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Purpose: To ascertain whether red light, known to enhance mitochondrial function, can blunt chemical insults to cell cultures and ischaemic insults to the rat retina., Methods: Raised intraocular pressure (IOP, 140 mmHg, 60 min) or ischaemia was delivered in complete darkness or in the presence of low intensity red light (16.5 watts/m(2) , 3000 lux, 625-635 nm) to one eye of each rat. Animals were killed at specific times after ischemia and retinas analysis for ganglion cell numbers, the localization of specific antigens or for changes in defined RNAs. RGC-5 cell cultures were also exposed to various chemical insults in the presence or absence of red light. Significant differences were determined by t-test and anova., Results: Elevation of IOP causes changes in the localization of glial fibrillary acid protein (GFAP), calretinin, calbindin, choline acetyltransferase, ganglion cell numbers and an elevation (GFAP, vimentin, HO-1 and mTORC1) or reduction (Thy-1 and Brn3a) of mRNAs in the rat retina. These negative effects to the rat retina caused by ischaemia are reduced by red light. Moreover, chemical insults to cell cultures are blunted by red light., Conclusions: Low, non-toxic levels of red light focussed on the retina for a short period of time are sufficient to attenuate an insult of raised IOP to the rat retina. Since mitochondrial dysfunctions are thought to play a major role in ganglion cell death in glaucoma, we propose the potential use of red light therapy for the treatment of the disease., (© 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Zinc levels are high in ocular tissues and the distribution is non-uniform. Zinc is particularly concentrated in the corneal epithelium and posterior stroma. Zinc is the most abundant trace metal in the retina. Bound-zinc is particularly located in the inner nuclear layer, (e.g. forming part of the structure of zinc finger transcription factors), while loosely-bound zinc is prominent in the retinal pigment epithelium and photoreceptor layers. Loosely-bound zinc ions in the photoreceptors might play a role in the phototransduction cascade and rhodopsin regeneration. Loosely-bound zinc is also found in presynaptic vesicles of photoreceptor cells in the outer plexiform and inner plexiform layers and can be synaptically released to affect both ionotropic and metabotropic receptors and also ion channels to modulate neurotransmission. The correct amount of loosely-bound zinc ions is maintained by regulating the function of zinc transporters, sensors and trafficking/storage proteins (i.e. metallothionein). The retinal homeostasis of zinc is dysregulated in systemic zinc depletion, aging and diseases such as age-related macular degeneration. Manipulation of retinal zinc metabolism in these situations might improve visual function.

Proton-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) was used to establish the distribution and concentration of trace elements within individual corneal and retinal areas in frozen sections from adult male Wistar rats (n = 6). The distribution of endogenous trace elements in the cornea and retina is non-homogenous. The most abundant metal in the cornea is calcium followed by zinc. Iron and copper are present in small amounts localised particularly to the epithelium. Iron is also identified in keratocytes. Relatively high levels of calcium occur in the corneal epithelial cell bodies. Zinc has a wide intense distribution across the corneal epithelium (with greater levels in the basal part) and posterior stroma. In the retina, zinc is the most common metal followed by iron and copper. Relatively high levels of zinc exist in the retinal pigment epithelium (RPE), photoreceptor inner segments (RIS) and inner nuclear layer (INL). Chelatable zinc was localised with fluorescent TSQ in the RPE, RIS and plexiform layers. It is interesting to note that the highest levels of total zinc and the greatest intensity of chelatable zinc staining do not coincide. In the RPE and corneal epithelium, zinc co-localised with the zinc-containing metallothioneins (MT). However, there was a clear mismatch between the localisation of the most intense levels of zinc in the neuroretina (i.e. INL) and corneal posterior stroma with that reported for MT. For example, the presence of zinc is not particularly associated with the retinal ganglion cells, retinal area that contains MTs in significant amounts. While high amounts of zinc are present in the INL and corneal posterior stroma, which are largely devoid of MTs. This probably represents pools of static, catalytic and structural zinc associated with substances other than the MTs.

The essential trace metals iron, zinc, and copper play important roles both in retinal physiology and disease. They are involved in various retinal functions such as phototransduction, the visual cycle, and the process of neurotransmission, being tightly bound to proteins and other molecules to regulate their structure and/or function or as unbound free metal ions. Elevated levels of "free" or loosely bound metal ions can exert toxic effects, and in order to maintain homeostatic levels to protect retinal cells from their toxicity, appropriate mechanisms exist such as metal transporters, chaperones, and the presence of certain storage molecules that tightly bind metals to form nontoxic products. The pathways to maintain homeostatic levels of metals are closely interlinked, with various metabolic pathways directly and/or indirectly affecting their concentrations, compartmentalization, and oxidation/reduction states. Retinal deficiency or excess of these metals can result from systemic depletion and/or overload or from mutations in genes involved in maintaining retinal metal homeostasis, and this is associated with retinal dysfunction and pathology. Iron accumulation in the retina, a characteristic of aging, may be involved in the pathogenesis of retinal diseases such as age-related macular degeneration (AMD). Zinc deficiency is associated with poor dark adaptation. Zinc levels in the human retina and RPE decrease with age in AMD. Copper deficiency is associated with optic neuropathy, but retinal function is maintained. The changes in iron and zinc homeostasis in AMD have led to the speculation that iron chelation and/or zinc supplements may help in its treatment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the total amount of trace elements in retina from adult male Sprague-Dawley rats (n = 6). Concentration of trace elements within individual retinal areas in frozen sections of the fellow eye was established with the use of two methodologies: (1) particle-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) and (2) synchrotron X-ray fluorescence (SXRF) microscopy. The most abundant metal in the retina was zinc, followed by iron and copper. Nickel, manganese, chromium, cobalt, selenium and cadmium were present in very small amounts. The PIXE and SXRF analysis yielded a non-homogenous pattern distribution of metals in the retina. Relatively high levels of zinc were found in the inner part of the photoreceptor inner segments (RIS)/outer limiting membrane (OLM), inner nuclear layer and plexiform layers. Iron was found to accumulate in the retinal pigment epithelium/choroid layer and RIS/OLM. Copper in turn, was localised primarily in the RIS/OLM and plexiform layers. The trace elements iron, copper, and zinc exist in different amounts and locations in the rat retina.

Purines (in particular, ATP and adenosine) act as neuro- and gliotransmitters in the sensory retina where they are involved in bidirectional neuron-glia signaling. This review summarizes the present knowledge about the expression and functional importance of P1 (adenosine) and P2 (nucleotide) receptors in Müller glial cells of the mammalian retina. Mammalian Müller cells express various subtypes of adenosine receptors and metabotropic P2Y receptors. Human Müller cells also express ionotropic P2X(7) receptors. Müller cells release ATP upon activation of metabotropic glutamate receptors and/or osmotic membrane stretching. The osmotic mechanism is abrogated under conditions associated with ischemia-hypoxia and inflammation, resulting in swelling of the Müller cells when the extracellular milieu is hypoosmotic. However, exogenous glutamate, which induces the release of ATP and adenosine, and thus activates P2Y(1) and A(1) adenosine receptors, respectively, prevents such osmotic swelling under pathological conditions, suggesting unimpaired receptor-induced release of ATP. In addition to the inhibition of swelling, which is implicated in regulating the volume of the extracellular space, purinergic signaling is involved in mediating neurovascular coupling. Furthermore, purinergic signals stimulate the proliferation of retinal precursor cells and Müller cells. In normal retinal information processing, Müller cells regulate the synaptic activity by the release of ATP and adenosine. In retinopathies, abrogation of the osmotic release of ATP, and the upregulation of ecto-apyrase (NTPDase1), may have neuroprotective effects by preventing the overactivation of neuronal P2X receptors that are implicated in apoptotic cell death. Pharmacological modulation of purinergic receptors of Müller cells may have clinical importance, e.g., for the clearance of retinal edema and for the inhibition of dysregulated cell proliferation in proliferative retinopathies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

It is concluded from immunohistochemical that all four types of prostaglandin-E(2) (PGE(2)) (EP1, EP2, EP3 and EP4) receptors are associated with specific cell-types in primary rat retinal cultures. Analysis specifically of EP2 receptor immunoreactivity shows it to coexist with some neurones expressing Thy-1 and calbindin immunoreactivities as well as with vimentin-positive Müller cells. Moreover, exposure of cultures to the EP2 specific agonist butaprost (100 nM) for a period of 24h results in a generation of cAMP thus providing support for the functionality of EP2 receptors. Cell survival was significantly affected in cultures where the serum concentration was reduced from 10 to 1% for 24h. This was reflected by a reduction in the number of GABA-positive neurons and an elevation of released lactate dehydrogenase (LDH) into the culture medium. Moreover, a number of cells displayed a clear generation of reactive oxygen species (ROS) and a staining for the breakdown of DNA by the TUNEL procedure as an indicator for apoptosis. These negative effects were attenuated when butaprost (100 nM) was present during the serum reduction and 30 min before the insult. The present studies provide evidence to show that all PGE(2) receptor types exist in the retina of rat pups, remain functional when the retinal cells are cultured and that specific activation of EP2 receptors with butaprost can attenuate a detrimental insult caused by insufficient serum that may occur in situ by reduced trophic support.

EGCG, a major component of green tea, has a number of properties which includes it being a powerful antioxidant. The purpose of this investigation was to deduce whether inclusion of EGCG in the drinking water of albino rats attenuates the effect of a light insult (2200lx, for 24h) to the retina. TUNEL-positive cells were detected in the outer nuclear layer of the retina, indicating the efficacy of the light insult in inducing photoreceptor degeneration. Moreover, Ret-P1 and the mRNA for rhodopsin located at photoreceptors were also significantly reduced as well as the amplitude of both the a- and b-waves of the electroretinogram was also reduced showing that photoreceptors in particular are affected by light. An increase in protein/mRNA of GFAP located primarily to Müller cells caused by light shows that other retinal components are also influenced by the light insult. However, antigens associated with bipolar (alpha-PKC), ganglion (Thy-1) and amacrine (GABA) cells, in contrast, appeared unaffected. The light insult also caused a change in the content of various proteins (caspase-3, caspase-8, PARP, Bad, and Bcl-2) involved in apoptosis. A number of the changes to the retina caused by a light insult were significantly attenuated when EGCG was in the drinking water. The reduction of the a- and b-waves and photoreceptor specific mRNAs/protein caused by light were significantly less. In addition, EGCG attenuated the changes caused by light to certain apoptotic proteins (especially at after 2 days) but did not appear to significantly influence the light-induced up-regulation of GFAP protein/mRNA. It is concluded that orally administered EGCG blunts the detrimental effect of light to the retina of albino rats where the photoreceptors are primarily affected.

Purpose: The cytokine osteopontin (OPN) has been localized to the retinal ganglion cell layer in the normal rodent retina, prompting the suggestion that it could serve as a useful marker for identifying and quantifying such neurons in models of retinal and optic nerve neurodegeneration. In the present study, we characterized the time course and cellular localization of OPN expression in the rat retina after excitotoxic and ischemic injuries., Methods: Excitotoxicity and ischemia-reperfusion experiments were performed by using standard techniques. Rats were killed at various time points, and the retinas were removed either for mRNA analysis or to be processed for immunohistochemistry., Results: In the normal retina, double-labeling immunofluorescence indicated that OPN is expressed by the majority of, if not all, RGCs, since OPN was associated with more cells than Brn-3, but was colocalized with Thy1.1. NMDA, kainic acid, and ischemia-reperfusion all caused decreases in the total retinal levels of Thy1 and Brn-3 mRNAs, reflecting injury to RGCs, but a dramatic, short-lived upregulation in OPN mRNA. The source of the increased OPN signal after excitotoxic-ischemic insults is unlikely to be injured RGCs, as no alteration in the intensity of OPN immunostaining in RGCs was apparent. Instead, additional cells, mostly contained within the IPL, were identified as positive for OPN. Double-labeling immunofluorescence showed that ED1 always colocalized with OPN in these cells, indicating their status as activated microglia., Conclusions: OPN is exclusively expressed by RGCs in the physiological retina, but in response to retinal neurodegeneration is synthesized de novo by endogenous, activated microglia.

Background: Diabetes inhibits dark adaptation and both processes alter the electroretinogram (ERG) in similar ways. This study aimed to investigate the relationship between oscillatory potentials (OPs) and the b-wave during dark adaptation and to determine if this relationship changes during the development of diabetes., Methods: Twenty-one rats were assigned to adaptation, control and diabetic groups. Rats were dark adapted for periods between 20 minutes and 4 hours, and ERGs recorded. Diabetes was induced with streptozotocin, and ERGs measured after 3, 6, 9 and 12 weeks after injection., Results: Increasing periods of dark adaptation led to a logarithmic increase in the amplitude of the b-wave and the OPs. This was accompanied by a decrease in the peak times of the OPs and b-wave. Total OP amplitude and b-wave amplitude were linearly related, allowing an empirical OP constant to be developed to describe the relationship between the two parameters. Diabetes led to a progressive decrease in the amplitude and increase in the peak time of all waves. The OP constant decreased in a linear fashion with increasing duration of diabetes., Conclusions: It is argued that OP masking of the b-wave could explain previous inconsistencies in reported ERG changes in diabetes and that a slowing of dark adaptation does not account for these ERG changes. The report concludes that the OPs and b-wave amplitudes and latencies are intimately related in the normal retina and that this correlation is lost predictably during the development of diabetes.

It has previously been shown that inhibitors of protein kinase C (PKC) attenuate retinal glutamate uptake in situ. The aim of the current study was to determine whether PKCdelta-mediated inhibition differentially reduces the transport of glutamate into retinal Müller cells when compared with retinal neurons. The influence of two different types of PKC inhibitors on the uptake of [3H]D-aspartate was therefore compared in the intact retina, mixed retinal cultures, and Müller cell-enriched retinal cultures. It was found that 25 microM of the pan-isoform PKC inhibitor, chelerythrine, reduced [3H]D-aspartate uptake by 78%, 71%, and 68% in isolated retinas, mixed neuronal/glial cultures, and Müller cell-enriched cultures, respectively. Importantly, 20 microM of the PKCdelta-selective inhibitor rottlerin also reduced the uptake of D-aspartate to similar extents in all three systems, and the reductions were statistically similar to those found for the pan-specific PKC inhibitor. Neither pan-isoform nor PKCdelta-selective activators stimulated glutamate uptake in either culture system or the intact retina. The current results suggest that specific PKC inhibitors are quantitatively similar in reducing the uptake of glutamate into retinal neurons and Müller cells.

Ciliary neurotrophic factor (CNTF) has been shown to protect ganglion cells in a variety of acute ischaemia models. Here we assess the efficacy of local CNTF gene transfer in protecting retinal ganglion cells when there is focal ischaemia combined with interruption of axoplasmic flow. This dual injury may be more representative of the pathological mechanisms operating in acute retinal diseases, such as vascular events acting at the optic nerve head. Fourteen rats received an intravitreal injection of an adeno-associated viral (AAV) vector expressing a secretable form of CNTF into the right eye and a control vector into the left eye. Three weeks later, each rat underwent a symmetrical small vertical 2mm standardised retinal crush injury approximately 2mm temporal to the optic disc. The injury also occluded the temporal retinal arteriole so that the axon crush was combined with an acute retinal infarction visible on fundoscopy. Changes in the damaged sector were compared histologically four weeks after injury and ganglion cell survival was estimated by comparing cell counts on retinal flat-mounts immunostained with RT-97 antibody. This mode of injury led to a profound loss of both the inner nuclear and ganglion cell layers, but was limited to the lesioned sector. In AAV.CNTF-treated eyes approximately 12% of ganglion cells survived compared with approximately 2% in control eyes (p=0.01). The scotopic electroretinogram (ERG), however, was reduced to about 50% in AAV.CNTF-treated eyes, both before and after injury. We therefore show that CNTF gene transfer confers neuroprotection to ganglion cells undergoing an acute ischaemic injury combined with interruption of axoplasmic flow. This approach may be relevant to optic nerve trauma and a variety of retinal vascular diseases that lead to swelling of the optic nerve head, provided CNTF can be delivered in a way that does not significantly suppress retinal function.

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.

Purpose: The diabetic retina exhibits decreases in endogenous nonangiogenic neurotrophins. This study hypothesized that deficiencies in systemic and retinal pigment epithelium-derived (RPE) neurotrophic factors also influence retinal changes in diabetes., Methods: Diabetes was established in Listar hooded rats with streptozotocin. Reverse transcriptase coupled polymerase chain reaction (RT-PCR) and immunoblotting were used to determine the expression of fibroblast growth factor-2 (FGF-2) in the retina and RPE, and glial fibrillary acid protein (GFAP) in the retina. In addition, primary human RPE cultures and a transformed Müller cell line were used to determine the effect of insulin, glucose, and insulin-like growth factor (IGF) on the expression of these substances., Results: FGF-2 and GFAP were increased in retina, but FGF-2 was decreased in the RPE of diabetic animals. Retinal GFAP correlated with RPE FGF-2 expression in these animals. Insulin produced a dose-dependent increase in FGF-2 in RPE cells and decrease in GFAP in Müller cells grown in 15 mM glucose. In 5 mM glucose, insulin had no effect on expression of either protein. Physiological levels of insulin inhibited changes induced by 15 mM glucose. The effect of 9 nM insulin on each culture was mimicked by 1 nM IGF, and blocked with an IGFR-1 inhibitor., Conclusions: It is suggested that decreased systemic insulin and high glucose levels contribute to decreased FGF-2 production in the RPE and increased glial cell activation in the diabetic retina. Addition of insulin and IGF act to reverse this effect through the IGFR-1. These mechanisms may contribute to the development of diabetic retinopathy.

Purpose: To determine the effect of diabetes on monocarboxylate transporter (MCT) expression in the rat retina., Methods: Diabetes was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (62.5 mg/kg). Rats were killed after 10 weeks, and the retinal levels of PKCalpha, bFGF, glial fibrillary acidic protein (GFAP), caspase-3, and MCT1, -2, and -4 were assessed with immunoblot analysis and RT-PCR. Hippocampal samples were used as a comparison. In other animals, the retinas were processed histologically and sections stained for the breakdown of DNA (TUNEL procedure) and for the distribution of MCT1, -2, and -4., Results: Diabetic rats exhibited cataract formation, elevated blood glucose levels, and polydipsia. Retinal levels and distribution of MCT1, 2, and -4 were similar in the diabetic and age-matched control groups; however, the early retinopathy markers bFGF and PKCalpha were significantly elevated in the diabetic retinas but not in the hippocampal samples. ERG recordings showed decreased oscillatory potentials in the diabetic group, and TUNEL staining was most evident in the photoreceptor layer. Activated caspase-3 levels were elevated in the diabetic retina., Conclusions: The expression of retinal MCT1, -2, and -4 is unaffected after 10 weeks of diabetes in rats. It appears unlikely that diabetic retinal neuropathy is a result of a hyperglycemic induction of altered MCT expression.

The process of metabolic coupling has been described as a means of providing additional fuel for neurons during periods of intense activity. This process has been suggested to occur in the mammalian retina, but whether retinal neurons can metabolise glial-derived monocarboxylates remains uncertain. The present study therefore sought to define the preferred energy substrates for maintenance of different retinal cells in culture, in order to clarify whether metabolic coupling can potentially occur in this tissue. All cells in rat retinal cultures were detrimentally affected by glucose deprivation. The effect on some neurons, however, could be partially reversed by 5 mm pyruvate or lactate. Furthermore, the glycolytic inhibitor, iodoacetic acid, caused a dose-dependent loss of all retinal cells in culture, whereas the mitochondrial inhibitor, 2,4-dinitrophenol, only led to a decrease in the number of neurons. Finally, inhibition of transporters for glucose or monocarboxylates caused the respective loss of glia or neurons from cultures. These data together demonstrate that, although cells do preferentially metabolise glucose, monocarboxylates such as lactate or pyruvate do play an important role in neuronal maintenance. These data therefore give partial support to the notion that metabolic coupling may occur in the retina.

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.

The recreational use of the psychoactive drug, methamphetamine has increased markedly over the last three decades. It has long been known that this drug has detrimental effects upon the mammalian brain monoaminergic system, but the long- or short-term effects on the retina, a neurological extension of the central nervous system, have received little attention. The aim of this study was, therefore, to determine whether intraocular injection of methamphetamine (MA) is toxic to the healthy adult rat retina and to analyse its effects on the compromised retina after an injection of the ionotropic glutamate receptor agonist, kainate, which is known to cause retinal neuropathology. The equivalent of 1 mM (in the vitreous humour) MA and/or kainate (40 microM) were injected intravitreally. Flash electroretinograms (ERGs) were recorded before and 2 and 4 days after treatment. Five days after treatment, animals were killed and the retinas analysed either for the immunohistochemical localisation of various antigens or for electrophoresis/Western blotting. Some animals were kept for 19 days after treatment and the retinas analysed for tyrosine hydroxylase immunoreactivity. No differences could be found between vehicle- and MA-treated retinas with respect to the nature or localisation of either tyrosine hydroxylase immunoreactivity after 5 or 19 days or other antigens after 5 days. Moreover, the normal ERG and GFAP and calretinin protein antigens were unaffected by MA. Kainate treatment, however, caused a change in the ERGs after 2 and 4 days, an alteration in every antigen localised by immunohistochemistry and an increase in the retinal levels of calretinin and GFAP proteins. Significantly, the changes seen in the b-wave amplitude and implicit time of the ERG after 4 days and the increased level of GFAP protein after 5 days following kainate treatment were enhanced when MA was co-injected. Intravitreal injection of methamphetamine had no detectable detrimental effect on the normal adult rat retina but exacerbated the damaging effects of kainic acid. Such data suggest that a neurotoxic effect of MA may be more obviously illustrated when the tissue is already compromised as occurs in, for example, ischemia.

Glaucoma is a chronic optic neuropathy in which retinal ganglion cells die over a number of years. The initiation of the disease and its progression may involve an ischaemic-like insult to the ganglion cell axons caused by an alteration in the quality of blood flow. Thus, to effectively treat glaucoma it may be necessary to counteract the ischaemic-like insult to the region of the optic nerve head. Studies on the isolated optic nerve suggest that substances that reduce the influx of sodium would be particularly effective neuroprotectants. Significantly, of the presently used antiglaucoma substances, only beta-blockers can reduce sodium influx into cells. Moreover, they also reduce the influx of calcium and this would be expected to benefit the survival of insulted neurones. Betaxolol is the most effective antiglaucoma drug at reducing sodium/calcium influx. Our electroretinographic data indicated that topical application of levobetaxolol to rats attenuated the effects of ischaemia/reperfusion injury. Timolol was also effective but to a lesser extent. Based on these data we conclude that beta-blockers may be able to blunt ganglion cell death in glaucoma, and that levobetaxolol may be a more effective neuroprotectant than timolol because of its greater capacity to block sodium and calcium influx.

Glial-derived monocarboxylate lactate is thought to be an important energy source for neurons during brain activation or in hypoxia-ischemia. Treatment with alpha-cyano-4-hydroxycinnamate (4-CIN), a monocarboxylate transporter inhibitor, has been recently reported to exacerbate delayed neuronal damage in a rat model of cerebral ischemia, an effect ascribed to inhibition of lactate/pyruvate transport. Since monocarboxylate transporters are abundant in the retina, we examined the effect of 4-CIN administration on the outcome of high intraocular pressure-induced retinal ischemia in rats. Retinal ischemic damage was assessed by changes in the electroretinogram (ERG), the retinal localization of choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) immunoreactivities, and the loss of retinal mRNA for Thy-1. Intraperitoneal or intravitreal administration of 4-CIN had no effect on the ERG or the localization of ChAT and nNOS immunoreactivities in either the control retina or a retina subjected to ischemia/reperfusion. In addition, intravitreal injection of 4-CIN had no effect on ischemia-induced reduction of retinal mRNA levels for Thy-1. These results provide no evidence to support the view that blockade of lactate uptake and/or pyruvate entry into mitochondria for oxidative metabolism has an influence on the outcome of retinal ischemia/reperfusion.

The main aim of this study was to investigate whether intraocular injection of low concentrations of zinc (no greater than 10 microM) aid the survival of ganglion cells in the rat retina after excitotoxic (NMDA) and ischemia/reperfusion injuries. We also determined whether low amounts of zinc cause any detectable retinal toxicity. Intraocular injection of NMDA caused substantial reductions in the mRNA levels of the ganglion cell-specific markers Thy-1 and neurofilament light (NF-L). Co-injection of 0.1 or 1 nmol zinc neither diminished nor exacerbated the effect of NMDA on the levels of these mRNAs. Likewise, ischemia/reperfusion caused significant decreases in the levels of Thy-1 and NF-L mRNAs and in the b-wave amplitude of the electroretinogram. These effects were not counteracted by injection of zinc. Intraocular injection of NMDA caused marked toxicological effects in retinal glial cells, including upregulations of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glial fibrial acidic protein (GFAP), basic fibroblast growth factor (FGF-2) and ciliary neurotrophic factor (CNTF). Interestingly, injection of 1 nmol zinc caused no changes in the levels of COX-2 and iNOS, yet produced similar, although quantitatively less pronounced, changes in FGF-2, GFAP and CNTF. The upregulations of FGF-2 and CNTF suggest that increasing zinc intake may benefit injured retinal neurons. However, this was not found to be the case in the present studies, perhaps due to the acute nature of the injury paradigms utilised.

Treatment with lipopolysaccharide (LPS) for 72 h was shown to dose-dependently increase nitric oxide production from 6-day-old retinal cultures. Cell death, as determined by lactate dehydrogenase (LDH) release and an increase in neuronal labelling for TUNEL, was elevated concurrently. During treatment there was an increase of both inducible nitric oxide synthase and glial fibrillary acidic protein labelling in glial cells and a reduction in the number of gamma-aminobutyric acid-positive neurones. The NOS inhibitors, N-nitro-L-arginine methyl ester, dexamethasone and indomethacin potently inhibited both nitric oxide stimulation and cell death caused by LPS. In this study, the beta(2)- (ICI-18551), beta(1)- (betaxolol) and mixed beta(1)/beta(2)- (timolol, metipranolol) adrenergic receptor antagonists were all shown to attenuate LPS-induced LDH release from these cultures, but to have no effect on LPS-stimulated nitric oxide production. This effect was mimicked by the calcium channel blocker, nifedipine. Interestingly, the beta-adrenergic receptor agonists, salbutamol, arterenol and isoproterenol were also able to attenuate cell death caused by LPS. Moreover, these compounds also inhibited LPS-stimulated nitric oxide release. These studies suggest that LPS stimulates nitric oxide release from cultured retinal glial cells and that this process leads to neurone death. beta-adrenergic receptor agonists prevent the effects of LPS by inhibiting the stimulation of nitric oxide production. The data also suggest that beta-adrenergic receptor antagonists can attenuate LPS-induced death of neurones, but that these compounds act in a manner that is neurone-dependent, is mimicked by blockade of calcium channels and is independent of the stimulation of nitric oxide release.

beta-adrenoceptor antagonists are used clinically to reduce elevated intraocular pressure in glaucoma which is characterised by a loss of retinal ganglion cells. Previous studies have shown that the beta(1)-selective adrenoceptor antagonist, betaxolol, is additionally able to protect retinal neurones in vitro and ganglion cells in vivo from the detrimental effects of either ischemia-reperfusion or from excitotoxicity, after topical application. The neuroprotective effect of betaxolol is thought not to be elicited through an interaction with beta-adrenoceptors, but by its ability to reduce influx of sodium and calcium through voltage-sensitive calcium and sodium channels. In the present study it is shown that the non-selective beta-adrenoceptor antagonists, metipranolol and timolol behave like betaxolol. When topically applied they all attenuate the detrimental effect of ischemia-reperfusion. Protection of the retina was determined by evaluating changes in the electroretinogram and by assessing the loss of mRNA for Thy-1, which is expressed in retinal ganglion cells. In addition, studies conducted on neurones in mixed retinal cultures demonstrated that metipranolol, betaxolol and timolol were all able to partially counteract anoxia-induced cell loss and viability reduction. The influence of timolol was, however, not significant. Within the confines of these investigations, an order of neuroprotective efficacy was delineated for the three beta-adrenoceptor antagonists: betaxolol>metipranolol>timolol. The ability of the beta-adrenoceptor antagonists to attenuate ligand-induced stimulation of calcium and sodium entry into neuronal preparations showed a similar order of effectiveness. In conclusion, the ability to confer neuroprotection to retinal neurones is a common feature of three ophthalmic beta-adrenoceptor antagonists (betaxolol, metipranolol and timolol). A comparison of the effectiveness of the individual compounds in protecting retinal cells in vivo was not possible in these studies. However, in vitro studies show that the capacity of the individual beta-adrenoceptor antagonists to act as neuroprotectants appears to relate to their capacity to attenuate neuronal calcium and sodium influx.

Purpose: To determine whether topical application of flunarizine reduces intraocular pressure (IOP) and acts as a retinal neuroprotectant and to compare the effectiveness of flunarizine with betaxolol and nifedipine at reducing the influx of calcium and sodium., Methods: Ischemia was delivered to the rabbit retina by raising the IOP. After 3 days, a flash electroretinogram (ERG) was recorded, and the retina processed for the localization of certain antigens. In the rat, N-methyl-D-aspartate (NMDA) was injected intravitreally, and 8 days later, the retinas were analyzed for the localization of Thy-1 or the relative amounts of mRNAs for antigens located to ganglion cells or photoreceptors. Rats and rabbits received topical flunarizine or vehicle before and after ischemia or NMDA. IOP was measured in rabbits after a single topical application of 2% flunarizine. Studies were conducted on isolated rat retinas, cortical cultures, and brain synaptosomes to compare the effectiveness of flunarizine with nifedipine and betaxolol at reducing the influx of calcium or sodium., Results: Changes in rabbit retinal choline acetyltransferase and parvalbumin immunoreactivities and the b-wave of the ERG caused by ischemia-reperfusion were blunted by topical treatment with flunarizine. Similarly, NMDA induced reductions in Thy-1 immunoreactivity and mRNA for rat ganglion cell antigens (Thy-1 and neurofilament light form) were counteracted by topical application of flunarizine. Topical application of 2% flunarizine significantly lowered the IOP in rabbits over a period of 5 hours. Flunarizine was more effective than betaxolol and much stronger than nifedipine at attenuating veratridine-induced influx of sodium into synaptosomes. Nifedipine, flunarizine, and betaxolol all reduced the NMDA-induced influx of calcium into the isolated retina or cortical neurons, but betaxolol was the least effective., Conclusions: Topically applied flunarizine reduces IOP and attenuates injury to the whole of the retina, including the ganglion cells. The neuroprotective action of flunarizine is to reduce the influx of calcium and sodium into stressed neurons. The potent effect of flunarizine on sodium influx would be particularly protective to axons.

Ischaemia was induced to the rat retina by raising the intraocular pressure above the systolic blood pressure for 45 min. After a reperfusion period of 5 days, alterations in the localisation of choline acetyltransferase (ChAT) and calretinin immunoreactivities, a reduction in the thickness of the inner retinal layers and a decline in the b-wave amplitude of the electroretinogram were recorded. These changes were blunted when clonidine was injected intraperitoneally before or after ischaemia or when applied topically by a specific regime. Other alpha(2)-adrenoceptor agonists, brimonidine and apraclonidine, acted in a similar way to clonidine when applied topically but because of the number of experiments carried out a comparison between the effectiveness of the different alpha(2)-adrenoceptor agonists was not possible. The protective effect of clonidine was attenuated when the alpha(2)-adrenoceptor antagonists yohimbine or rauwolscine were co-administered, suggesting that the mechanism of action of the drug is to stimulate alpha(2)-adrenoceptors. In addition, the imidazoline receptor ligands, BU-226 and AGN-192403 did not blunt the effect of ischaemia/reperfusion, supporting the notion that the protective action of the alpha(2)-adrenoceptor agonists does not involve imidazoline sites but rather the activation of alpha(2)-adrenoceptors. The protective effect of 0.5% clonidine appeared to be greater when topically applied to the eye that received ischaemia than when applied by the same regime to the contralateral eye. These studies suggest that while most of topically applied clonidine reaches the retina by a systemic route one cannot rule out additional pathways.

Experimental evidence exists to suggest that zinc can have positive and negative effects on the physiology of cells depending on the "local" concentration, localisation (extracellular vs. intracellular) and/or state (bound vs. free). The retina contains particularly high amounts of zinc suggesting a pivotal role in the tissue. There is also suggestive evidence that zinc deficiency in humans may result in abnormal dark adaptation and/or age-related macular degeneration. The purpose of this article is to provide an overview of various proposed functions for zinc, particularly in the retina. Endogenous chelatable zinc in the retina is localised mainly to the photoreceptors and retinal pigment epithelial cells. Moreover, the zinc localisation in the photoreceptors varies in dark and light, suggesting a role for zinc in a light-regulated process. Some zinc is also located to other areas of the retina but clearly defined zinc-enriched neurones could not be identified as has been shown to occur in certain areas of the brain. Neurones post-synaptic to zinc-enriched neurones in the brain have been suggested to be particularly vulnerable in ischaemia. The role of zinc in retinal ischaemia has been investigated to determine how it is involved in the process. It would appear that when zinc is administered in low concentrations it generally has a positive effect on an insulted retina as in ischaemia. However, higher concentrations of zinc exacerbates the influence of the insult and also acts as a toxin. Use of zinc supplements in diet must, therefore, be taken with caution.

Rises in intracellular Ca2+ induced by activation of glutamate receptors are of ultimate importance for neuronal excitability and pathophysiological processes. In the present study, we aimed to elucidate the types of voltage-dependent Ca2+ channels involved in the NMDA-stimulated influx of Ca2+ into the isolated rat retina by using selective blockers. Additionally, the number of binding sites for radioligands labelling L- ([3H]nitrendipine), N- ([125I]omega-conotoxin MVIIA) and P/Q-type ([125I]omega-conotoxin MVIIC) Ca2+ channels was assessed in the rat retina and, for further comparison, in the rat cortex. Incubation of isolated rat retinas with 100 microM NMDA produced a three-fold increase in the influx of 45Ca2+ that was completely blunted by MK-801, a NMDA receptor antagonist, and partially attenuated (approximately 20%) by tetrodotoxin, a Na+ channel blocker. The L-type Ca2+ channel blocker nifedipine reduced NMDA-stimulated Ca2+ influx in a dose-related fashion, with a maximum reduction of approximately 50%. Similar effects were observed with verapamil and diltiazem. Blockers of N- and P/Q-type Ca2+ channels had no significant effect on the influx of Ca2+ evoked by NMDA. Co2+, a non-specific Ca2+ channel blocker, caused an inhibition of NMDA-stimulated Ca2+ influx similar to that of nifedipine. Therefore, of all voltage-dependent Ca2+ channels, L-type channels appear to make the greatest contribution (up to 50%) to the NMDA-stimulated influx of Ca2+ into the isolated rat retina. This finding contrasts with evidence obtained in brain neurones supporting a role for L-, N- and P/Q-type channels in NMDA-evoked Ca2+ signals. A comparison of the number of radioligand binding sites associated with L-, N- or P/Q-type Ca2+ channels in the rat cortex and retina revealed that such a difference cannot be ascribed to a distinct expression pattern of these channels in both tissues, although some variations were found. Interestingly, a different affinity of [3H]nitrendipine for L-type Ca2+ channels in the rat retina and cortex was observed which may reflect the expression of different classes of L-type channels in these tissues. The ability of L-type Ca2+ channel blockers to attenuate NMDA-stimulated Ca2+ influx may underlie their neuroprotective effects in the retina.

It has previously been reported that the beta(1)-adrenoceptor antagonist, betaxolol, can protect retinal neurones from ischaemia when applied topically. It has further been shown that betaxolol can reduce influx of both sodium or calcium into neurones through interaction at neurotoxin site 2 of the sodium channel and the L-type calcium channel, respectively. The present study sought to further investigate the neuroprotective mode of action of betaxolol in the rat retina. Rats were treated topically with L-betaxolol for 10, 5 and 1 min before ischaemia, induced by raising the intraocular pressure above systolic blood pressure for 45 min. This was followed by reperfusion of 3 or 5 days where L-betaxolol was applied topically twice daily. Ischaemia plus reperfusion caused both a loss of immunoreactivity for choline acetyl transferase (ChAT) and a marked reduction of the b-wave of the electroretinogram (ERG). Treatment, as described, with topical L-betaxolol, completely blunted the effects upon ChAT immunoreactivity and caused a significant reversal of the ERG changes. Furthermore, other rats treated topically with commercially available racemic betaxolol (Betoptic Solution, 0.5%) for 6 hr had raised levels of mRNA for brain derived neurotrophic factor (BDNF) but not for basic fibroblast growth factor (bFGF) in their retinas. The combined data provide further evidence that betaxolol can blunt the effects of ischaemia to the rat retina when applied topically just before the insult. Furthermore, the finding that retinal levels of BDNF mRNA are raised following topical betaxolol treatment shows that not only can this drug reach the retina but that it can also induce changes in expression of factors which are known, themselves, to provide neuroprotection to retinal neurones., (Copyright 2001 Academic Press.)

alpha(2)-adrenoceptor agonists, such as clonidine, attenuate hypoxia-induced damage to brain and retinal neurones by a mechanism of action which likely involves stimulation of alpha(2)-adrenoceptors. In addition, the neuroprotective effect of alpha(2)-adrenoceptor agonists in the retina may involve stimulation of bFGF production. The purpose of this study was to examine more thoroughly the neuroprotective properties of clonidine. In particular, studies were designed to ascertain whether clonidine acts as a free radical scavenger. It is thought that betaxolol, a beta(1)-adrenoceptor antagonist, acts as a neuroprotective agent by interacting with sodium and L-type calcium channels to reduce the influx of these ions into stressed neurones. Studies were therefore undertaken to determine whether clonidine has similar properties. In addition, studies were undertaken to determine whether i.p. injections of clonidine or betaxolol affect retinal bFGF mRNA levels. In vitro data were generally in agreement that clonidine and bFGF counteracted the effect of NMDA as would occur in hypoxia. No evidence could be found that clonidine interacts with sodium or L-type calcium channels, reduces calcium influx into neurones or acts as a free radical scavenger at concentrations below 100 microM. Moreover, i.p. injection of clonidine, but not betaxolol, elevated bFGF mRNA levels in the retina. The conclusion from this study is that the neuroprotective properties of alpha(2)-adrenoceptor agonists, like clonidine, are very different from betaxolol. The fact that both betaxolol and clonidine blunt hypoxia-induced death to retinal ganglion cells suggests that combining the two drugs may be a way forward to producing more effective neuroprotection.

Age- and sex-matched pigmented (Lister Hooded) and albino (Wistar) rats were used in this study. The retinas of the animals were subjected to pressure-induced ischaemia (35 min, 120 mmHg) and reperfusion (3 days) in precisely the same way. The b-wave of the electroretinogram (ERG) in the pigmented animals recovered to normal levels while those of the albino rats were reduced by more than 80%. Moreover, the choline acetyltransferase (ChAT) immunoreactivity associated with a sub-set of amacrine cells was almost completely obliterated in the retinas from the albino rats but unaffected in the retinas of the pigmented rats. Also, in certain areas of the retina from albino rats there was a suggestion that the calretinin-immunoreactivity was affected. This was never seen in the retinas of the pigmented animals. The GABA-immunoreactivity in the retina of both albino and pigmented rats appeared to be unaffected by ischaemia/reperfusion. The data presented show that retinas from albino rats are more susceptible to ischaemia/reperfusion than retinas from pigmented animals. The results also show that reduction of the b-wave of the ERG and changes in the nature of the ChAT immunoreactivity represent sensitive markers to detect the effect of ischaemia/reperfusion to the retina.

The effect of an intravitreal injection of NMDA on the expression of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells was investigated in rats. Forty-eight hours after intravitreal injection of NMDA retinal ganglion cell BDNF immunoreactivity was practically obliterated, as was the choline acetyltransferase (ChAT) immunoreactivity associated with a subset of amacrine cells. However, 2h following treatment with NMDA the BDNF immunoreactivity and BDNF mRNA associated with the ganglion cells was enhanced while the amacrine cell ChAT immunoreactivity was clearly reduced and the levels of mRNA coding for rhodopsin and Thy-1 did not change. However, 4h after NMDA injection the increase in BDNF mRNA was now no longer apparent. The results show that synthesis of BDNF is increased in the ganglion cells immediately following an insult by NMDA. It is suggested that this is a natural protective mechanism of rat retinal ganglion cells.

The aim of the study was to determine whether memantine could slow down the changes seen in the rabbit and rat retina following ischemia/reperfusion. A "suction cup procedure," which raises the intraocular pressure, was used to give an ischemic insult to the rabbit retina. The electroretinogram was recorded before ischemia and after 2 days of reperfusion. Memantine or saline (10 microl) was injected into the eye before ischemia. Immunohistochemistry was used to study the effect of ischemia/reperfusion on the GABA, ChAT, and alphaPKC immunoreactivities. Ischemia/reperfusion injury to the rat retina was induced by raising the intraocular pressure above the systolic blood pressure for 60 min, followed by reperfusion of 3-14 days. Memantine (5 mg/kg) or saline was injected i.p. at the onset of ischemia or reperfusion. Immunohistochemistry was used to study the effect of ischemia/reperfusion on the ChAT, alphaPKC, and Thy-1 immunoreactivities. In addition, morphometric analysis was carried out to determine the effects of ischemia/reperfusion on the thickness of the retina. Ischemia for 75 min caused a change in the nature of the normal GABA and ChAT immunoreactivities in the rabbit retina and a reduction in the b-wave of the electroretinogram. When memantine was injected into the vitreous humour at the onset of an ischemic insult, the changes in the GABA and ChAT immunoreactivities were reduced and the recovery of the reduced b-wave of the electroretinogram after 2 days reperfusion was enhanced significantly. Ischemia for 60 min followed by 3 days reperfusion showed a clear change in ChAT immunoreactivity in the rat retina. The Thy-1 immunoreactivity was only clearly altered after a reperfusion period of 7 days. Moreover, a measurable change in the thickness of the inner retinal layers was detected after 14 days of reperfusion. When given at the onset of ischemia, memantine counteracted the effect of ischemia/reperfusion to varying degrees. However, when memantine was given at the onset of the reperfusion this was not the case. The combined data show that a single injection of memantine given i.p. or intravitreally will protect the retina from a subsequent ischemic insult.

Serotonin is thought to play a physiological role in various tissues of the rabbit eye, yet little is known about the relative distribution of the different serotonin receptors. Demonstration of the receptor subtypes present in the various ocular tissues is essential in order to understand the function of serotonin in the eye. Using a combination of in situ hybridization histochemistry, in vitro receptor autoradiography and polymerase chain reaction studies, we have explored the distribution of the 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in the rabbit eye. As these receptors have not been sequenced in the rabbit, we initially established the suitability of the oligonucleotide probes by analysis of brain tissue. The distributions of 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptor messenger RNAs in rabbit brain correlated well with those in other species, confirming the specificity of the probes for detection of the messenger RNAs in rabbit tissues. In the eye, the expression of 5-hydroxytryptamine1A receptors appears to be restricted to the epithelial cell layer of the ciliary processes, although very low levels may appear in the retina. In contrast, the expression of 5-hydroxytryptamine7 receptor messenger RNA is more widespread with positive signals evident in the ciliary processes, retina and iris. The results confirm the existence of 5-hydroxytryptamine1A receptors in the ciliary body and their localization in the ciliary epithelium supports the hypothesis that they are involved in the secretion of aqueous humour. Unexpectedly, there was little evidence to support the idea that 5-hydroxytryptamine1A receptors are present in the retina and iris sphincter. However, the subsequent finding of 5-hydroxytryptamine7 receptor messenger RNA in the retina and iris may explain the apparent absence of 5-hydroxytryptamine1A receptors in these tissues. The presence of both 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in the ciliary processes may account for the complex intraocular pressure response of the rabbit to serotonin.

1. Neurotrophins are molecules that regulate the survival, development and maintenance of specific functions in different populations of nerve cells. 2. In the present work, we studied the localization, at the cellular level, of the different neurotrophins and their receptors within the rat retina in control and after ischemia-reperfusion of the retina. We found variations in the localization of some of these molecules depending on the reperfusion time of the retina after the ischemic lesion. 3. Thus it is suggested that the changes in the distribution and concentration of neurotrophins and their receptors caused by ischemia are protective reactions related to neuronal damage and synaptic reorganization.

1. Endogenous zinc is localized mainly in the retinal photoreceptors and retinal pigment epithelial cells in the mammalian retina. No other types of retinal neurons contain large amounts of zinc. 2. Low concentrations of exogenous zinc, like the N-methyl-D-aspartate (NMDA) antagonist MK-801, counteract the NMDA-induced changes in the gamma-aminobutyric acid (GABA) immunoreactivity in the rabbit retina. However, greater concentrations of zinc exacerbate the effects of NMDA and ischemic-like insults (lack of glucose and oxygen) on GABA immunoreactivity. The data suggest that low concentrations of zinc are neuroprotective, but higher concentrations of zinc have a negative effect. 3. When low concentrations of zinc are present during ischemic-like insults to the retina, the GABA immunoreactivity is localized to the Müller cells, suggesting that the metabolism of GABA in the Müller glial cells is prevented. 4. Ascorbate/iron-induced generation of reactive oxygen species (ROS) in the retina is prevented by deferoxamine but not by zinc. High concentrations of zinc potentiate the ascorbate/iron induced formation of ROS.

1. Flupirtine (Katadolon) is a member of a class of triaminopyridines and is used as a nonopioid analgesic agent with muscle relaxant properties. 2. In situ experiments have revealed that flupirtine protects against ischemic-induced insults to the retina and brain. 3. Data derived from in vitro and in vivo studies suggest that flupirtine functions as a weak N-methyl-D-aspartate (NMDA) antagonist with little evidence that it acts on AMPA-kainate type glutamate receptors. 4. No evidence could be found from binding studies to suggest that flupirtine has an affinity for any of the characterized binding sites associated with the NMDA receptor. 5. Studies on cultured cortical neurons show that the NMDA-induced influx of 45Ca2+ is more readily decreased by flupirtine when a reducing agent (dithiothreitol) is present. However, when N'-ethylmaleimide, which is thought to alkylate the NMDA receptor redox site, is present, no obvious effect on the NMDA-induced influx of 45Ca2+ is produced by flupirtine. 6. Flupirtine is also known to counteract the production of reactive oxygen species caused by ascorbate/iron as well as to prevent apoptosis in cells lacking NMDA receptors induced by oxidative stress. 7. To explain all the experimental data, it is suggested that flupirtine affects the redox state/pH/electrons in the cell. The specific way by which flupirtine antagonizes the NMDA receptor might be by an action on the known redox site of the receptor.

The present experiments were carried out on three types of neurone in primary rabbit retinal cultures. One cell-type, bipolar neurones, have glutamate APB-type metabotropic receptors and can be identified by the presence of thetaPKC-immunoreactivity. The other two cell-types are primarily amacrine cells and can be 'stained' for the localisation of GABA immunoreactivity or for serotonin taken up from the medium. Most of the serotonin-accumulating and GABA-containing neurones contain glutamate kainate-type receptors. Exposure of the cultures to treatment of kainate (50 microM) or experimental ischaemia (8 h followed by 16 h reoxygenation) produced essentially similar findings. The serotonin-accumulating and GABA cells were affected as they were drastically reduced in numbers while the numbers of thetaPKC-containing cells were unaffected. Inclusion of the kainate/AMPA antagonist CNQX (100 microM) or melatonin (100 microM) to the medium during kainate or ischaemia treatments largely prevented the detrimental influences on the serotonin-accumulating and GABA cells. It is concluded that during experimental ischaemia excessive glutamate is released to influence cells which contain kainate and APB-type receptors. However, only the neurones containing the kainate receptors are negatively affected with the generation of free radicals. Melatonin or CNQX protects against this effect by scavenging free radicals or acting at the receptor level, respectively.

The protein kinase C (PKC) family of serine/threonine kinase isoenzymes are universally expressed in vertebrate tissues where they control vital cellular functioning. PKC comprises twelve currently identified mammalian isoenzymes, described in three distinct groups according to their need for different effector stimulation. Immunological localisation studies in various vertebrate retinas have indicated the presence, so far, of eight of the PKC subspecies, each with a unique cellular distribution in this tissue. Use of these immunological probing techniques with antibodies raised to the individual PKC family members by immunohistochemistry and western blotting, along with biochemical tools such as the potent activators, the tumour-promoting phorbol esters can hopefully lead to elucidation of the roles of these enzymes in the neural retina. Research work to date has pinpointed a number of roles for PKC in this tissue including control of dopamine release, modulation of glutamate receptor function (probably by a process of direct receptor phosphorylation), phosphorylatory modulation of GABAC-receptor function, an involvement in the retinal ischaemic cascade process (the relevance of which is unknown as yet), involvement in control of cytoskeletal interactions by cytoskeletal element-kinase action and feedback control of enzymes involved in the process of inositol phosphate signalling. PKC has been shown to have an important regulatory role in the process of phototransduction: many of the enzymes and proteins making up the phototransduction cascade act as in vitro and in vivo substrates for PKC-dependent phosphorylation and can have their normal function modified in this way. Also, PKC has been implicated in the control of spinule formation in the retina, a process involved in retinal synaptic plasticity and functioning. All of this work has been described, herein. Collation and utilisation of knowledge of all of the work described here may help us to determine the exact roles for individual isoenzymes in the retina. This in turn may help us to understand and further to prevent pathological conditions leading to inappropriate retinal functioning and possible blindness. Furthermore, understanding the roles of PKC in the neural retina may lead us to vital clues in the understanding of the functioning of this important group of enzymes in the nervous system as a whole and eventually to the prevention of many major neuropathological disorders.

Raising the rat's intraocular pressure above the systolic blood pressure for 60 min followed by a reperfusion of 7-10 days caused an ischaemic insult to the retina. The b-wave of the electroretinogram was abolished and the retinal thickness was much reduced, the greatest influence being associated with the inner retinal layers. No obvious histological damage was apparent at the light microscopy level. Immunocytochemistry, however, revealed a clear change in the nature of certain antigens associated with specific cell-types. Thy-1 antigen located to ganglion cell membranes was much reduced, suggesting that the ganglion cells are affected by ischaemia. Calretinin-immunoreactivity associated with amacrine cells is drastically reduced by ischaemia. In contrast, Ret-P1, located to the outer segments of the photoreceptors is unaffected by ischaemia. Ischaemia also caused GFAP-immunoreactivity to be expressed in the Müller cells, which is normally only associated with astrocytes in the ganglion/nerve fibre layer. Injection of a mixture of CNQX and MK-801, kainate and NMDA receptor antagonists, respectively, into the eye just before ischaemia failed to reverse the changes induced by the insult. However, analysis 3 days after reperfusion revealed that when the ischaemic insult was reduced to 45, rather than 60 min, the changes in the calretinin-immunoreactivity were reversed. The results show that immunocytochemistry provides a powerful way of following biochemical changes associated with specific cell types caused by ischaemia.

Purpose: The aim of this study was to determine wether flupirtine can slow down the changes seen in the rabbit retina after ischemia--reperfusion., Methods: A suction-cup procedure, which raises intraocular pressure, was used to give an ischemic insult to the rabbit retina. Electroretinograms were recorded before ischemia and at different periods after ischemia. In some instances, flupirtine was injected into the eye before ischemia. Immunohistochemistry was used to study the effect of ischemia-reperfusion on the gamma-aminobutyric acid (GABA) immunoreactivity and uptake of serotonin by the retina. The effect of flupirtine and ischemia on retinal adenosine triphosphate (ATP) levels were determined in in vivo and in vitro experiments., Results: Ischemia for 75 minutes causes a change in the nature of normal GABA immunoreactivity and reduction in the b-wave of the electroretinogram. When flupirtine is injected into the vitreous humor at the onset of ischemic insult, the changes in GABA immunoreactivity are reduced and the recovery of the reduced b-wave of the electroretinogram after defined reperfusion times is enhanced significantly. Rat retinas incubated in vitro in physiological solution containing flupirtine caused a significant rise in the tissues' ATP content compared with control samples. However, incubation of the tissue in physiological solution saturated with nitrogen caused a drop in retinal ATP levels. Addition of flupirtine prevented this decrease from taking place. Serotonin injected into the vitreous humor of the rabbit eye is taken up by certain amacrine cells. The amount of serotonin taken up is reduced greatly in retinas, as judged by immunohistochemistry when tissues are subjected to ischemia. Because the ischemia was shown to cause a drop in the tissue ATP level, it is concluded that this is the cause of the reduced uptake of exogenous serotonin. Injection of flupirtine into the vitreous humor during ischemia enhanced the uptake of serotonin., Conclusions: Combined data show that flupirtine is a neuroprotective agent in retinal ischemia and that one mode of its mechanism of action is to influence ATP levels. Flupirtine may lower the activity of NMDA receptors, thus causing ATP levels to be less affected in the presence of the drug as a secondary effect.

The localization and immunochemical identification of the novel protein kinase C theta (nPKC theta) and the atypical protein kinase C lambda (aPKC lambda) isoforms in retinas of different species were analyzed by immunohistochemistry and SDS-PAGE/Western blotting. nPKC theta immunoreactivity is associated with bipolar cells of mammalian (rabbit, rat and guinea pig) retinas but not the non-mammalian goldfish retina which has a lower concentration of nPKC theta. However, SDS-PAGE and Western blotting data indicate the antigen recognized by the nPKC theta monoclonal antibody in the retina is of a lower molecular weight than that expected for nPKC theta. This would suggest nPKC theta is more susceptible to degradation/breakdown than other PKC isoforms found in the retina or that the nPKC theta antibody may be recognizing an unknown retinal antigen. A comparison of nPKC theta and cPKC alpha immunoreactivities in bipolar cells shows unique distributions exist for the two isoforms. nPKC theta is present in the developing retina at an earlier stage than cPKC alpha. The typical 'transport' of cPKC alpha toward axonal terminals by phorbol-12,13-dibutyrate does not occur for nPKC theta yet both are translocated from the cytosolic to membrane compartments. The inner plexiform layer and the inner nuclear layer (putative horizontal cells) of all species examined (rabbit, rat, guinea pig and goldfish) exhibited positive immunoreactivity for aPKC lambda as confirmed by SDS-PAGE/Western blotting.

Pressure-induced ocular ischaemia followed by 25-28 hr of reperfusion to the rabbit retina drastically reduces or eliminates the b-wave of the electroretinogram and results in all the GABA from the amacrine cells being released, as judged by immunohistochemistry. Some of these GABA cells have the capacity to take-up exogenous serotonin and these GABA/serotonin cells have kainate/AMPA receptors. Previous studies have shown that an ischaemic insult causes these receptors to be stimulated to produce a release of the cells' GABA. The majority of the GABA/serotonin cells are also incapable of taking-up exogenous serotonin after ischaemia, which suggests that they are irreversibly damaged. However, there was still a minority of the cells which accumulated serotonin, which shows that neurones containing kainate/AMPA receptors are not irreversibly damaged at the same rate by ischaemia. The "staining" patterns for GABAA-receptor and GABA immunoreactivities in the rabbit retina are very similar and following ischaemia the GABAA-receptor immunoreactivity was reduced in intensity and became patchy in nature. It is not known whether this result reflects a down-regulation of the GABAA-receptors caused by the released GABA or a destruction of cells containing the GABAA-receptors. The ischaemic conditions used caused patchy, irregular and inconsistent signs of histological damage to the retina, even in areas of similar eccentricity, suggesting this parameter should be used with caution when judging the severity of an ischaemic insult. alpha-Protein kinase C (alpha PKC) present in the on-bipolar cells which have glutamate metabotropic APB receptors is both reduced or down-regulated and translocated by ischaemia. This is also the case for delta PKC which is absent from the on-bipolar cells. These data were established by a combination of immunohistochemistry and electrophoresis/blotting experiments. Enzyme analysis also showed that all PKC calcium-dependent and -independent isoenzymes, are translocated and reduced by ischaemia making it difficult to judge whether PKC inhibitors may be appropriate anti-ischaemic agents.

Purpose: To compare the effects of glutamate agonists and different types of ischemic insult on choline acetyltransferase (ChAT) immunoreactivity in the rat retina., Methods: Rat retinas were exposed to different glutamate agonists in vivo or in vitro for specific periods of time, and the retinas were then fixed and processed for the localization of ChAT immunoreactivity. In other experiments, rats were administered an ischemic insult either by ligaturing the carotids (two-vessel occlusion [2-VO] procedure), cannulating the anterior chamber, and raising the intraocular pressure (high intraocular pressure [HIOP] procedure) or placing a ligature around the optic nerve sufficiently tightly to prevent blood flow through the central retinal artery (vascular ligation [VL] procedure). The electroretinogram was recorded, and, after a specific period of time, reperfusion was allowed to occur. Thirty to 36 hours after reperfusion, the retinas were dissected and processed for the localization of ChAT, as well as for parvalbumin, Thy-1, and alpha PKC immunoreactivities., Results: Of the glutamate agonists tested, only kainate reduced ChAT immunoreactivity significantly in vivo and in vitro. This effect of kainate could be counteracted by the antagonist CNQX (6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione). The ChAT immunoreactivity was unaffected in retinas in which ischemia was induced by the 2-VO procedure. In contrast, ChAT immunoreactivity was obliterated in retinas in which the HIOP was used and drastically reduced when the VL procedure was used. Interestingly, neither alpha PKC nor Thy-1 immunoreactivities were affected in retinas subjected to HIOP or VL methods. However, parvalbumin immunoreactivity was reduced in the HIOP model but only slightly altered in the VL model., Conclusions: The current results suggest that kainate receptors are associated with the cholinergic retinal neurones in the rat retina. Activation of these receptors by kainate causes a reduction in the neurones' ChAT content. This effect can be mimicked by subjecting the retina to a sufficiently harsh ischemic insult, as occurs in the VL and HIOP procedures. When the ischemic insult is mild, as in the 2-VO procedure, no obvious change in ChAT immunoreactivity is apparent. The HIOP procedure for inducing an ischemic insult was found to be the most severe of the three procedures used, because ChAT immunoreactivity was obliterated and clear changes in the parvalbumin immunoreactivity also were recorded. Interestingly, neither the HIOP nor the VL procedures caused a change in the Thy-1 and alpha PKC immunoreactivities.

The purpose of this study was to investigate the effect of kainate on protein kinase C (PKC), gamma-aminobutyrate (GABA) and serotonin uptake in the rabbit retina. Kainate when injected into the vitreous humour produces a change in the GABA immunoreactivity within 6 hours. After 3 days, remnants of the normal GABA immunoreactivity still persist and additionally astrocyte and microglia-like elements "stain" positively for GABA. After 7 days exposure to kainate none of the normal GABA immunoreactivity is apparent, instead a number of round-shaped elements which may be reactive astrocytes and/or microglia stain positively for GABA. During these stages kainate does not affect the alpha PKC immunoreactivity associated with the on-bipolar cells. Six hours following kainate treatment the ability of certain GABA amacrine cells to take up exogenous serotonin is unaffected. After three days only a few of these cells can still take up exogenous serotonin and then not avidly. After seven days the GABA/serotonin amacrine cells cannot take up exogenous serotonin suggesting that all of these neurons are irreversibly damaged. One hour after treatment with kainate both calcium-dependent and -independent PKC species are translocated from the cytosolic to membrane compartments. After 5 hours and 7 days there was also evidence from the enzyme assay experiments that kainate caused the calcium-dependent and -independent PKC enzymes to be translocated but because the total enzyme activity was reduced due perhaps to down-regulation of the enzyme this was difficult to assess precisely.(ABSTRACT TRUNCATED AT 250 WORDS)

When rabbit retinas are exposed in vitro to specific excitatory amino acid receptor agonists certain GABAergic amacrine cells are activated to cause a release of GABA. The GABA that is not released can be detected by immunohistochemistry. Exposure of tissues to kainate or NMDA each caused a characteristic change in the GABA immunoreactivity. CNQX antagonised the kainate effect specifically while MK-801 counteracted the influence of NMDA. The effect produced by kainate was mimicked by domoic acid while the influence of homocysteic acid was identical with NMDA. Flupirtine alone did not influence the nature of the GABA immunoreactivity and so did not act as a kainate or NMDA agonist. However, flupirtine counteracted the influence produced by NMDA and homocysteic acid but had no effect on the kainate and domoic acid responses. Thus in this system flupirtine acts as an NMDA antagonist.

The aim of the described experiments was to use immunohistochemistry to visualize the release of GABA from specific retinal amacrine cells following ischaemia and to establish the involvement of defined glutamatergic receptors. In initial experiments, rabbit retinas were exposed in vitro to excitatory amino acid agonists alone or in combination with a putative antagonist, or in physiological solution lacking oxygen and glucose, or in solution containing potassium cyanide for 45 min at 37 degrees C. The nature of the GABA immunoreactivity was then examined by immunohistochemistry. In other in vitro experiments, retinas were first allowed to accumulate exogenous serotonin before exposing the tissues to the combinations as described. These tissues were then processed immunohistochemically for the localization of serotonin. In yet other experiments, the intraocular pressure of a rabbit's eye was raised to about 110 mmHg for 60 min and a reperfusion time of 45 min allowed before dissecting the retina and processing for the localization of GABA immunoreactivity. The other eye served as a control. Of the excitatory amino acid agonists tested, only N-methyl-D-aspartate, kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused a change in the GABA immunoreactivity. The N-methyl-D-aspartate effect was specifically antagonized by dizocilpine maleate, dextromethorphan and memantine, and was characterized by a reduction in the number of GABA-immunoreactive perikarya. The GABA "staining" in the inner plexiform layer also appeared as four clear bands. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-induced effects were both antagonized by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione and partially by kynurenic acid at the concentrations used. Here, the amount of GABA-positive perikarya was greatly reduced and three immunoreactive bands appeared in the inner plexiform layer. However, for low concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid four GABA-immunoreactive bands could be identified in the inner plexiform layer. The normal GABA immunoreactivity of the inner plexiform layer also appeared to be in defined bands in retinas which received an ischaemic insult either by reducing the availability of glucose and oxygen, exposing the tissue to potassium cyanide or raising the intraocular pressure of an eye. In these cases the number of GABA-positive perikarya was also reduced. Only alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate of the excitatory amino acid agonists tested caused a release of serotonin and this process was antagonized by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione and partially by kynurenic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

The localisation and immunochemical identification of three different forms of calcium-independent protein kinase C (PKC-epsilon, PKC-delta and PKC-zeta) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE/Western blotting, respectively. More than one component of different molecular weights reacted with the polyclonal antibodies in all retinal samples though in all instances a component of molecular weight corresponding to the individual PKCs was recognised and could be eliminated or reduced by preincubating the primary antibodies with the peptides used to generate the antibodies. PKC-zeta immunoreactivity was exclusively associated with the inner segments of the photoreceptors in both mammalian (guinea-pig, rabbit, rat) and non-mammalian (goldfish, chick) retinas. PKC-epsilon immunoreactivity is present in bipolar cells, particularly in their terminals of mammalian and goldfish retinas. In the chick retina immunoreactivity for this enzyme and for PKC-delta was with the inner segments of the photoreceptors. The Müller cells in mammalian retinas and a sub-population of ganglion cells in the goldfish retina exhibited positive immunoreactivity for PKC-delta. The immunoreactivities for all the PKC isoenzymes were eliminated or drastically reduced when the primary antibodies were first preincubated with the peptides used to generate the antibodies.

Retinal ganglion cells, cone photoreceptor cells, and horizontal cells arise earlier in ontogenesis than the other retinal cell types. Although during the first postnatal week of life much cell proliferation occurs in the rabbit retina, 3H-thymidine labeling shows that these particular neurons are already postmitotic in neonatal animals. We show here that, in the adult retina, these early-born neurons express antigens as neuron-specific enolase, HNK-1 epitope of N-CAM, and PGP 9.5 antigen, which are not expressed by the neurons later born. It is concluded that the mammalian retina contains two genotypically different sets of neuronal cells which can be distinguished by immunocytochemistry.

The distribution of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase was examined histochemically in the retina, iris, ciliary processes, cornea and conjunctiva of the rabbit eye. The epithelial cells of the ciliary process, iris, conjunctiva and, to a lesser extent, the cornea all showed intense staining. In the retina, staining for NADPH diaphorase was intense in the inner segments of the photoreceptors and a sparsely distributed population of amacrine cells. In addition, another population of amacrine cells, some presumed ganglion cells as well as a number of horizontal cells, stained less intensely for the enzyme. The retina, ciliary processes and, as a comparison, the cerebellum of the rabbit all contain nitric oxide synthetase (NOS) activity, as each tissue can metabolize citrulline from arginine. This process is Ca2+ dependent and is reduced by the NOS inhibitor, NG-monomethyl-L-arginine. The presence of NOS activity in the ciliary processes and the localization of NADPH diaphorase in the ciliary epithelial cells are of significance as they suggest that the ciliary epithelial cells may contain NOS which would imply a role for nitric oxide in aqueous humour production.