Primary Retinal Cultures Research Articles

Visible light affects mitochondrial function and induces neuronal death in retinal cell cultures

The aim of this study was to provide “proof of principle” for the hypothesis that light would have a detrimental influence on ganglion cells in certain situations, like in glaucoma, by directly impinging on the many mitochondria in their axons within the globe. In this study primary rat retinal cultures and freshly isolated liver mitochondria were exposed to light (400–760 nm; 500–4000 lux) as entering the eye. For culture assessment, 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4-[3-(-iodophenyl)-2-(4-nitrophenyl)-2 H-5-tetzolio]-1,3-benzene disulfonate (WST-1) reduction assays were used to assess cell and mitochondrial viability, respectively. Furthermore, cultures were stained for reactive oxygen species (ROS), DNA breakdown, numbers of GABA-immunoreactive (IR) cells and caspase-3 content to provide information concerning the effect of light on neuronal survival. Uptake of 3H-GABA by autoradiography was also used, to assess the effects of light on the energy status of neurons. Light, in an intensity-dependent and trolox-inhibitable manner, reduced cell viability, affected mitochondrial function, increased the number of TUNEL-positive cells, decreased the numbers of GABA-IR neurons and enhanced labelling for ROS. These effects were all exacerbated by the absence of serum. There was also an increased caspase-3 protein content and a reduction of 3H-GABA uptake in light- compared with dark-treated cultures. These findings support the hypothesis that light can affect mitochondria which could lead to neuronal apoptosis if the energetic status of these neurons is already compromised.

Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

Hierarchical Disabled-1 Tyrosine Phosphorylation in Src family Kinase Activation and Neurite Formation

There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons.

Partial mitochondrial complex I inhibition induces oxidative damage and perturbs glutamate transport in primary retinal cultures.

Leber Hereditary Optic Neuropathy (LHON) is a maternally inherited form of visual loss, due to selective degeneration of retinal ganglion cells. Despite the established aetiological association between LHON and mitochondrial DNA mutations affecting complex I of the electron transport chain, the pathophysiology of this disorder remains obscure. Primary rat retinal cultures were exposed to increasing concentrations of rotenone to titrate complex I inhibition. Neural cells were more sensitive than Müller glial cells to rotenone toxicity. Rotenone induced an increase in mitochondrial-derived free radicals and lipid peroxidation. Sodium-dependent glutamate uptake, which is mostly mediated by the glutamate transporter GLAST expressed by Müller glial cells, was reduced dose-dependently by rotenone with no changes in GLAST expression. Our findings suggest that complex I-derived free radicals and disruption of glutamate transport might represent key elements for explaining the selective retinal ganglion cell death in LHON.

Neuroprotection of Insulin against Oxidative Stress-induced Apoptosis in Cultured Retinal Neurons: Involvement of Phosphoinositide 3-kinase/Akt Signal Pathway

In order to investigate the neuroprotection of insulin in retinal neurons, we used retinal neuronal culture as a model system to study the protective effects of insulin against H2O2-induced cytotoxicity and apoptotic death. Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawley rats. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay, and by DNA laddering analysis. Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide 4,5-bisphophate and [gamma-32P]ATP as substrate. Western blot analysis with anti-phospho-Akt (pS473) antibody was performed to examine the level of phosphorylated Akt. We observed that treatment with 100 microM H2O2 for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons, and that pretreatment with 10 nM insulin significantly inhibited or attenuated H2O2-induced cytotoxicity and apoptosis. Pretreatment with LY294002, a specific PI3K inhibitor, abolished the cytoprotective effect of insulin. Insulin also strongly activated both PI3K and the downstream effector Akt. These results suggest that insulin protects retinal neurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulin-mediated retinal neuroprotection.

Sulfisoxazole, an endothelin receptor antagonist, protects retinal neurones from insults of ischemia/reperfusion or lipopolysaccharide

Endothelins exert pathological effects in the eye and much interest centres on their role in causing retinal neuronal death in ischemic diseases like glaucoma. In the present study the influence of the non-selective endothelin antagonist, sulfisoxazole on raised intraocular pressure-induced ischemia to the rat retina was investigated. Moreover, in vitro studies on primary rat retinal cultures were undertaken to see whether sulfisoxazole is able to blunt the toxic effect of lipopolysaccharide (LPS) to retinal neurones. In order to determine whether sulfisoxazole provides protection to the retina the a- and b-wave amplitudes of the electroretinogram (ERG), the localisation of retinal choline acetyltransferase (ChAT), nitric oxide synthase (nNOS) and Thy-1 and the retinal mRNA levels of Thy-1 and FGF-2 were deduced in retinas subjected to ischemia in the absence or presence of sulfisoxazole. The results showed that the ischemia-induced changes to the a- and b-wave amplitudes of the ERG and changes associated with the localisation of ChAT, nNOS and Thy-1 to be significantly blunted by sulfisoxazole. However, while the ischemia-induced changes to Thy-1 and FGF-2 mRNAs were reduced by sulfisoxazole, the reduction was non-significant. The in vitro studies provided support for the protective effect of sulfisoxazole. Here, it was clearly shown that sulfisoxazole attenuated the elevation of nitric oxide (deduced by measuring nitrite) and the reduction in numbers of GABA-containing neurones caused by LPS. The present study provides evidence for the first time that endothelin antagonist can protect the retina from ischemic-like insults as occurs in glaucoma.

Vaccinia virus infection and gene transduction in cultured neurons

The study of neurons in culture would benefit from the development of a gene transduction system capable of delivering foreign genes at high efficiency, as transduction of primary neurons with existing systems is inefficient. The efficacy of lytic vaccinia virus (VV) infection of primary retinal cultures and PC12 cells (a model of neuronal differentiation) was examined in order to determine the efficiency of gene transduction using VV in neuronal primary culture. VV was able to infect retinal cells and PC12 cells and express transgenes of Escherichia coli β-galactosidase ( lacZ) and epithelial fatty acid binding protein (E-FABP) in a virus dose-dependent manner. Most (50–100%) of the retinal cells were positive for transgene protein at multiplicities of infection (MOI) between 10 and 100 plaque-forming units (PFU), while over 50% of VV-infected PC12 cells expressed the virus encoded gene at an MOI = 10. The production of foreign mRNA and protein by VV following infection was verified by PCR and Western blot. Because VV is a lytic virus, cytopathic effects were examined. Retinal cultures maintained for 0.5 days in vitro showed greater than 90% survival at 24 h post-infection, while 14-day cultures were equally viable for 48 h. Retinal ganglion cells and differentiated PC12 cells appear to be more protected against lytic VV infection than proliferating glial and undifferentiated PC12 cells. These data suggest that VV may be a useful vector for delivering foreign genes to neuronal cells with an efficient transient transgene expression.

Transcriptional Activity of the Paired-like Homeodomain Proteins CHX10 and VSX1

CHX10 and VSX1 are homeodomain (HD) proteins essential for normal retinal development. CHX10 is required first for retinal progenitor cell proliferation and later for bipolar cell differentiation, whereas VSX1 is important in the terminal differentiation of a subset of bipolar cells. Elucidating the transcriptional activity of CHX10 and VSX1 is required to understand how these factors control retinal development. We show that CHX10 and Vsx1 can function as transcriptional repressors. When tethered to a promoter by a heterologous LexA DNA-binding domain or its HD, CHX10 repressed multiple classes of activators in different immortalized cell lines. CHX10 blocked TATA-containing and TATA-less promoters, repressed at a distance, and inhibited a complex enhancer positioned upstream or downstream of the reporter gene, whereas retinoblastoma protein (RB) inhibited the downstream enhancer only. Interestingly, CHX10 mildly potentiated a subset of activators in chick neuronal cultures. Thus, CHX10 is both a versatile repressor and a context-specific weak activator. The CHX10 HD and CVC domains were sufficient for DNA binding and repression. VSX1 contains closely related homeo and CVC domains and, like CHX10, also repressed transcription. A VSX1 HD mutation, R166W, that impairs DNA binding and causes keratoconus in humans, hindered repressor function. Therefore, CHX10 and VSX1 may control retinal bipolar cell specification or differentiation by repressing genes required for the development of other cell types.

Bunazosin, a Selective α1 ‐Adrenoceptor Antagonist, as an Anti‐glaucoma Drug: Effects on Ocular Circulation and Retinal Neuronal Damage

Bunazosin hydrochloride is a potent and selective alpha1-adrenoceptor antagonist that has been clinically used both as a systemic antihypertensive as well as an ocular hypotensive drug. In a number of studies, we have examined some effects of bunazosin hydrochloride that might indicate its potential as an anti-glaucoma drug. In normal rabbit eyes, topically instilled bunazosin hydrochloride reached the posterior retina by local penetration at concentrations sufficient to attenuate the phenylephrine- or endothelin-1 (ET-1)-induced constriction of retinal arteries. Furthermore, bunazosin hydrochloride improved the impairment of optic nerve head (ONH) blood flow, the prolongation of visual-evoked potentials (VEP) implicit time, the enlargement of the optic disk cup, and the decrease in the number of retinal ganglion cell layer cells induced by repeated injections of ET-1 in rabbits. Topically instilled bunazosin hydrochloride improved the reductions in ONH capillary blood flow and VEP amplitude induced in rabbit eyes by nitric oxide synthase inhibition. In rat primary retinal cultures, bunazosin hydrochloride reduced glutamate-induced neuronal cell death, presumably through a Na+ channel blocking effect. In healthy humans, topically instilled bunazosin hydrochloride reportedly increases blood velocity in the ONH, retina and choroid, without significantly altering either blood pressure or heart rate. These results indicate that bunazosin hydrochloride exerts both an improvement effect within the ocular circulation and a direct neuroprotective effect. Hence, bunazosin hydrochloride may be useful as a therapeutic drug against ischemic retinal diseases (such as glaucoma and retinal vascular occlusive diseases) that are associated with disturbances of the ocular circulation.

Mutations in PRPF31 inhibit pre-mRNA splicing of rhodopsin gene and cause apoptosis of retinal cells.

Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes.

Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose‐sensitive manner

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

Involvement of phosphatidylinositol 3-kinase and insulin-like growth factor-I in YXLST-mediated neuroprotection

In the present study, we examine the neuroprotective role of the external Qi of YXLST in cultured retinal neurons. Primary retinal neuronal cultures were grown from retinas of 0–2-day-old Sprague–Dawley rats. Cultures were treated directly with external Qi of YXLST 30 min prior to H 2O 2 exposure in most experiments. Cell viability was measured by 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling TUNEL assay, and by DNA laddering analysis. Northern blot analysis was performed to examine the level of insulin-like growth factor-I (IGF-I) gene expression. Phosphatidylinositol 3-kinase (PI3K) assay was performed to study the PI3K activity. The results showed that treatment of external Qi of YXLST significantly attenuated neuronal death that was induced by 24-h exposure to hydrogen peroxide, and greatly inhibited hydrogen peroxide-induced apoptosis. External Qi of YXLST also upregulated IGF-I gene expression and increased PI3K activity. These observations indicate that external Qi-mediated IGF-I expression and PI3K signaling could be one of the mechanisms in neuroprotection by YXLST.

Bunazosin hydrochloride reduces glutamate-induced neurotoxicity in rat primary retinal cultures

To study neuroprotective effects of bunazosin hydrochloride which is an α 1-adrenoceptor antagonist used as an ocular hypotensive drug compared to other α 1-adrenoceptor antagonists, and its mechanism of action. We evaluated the neuroprotective effects of bunazosin hydrochloride or seven other α 1-adrenoceptor antagonists against glutamate-induced cell death in rat primary retinal cultures. We also evaluated the binding inhibition of bunazosin hydrochloride for 24 different receptors/channels and its effects on the Na + influx into cells induced by veratridine or glutamate. Bunazosin hydrochloride significantly inhibited glutamate-induced cell death at concentrations of 1 and 10 μM. Cells were also protected when treated with some α 1-adrenoceptor antagonists, but not by the others. Bunazosin hydrochloride showed a high inhibition for Na + channels and inhibited the Na + influx induced by veratridine or glutamate. These findings indicate that in retinal cultures bunazosin hydrochloride has a neuroprotective effect against glutamate-induced cell death and that the inhibition of Na + channels by bunazosin hydrochloride may be partly responsible for this effect.

Ciliary neurotrophic factor protects rat retina cells in vitro and in vivo via PI3 kinase

Purpose. Neurotrophic factors and neurotrophins are well-known to have neuroprotective efficacy against retinal injury. The aim of this experiment is to investigate the signal transduction pathway of ciliary neurotrophic factor (CNTF) on the upregulation of viability of retinal primary culture and retinal protection against constant light damage in vivo. CNTF is known to enhance the viability of retinal culture and provide protection under constant light exposure conditions, but little is known about how the signal transduction pathways of CNTF affect retina function. Methods. Primary retinal cultures were prepared from 7-day-old Wistar rats. Brain-derived neurotrophic factor (BDNF) (0.1, 1, 10 ng/ml), CNTF (0.1, 1, 10 ng/ml), PD98059 (10, 100, 1000 nM), or LY294002 (10, 100, 1000 nM) was added to these cultures at the time of cell preparation. After 3 days, the percentage of cells surviving was assessed using alamarBlue. For the in vivo experiment, inhibitors for the MAPKK (PD98059, 10 µg/eye) or PI3K (LY294002, 10 µg/eye) pathways were injected into the vitreous together with CNTF (1 µg/eye) 2 days before constant light exposure. Electroretinogram (ERG) analysis was performed to investigate which pathway was used by CNTF. Results. CNTF at 1, 10, or 100 ng/ml enhanced cell viability in retinal cultures. The cell-survival activity of CNTF was blocked by 10 ng/ml LY294002 (Dunnet's test, p < 0.05). In vivo, the neuroprotective activity of CNTF in constant-light conditions was attenuated by 10 µg/eye LY294002 (Dunnet's test, p < 0.05). Conclusions. These data suggest that CNTF promotes cell survival via the PI3K signaling pathway in vitro and in vivo.

Perfluorohexyloctane (F6H8) induces structural modifications and increases apoptosis in rat primary retinal cultures.

The effects of perfluorohexyloctane (F6H8), recently investigated as a long-term artificial vitreous substitute, were studied in vitro, with the use of rat retinal cultures seeded on microporous inserts that allow the cell layer to be in contact with the material to be tested, on the apical side, and with the nutrient medium, on the basal side. After 72 h of treatment with F6H8, retinal cultures lost the characteristic two-layered organization with glial cells at the bottom and neuronal cells on top of them. They appeared to be composed of only one layer of polyhedrical, flattened, and disconnected cells. TUNEL assay revealed an evident increase in the percentage of apoptotic cells in F6H8-treated cultures (30.1 +/- 4.5), compared to control (10.3 +/- 2.6) and perfluoroctane-treated cultures (10.1 +/- 1.7). Immunolabeling of MAP-2, a protein of neuronal cytoskeleton, evidenced a marked loss of neurites. The results suggest that F6H8 is harmful to retinal cells in vitro and can therefore be potentially noxious to the retina as an artificial vitreous substitute.

Retinal Neuron Activity of ETS Domain-binding Sites in a Nicotinic Acetylcholine Receptor Gene Cluster Enhancer

Nicotinic acetylcholine receptors (nAchRs) mediate amacrine to ganglion cell synaptic transmission in the developing mammalian retina. The clustered neuronal nAchRs subunit genes, alpha 3 and beta 4, are expressed in amacrine and ganglion cells where they are used to assemble functional receptor subtypes. The transcriptional mechanisms underlying expression of these subunits in retina are not yet known but may involve enhancers that are selectively active in retinal neurons. We previously identified a neuron-selective enhancer, beta 43', whose activity in neural cell lines is dependent on ETS domain-binding sites. To determine whether beta 43' is active in retinal neurons that express the alpha 3 and beta 4 genes, we investigated beta 43' activity in primary dissociated rat retinal cultures. We found that beta 43' is selectively active in retinal neurons compared with retinal non-neuronal cells. This activity was derived primarily from amacrine and ganglion neurons, which are the retinal neuron cell types that express the clustered genes. Moreover, beta 43' was selectively active in retinal neurons compared with cerebral cortical neurons suggesting that it is not a pan-neuronal enhancer. ETS factor-binding sites in the enhancer are required for its retinal neuron activity. These findings suggest that ETS factor interactions with beta 43' control retinal neuron expression of certain nAchR subtypes.

Ethambutol-Induced Vacuolar Changes and Neuronal Loss in Rat Retinal Cell Culture: Mediation by Endogenous Zinc

Ethambutol is an efficacious antituberculosis agent. However, its use has been limited by the occurrence of ocular toxicity. To investigate characteristics and possible mechanisms of ethambutol ocular toxicity, we used primary rat retinal cultures as a model. Primary rat retinal cultures were obtained from newborn Sprague–Dawley rats and used for experiments after maturation (DIV ≥ 10). Cytopathologic changes were examined under light and electron microscopes. Thy-1 (a membrane glycoprotein expressed by retinal ganglion neurons)-containing neurons and γ-aminobutyric acid (GABA)-ergic neurons were identified immunocytochemically. Exposure of retinal cultures for 24–48 h to ethambutol induced cytoplasmic vacuolar changes and neuronal loss. Vacuolar changes were partially reversible with the termination of ethambutol exposure. Of neurons, Thy-1(+) ganglion neurons were more vulnerable than GABA(+) neurons. Glutamate antagonists, an antioxidant (trolox), or cycloheximide, did not attenuate either vacuolar changes or neuronal loss. A cell-permeant zinc chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) markedly attenuated vacuolar degeneration and neuronal loss, while the addition of zinc augmented both. In rat retinal cultures, ethambutol induces reversible vacuolar degeneration as well as irreversible neuronal loss, more preferentially of Thy-1(+) ganglion neurons. Contrary to the current theories, ethambutol-induced retinal cytotoxicity in the present study is mediated not by excitotoxicity or zinc deficiency but by a mechanism requiring intracellular zinc. In addition, features of the ethambutol-induced cell death were not consistent with those of apoptosis.

Hypoxic Repression of Lactate Dehydrogenase-B in Retina

In order to elucidate mechanisms controlling hypoxia induced gene repression in the retina, we studied expression of the lactate dehydrogenase gene LDH-B. RT/PCR was used to isolate cDNA fragments of the LDH-A and LDH-B genes from both rat and chick. Northern analysis was used to measure LDH-B mRNA expression and in situ hybridization to localize LDH-B mRNA during development of the avascular chick retina. Primary retinal cultures of rat and chick were subjected to hypoxia and reperfusion paradigms, and levels of LDH-A and LDH-B mRNA expression were measured by Northern and semi-quantitative RT/PCR analyses. Northern analysis demonstrated that LDH-B mRNA is developmentally regulated in the chick retina according to a time course that correlates with oxygen availability. In developing chick retina in situ hybridization localized LDH-B mRNA to aerobic regions of the retina, coincident with the distribution of active mitochondria. Northern and semi-quantitative RT/PCR analyses demonstrated that LDH-B mRNA levels decrease in primary rat and chick retinal cells in response to hypoxia. LDH-B mRNA increased to control levels in retinal cells exposed to hypoxia then reperfused with oxygen, while the opposite was true for LDH-A, an hypoxia inducible gene. These data demonstrate that LDH-B gene repression after hypoxia and reactivation after oxygen reperfusion occurs in both vascular and avascular retinal cells. Oxygen regulated expression of LDH-B is opposite and complementary to that of LDH-A and provides a system to elucidate the mechanisms underlying hypoxic gene repression and reactivation after reperfusion. Mechanisms controlling gene repression and reactivation based on oxygen availability are likely to be critically involved in ischemic damage and neovascular retinopathy.

Differential expression of AP-2alpha and AP-2beta in the developing chick retina: repression of R-FABP promoter activity by AP-2.

Retinal fatty acid binding protein (R-FABP) is the avian counterpart of murine brain FABP implicated in glial cell differentiation and neuronal cell migration. R-FABP is highly expressed in the undifferentiated retina and brain of chick embryos. We have previously shown by in vitro studies that the AP-2 transcription factor binds to a consensus AP-2 binding site in the R-FABP promoter region. Based on the expression pattern of AP-2 in the developing retina and on mutational analysis of the AP-2 binding site in DNA transfection experiments, we proposed that AP-2 could be involved in the down-regulation of R-FABP transcription. Here, we describe the cDNA isolation of two members of the AP-2 family expressed in the chick retina, AP-2alpha and AP-2beta. We show that R-FABP mRNA and the AP-2 factors are expressed in mutually exclusive patterns in the differentiating retina: whereas AP-2alpha and AP-2beta are selectively expressed either in amacrine, or in amacrine and horizontal cells, respectively, R-FABP mRNAis found in Muller glial cells and/or bipolar cells. Furthermore, a decrease in R-FABP-dependent expression is obtained upon cotransfection of primary retinal cultures with AP-2 expression vectors and a CAT reporter construct. The early and cell-specific expression of AP-2alpha and AP-2beta in the developing retina suggest a role for this transcription factor family in the early steps of amacrine and horizontal cell differentiation. Repression of the R-FABP gene in these cells may be an important component of their developmental program.

Protection Against Glutamate Neurotoxicity in Retinal Cultures by Acidic Conditions

We evaluated the effects of extracellular acidic conditions on glutamate-induced death in cultured retinal neurons. Primary retinal cultures, obtained from 3- to 5-day-old Wistar rats, were estimated to be consisted of mainly amacrine cells (90%) together with a small population of horizontal (8%) and ganglion cells (2%). We examined the effects of acidic pH (pH 6.0 to 7.0) on glutamate neurotoxicity by monitoring the delayed death of retinal neurons induced by brief (10 min) exposure to 1 mM glutamate followed by a 24-hr incubation. The glutamate-induced delayed death of cultured retinal neurons was attenuated with an acidic pH between 6.0 and 7.0. Furthermore, whole-cell patch-clamp recordings were taken from retinal neurons to examine the effects of acidic pH on N-Methyl-D-aspartate (NMDA) or kainate receptor-mediated currents. NMDA- and kainate-induced currents were suppressed at pH 6.0 to 7.0 and pH 6.0 to 6.5, respectively. The acidity of the medium protected the retinal neurons from glutamate-induced delayed death, probably by inhibiting NMDA and/or kainate receptor activation.