methods included DNA isolation from peripheral blood leucocytes, polymerase chain reaction amplification and direct nucleotide sequencing analysis of the complete coding region and exon-intron junctions of NDP. Haplotype analysis using NDP-linked microsatellites markers was performed in both ND families. RESULTS: A novel Norrin missense mutation, p.Arg41Thr, was identified in two apparently unrelated families with ND. Haplotype analysis demonstrated that affected males in these two families shared the same ND-linked haplotype, suggesting a common origin for this novel mutation. The previously reported p.Arg121Trp and p.Arg121Gln Norrin mutations were identified in the two families with XL-FEVR. CONCLUSION: Our results expand the mutational spectrum in ND. This is the first report of ND resulting from mutation at arginine position 41 of Norrin. Interestingly, mutations at the same residue but resulting in a different missense change were previously described in subjects with XL-FEVR (p.Arg41Lys) or persistent fetal vasculature syndrome (p.Arg41Ser), indicating that the novel p.Arg41Thr change causes a more severe retinal phenotype. Preliminary data suggest a founder effect for the ND p.Arg41Thr mutation in these two Mexican families.

Effect of bacterial lipopolysaccharide on ischemic damage in the rat retina.

PURPOSE: The purpose of this study was to investigate whether bacterial lipopolysaccharide (LPS) induces ischemic preconditioning in the rat retina, and, if so, whether nitric oxide (NO) is involved in this process. METHODS: Rats were intravitreously injected with different doses of LPS (0.1, 1, or 5 microg) in one eye and vehicle in the contralateral eye 24 hours before retinal ischemia induced by increasing intraocular pressure to 120 mm Hg for 40 or 60 minutes. Subsequently, 7 or 14 days after ischemia, the rats were subjected to electroretinography and histologic analysis. One group of animals received intraperitoneal injections of NOS inhibitors, N-nitro-L-arginine methyl ester (L-NAME) aminoguanidine or N-(3-(aminomethyl)benzyl)acetamidine (W1400) before the injection of LPS or vehicle. Retinal nitric oxide synthase (NOS) activity was assessed through the conversion of (3)H-L-arginine to (3)H-L-citrulline. RESULTS: One microgram (but not 0.1 or 5 microg) LPS afforded significant morphologic and functional protection in eyes exposed to ischemia-reperfusion injury. The beneficial effect of LPS was reversed by treatment with L-NAME, aminoguanidine, or W1400. LPS (1 and 5 microg, but not 0.1 microg) significantly increased retinal NOS activity. CONCLUSIONS: These results indicate that LPS provides retinal protection against ischemia-reperfusion injury in a dose-dependent manner, probably through an inducible NOS-dependent mechanism.

Distribution of calbindin-28kD and parvalbumin in V1 in normal adult Cebus apella monkeys and in monkeys with retinal lesions.

Several proteins have their normal patterns of distributions altered by monocular visual deprivation. We studied the distribution of the calcium-binding proteins calbindin-28kD (Cb) and parvalbumin (Pv) in V1 in normal adult Cebus apella monkeys and in monkeys with monocular retinal lesions. In normal monkeys, the interblobs regions in layers 2/3 and the layer 4B are intensely labeled for Cb, while Pv reaction showed a complementary labeling pattern with a stronger staining in layers 4A, 4C and in the blob regions in layers 2/3. In monkeys with monocular retinal lesion, the laminar distribution of these proteins was differentially affected, although both reactions resulted in stronger labeling in non-deprived ocular dominance columns. While Cb reaction resulted in stronger labeling in layers 1 through 5, Pv labeling was heavier in layers 2/3, 4A and 4C. There was a clear reduction in the intensity of neuropil staining for both Pv and Cb in deprived ocular dominance columns with little or no reduction in number of labeled cells. This reduction could thus be attributed to activity-dependent changes at synapses level.

The retinal anatomy and function of the myelin mutant taiep rat.

PURPOSE: To study the histology and the physiological function of the retina in the neurological myelin mutant, taiep rats during the postnatal developmental period (P20-P360). METHODS: Electroretinography (ERG) was applied to evaluate intensity dependence and spectral sensitivity of the responses to light. Retinal histology, morphometry, and immunocytochemistry were used to characterize the structure of the retina, with particular emphasis on the Müller (glial) cells. RESULTS: In the taiep rats of all ages studied, the scotopic ERG showed normal a- and b-wave amplitudes and latencies; likewise, the scotopic spectral sensitivity function was the same for control and taiep animals, with a maximal sensitivity (lambda(max)) at 500 nm. However, in adult taiep rats (P90 to P360) a secondary cornea-positive wave ('b(2)') was observed in response to high stimulus intensities, which never occurred in controls. This correlated with the observation that in the photopic ERG responses of the taiep rats, the b-wave was reduced in amplitude, and was followed by a rapid cornea-negative after-potential. After 1 year of life, in taiep rats the outer plexiform layer (OPL) became slightly thinner and the inner plexiform/ganglion cell layers (IPL/GCL) appeared to be swollen, and increased in thickness; in addition, the number of retinal neurons (particularly, of photoreceptor cells) slightly decreased. Increased GFAP immunoreactivity revealed a hypertrophy and reactivity of the Müller cells in 1-year-old taiep rats. CONCLUSIONS: The present results suggest the occurrence of a relatively mild and slowly progressing neural retinal alteration in taiep rats, which becomes histologically and functionally evident at the end of the first year of life, and mainly affects the circuit(s) of the photopic ON-response. It is speculated that this alteration is due to missing/altered signals from demyelinated optic nerve.

Y1 receptor of neuropeptide Y as a glial marker in proliferative vitreoretinopathy and diseased human retina.

The Y1 receptor of neuropeptide Y (NPY) has been demonstrated in glial cells of astrocytic lineage in vitro. We have studied the immunohistochemical expression of Y1 receptors in the glia of the diseased human retina, in tissue samples obtained after surgery for proliferative vitreoretinopathy. In this condition, glia and other cell types migrate and form epi- or subretinal membranes. Both diseased retinas (n = 8) and PVR membranes (n = 43) contained numerous Y1-immunoreactive cells. In the diseased retina, the Y1 antiserum labeled cells with the morphological radial pattern characteristic of Müller cells, whereas in the membranes, label appeared in a large population of elongate cells, measuring up to 250 microm. In both retina and membranes, double labeling demonstrated that the vast majority of Y1-immunoreactive cells were also labeled by a glial fibrillary acidic protein (GFAP) antibody, indicating their glial origin. Retinal regions devoid of GFAP immunoreactivity also lacked the Y1 label. None of these markers was detected in Müller cells of normal retina. Y1 immunoreactivity did not co-localize with smooth muscle actin immunoreactivity, a marker of myofibroblasts. Expression of Y1 receptors would characterize reactive and proliferating glial cells of the diseased retina and could perhaps be involved in the proliferation of injured glial cells causing regrowth of PVR membranes and the consequent secondary retinal detachments.

Glutamate release in experimental ischaemia of the retina: an approach using microdialysis.

A rabbit eye model of neural ischaemia is described that uses an increased pressure in the anterior eye chamber to block the capillary supply to the retina. A microdialysis probe placed very close to the retinal surface was used to monitor release of amino acids during ischaemia. A large (two- to threefold) increase in the release of glutamate and O-phosphoserine (twofold), but not of six other amino acids monitored, occurred during initial ischaemia. During reperfusion after release of intraocular pressure, much larger (five- to 10-fold) increases in the release of these amino acids were observed. Parallel ischaemic retinal tissue damage was observed. This damage was prevented by ketamine applied locally via a superfusion needle, suggesting that glutamate released during ischaemia, and particularly during reperfusion, was responsible for cell death.