Blockade of microglial adenosine A2A receptor impacts inflammatory mechanisms, reduces ARPE-19 cell dysfunction and prevents photoreceptor loss in vitro.

Age-related macular degeneration (AMD) is characterized by pathological changes in the retinal pigment epithelium (RPE) and loss of photoreceptors. Growing evidence has demonstrated that reactive microglial cells trigger RPE dysfunction and loss of photoreceptors, and inflammasome pathways and complement activation contribute to AMD pathogenesis. We and others have previously shown that adenosine A2A receptor (A2AR) blockade prevents microglia-mediated neuroinflammatory processes and mediates protection to the retina. However, it is still unknown whether blocking A2AR in microglia protects against the pathological features of AMD. Herein, we show that an A2AR antagonist, SCH58261, prevents the upregulation of the expression of pro-inflammatory mediators and the alterations in the complement system triggered by an inflammatory challenge in human microglial cells. Furthermore, blockade of A2AR in microglia decreases the inflammatory response, as well as complement and inflammasome activation, in ARPE-19 cells exposed to conditioned medium of activated microglia. Finally, we also show that blocking A2AR in human microglia increases the clearance of apoptotic photoreceptors. This study opens the possibility of using selective A2AR antagonists in therapy for AMD, by modulating the interplay between microglia, RPE and photoreceptors.

Adenosine A2A receptor regulation of microglia morphological remodeling-gender bias in physiology and in a model of chronic anxiety.

Developmental risk factors, such as the exposure to stress or high levels of glucocorticoids (GCs), may contribute to the pathogenesis of anxiety disorders. The immunomodulatory role of GCs and the immunological fingerprint found in animals prenatally exposed to GCs point towards an interplay between the immune and the nervous systems in the etiology of these disorders. Microglia are immune cells of the brain, responsive to GCs and morphologically altered in stress-related disorders. These cells are regulated by adenosine A2A receptors, which are also involved in the pathophysiology of anxiety. We now compare animal behavior and microglia morphology in males and females prenatally exposed to the GC dexamethasone. We report that prenatal exposure to dexamethasone is associated with a gender-specific remodeling of microglial cell processes in the prefrontal cortex: males show a hyper-ramification and increased length whereas females exhibit a decrease in the number and in the length of microglia processes. Microglial cells re-organization responded in a gender-specific manner to the chronic treatment with a selective adenosine A2A receptor antagonist, which was able to ameliorate microglial processes alterations and anxiety behavior in males, but not in females.

The Adenosinergic System in Diabetic Retinopathy.

The neurodegenerative and inflammatory environment that is prevalent in the diabetic eye is a key player in the development and progression of diabetic retinopathy. The adenosinergic system is widely regarded as a significant modulator of neurotransmission and the inflammatory response, through the actions of the four types of adenosine receptors (A1R, A2AR, A2BR, and A3R), and thus could be revealed as a potential player in the events unfolding in the early stages of diabetic retinopathy. Herein, we review the studies that explore the impact of diabetic conditions on the retinal adenosinergic system, as well as the role of the said system in ameliorating or exacerbating those conditions. The experimental results described suggest that this system is heavily affected by diabetic conditions and that the modulation of its components could reveal potential therapeutic targets for the treatment of diabetic retinopathy, particularly in the early stages of the disease.

Obesity and brain inflammation: a focus on multiple sclerosis.

The increase in prevalence of obesity in industrialized societies is an indisputable fact. However, the apparent passive role played by adipocytes, in pathophysiological terms, has been gradually substituted by a metabolically active performance, relevant to many biochemical mechanisms that may contribute to a chronic low-grade inflammatory status, which increasingly imposes itself as a key feature of obesity. This chronic inflammatory status will have to be integrated into the complex equation of many diseases in which inflammation plays a crucial role. Multiple sclerosis (MS) is a chronic inflammatory condition typically confined to the central nervous system, and many work has been produced to find possible points of contact between the biology of this immune-mediated disease and obesity. So far, clinical data are not conclusive, but many biochemical features have been recently disclosed. Brain inflammation has been implicated in some of the mechanisms that lead to obesity, which has also been recognized as an important player in inducing some degree of immune dysfunction. In this review, we collected evidence that allows establishing bridges between obesity and MS. After considering epidemiological controversies, we will focus on possible shared mechanisms, as well as on the potential contributions that disease-modifying drugs may have on this apparent relationship of mutual interference.

Tauroursodeoxycholic acid protects retinal neural cells from cell death induced by prolonged exposure to elevated glucose.

Diabetic retinopathy is one of the most frequent causes of blindness in adults in the Western countries. Although diabetic retinopathy is considered a vascular disease, several reports demonstrate that retinal neurons are also affected, leading to vision loss. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has proven to be neuroprotective in several models of neurodegenerative diseases, including models of retinal degeneration. Since hyperglycemia is considered to play a central role in retinal cell dysfunction and degeneration, underlying the progression of diabetic retinopathy, the purpose of this study was to investigate the neuroprotective effects of TUDCA in rat retinal neurons exposed to elevated glucose concentration. We found that TUDCA markedly decreased cell death in cultured retinal neural cells induced by exposure to elevated glucose concentration. In addition, TUDCA partially prevented the release of apoptosis-inducing factor (AIF) from the mitochondria, as well as the subsequent accumulation of AIF in the nucleus. Biomarkers of oxidative stress, such as protein carbonyl groups and reactive oxygen species production, were markedly decreased after TUDCA treatment as compared to cells exposed to elevated glucose concentration alone. In conclusion, TUDCA protected retinal neural cell cultures from cell death induced by elevated glucose concentration, decreasing mito-nuclear translocation of AIF. The antioxidant properties of TUDCA might explain its cytoprotection. These findings may have relevance in the treatment of diabetic retinopathy patients.

Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate.

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 µM) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy.

Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus.

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

Contribution of TNF receptor 1 to retinal neural cell death induced by elevated glucose.

Diabetic retinopathy (DR), a leading cause of vision loss and blindness among working-age adults, holds several hallmarks of an inflammatory disease. The increase in cell death in neural retina is an early event in the diabetic retina, preceding the loss of microvascular cells. Since tumor necrosis factor-α (TNF-α) has been shown to trigger the death of perycites and endothelial cells as well as the breakdown of the blood-retinal barrier, we set out to investigate whether TNF-α acting through tumor necrosis factor receptor 1 (TNFR1), the major receptor responsible for mediating TNF-induced cell death, could also be responsible for the early neuronal cell death observed in DR. We used retinal neural cell cultures exposed to high glucose conditions, to mimic hyperglycaemia, and evaluated the contribution of TNFR1 in neural cell death. TNFR1 was found to be present to a great extent in retinal neurons and the levels of this receptor were found to be altered in cells cultured in high glucose conditions. High glucose induced an early decrease in cell viability, an increase in apoptosis and a higher immunoreactivity for the cleaved caspase-3, indicating a high glucose-induced caspase-dependent cell death. These observations were correlated with an increase in TNF-α expression. Nonetheless, inhibiting the activation of TNFR1 was sufficient to prevent the decrease in cell viability and the increase in retinal cell death by apoptosis. In conclusion, our data indicate that TNF-α acting through TNFR1 is responsible for the high glucose-induced cell death and that blocking the activity of this receptor is an adequate strategy to avoid cell loss in such conditions.

Protective effects of the dipeptidyl peptidase IV inhibitor sitagliptin in the blood-retinal barrier in a type 2 diabetes animal model.

AIM: The aim of this study was to evaluate the efficacy of sitagliptin, a dipeptidyl peptidase IV inhibitor (DPP-IV), in preventing the deleterious effects of diabetes on the blood-retinal barrier in male Zucker Diabetic Fatty (ZDF) rats. METHODS: ZDF rats at 20 weeks of age were treated with sitagliptin (10 mg/kg/day) during 6 weeks. The effect of the drug on glycaemia was assessed by evaluating glycated haemoglobin (HbA1c). The content and/or distribution of tight junction (TJ) proteins occludin and claudin-5, as well as nitrotyrosine residues, interleukin (IL)-1ß, BAX and Bcl-2 was evaluated in the retinas by western blotting and/or immunohistochemistry. Retinal cell apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The number of CD34+ cells present in peripheral circulation was assessed by flow cytometry, and endothelial progenitor cells (EPC) adhesion ability to the retinal vessels was evaluated by immunohistochemistry. RESULTS: Sitagliptin improved glycaemic control as reflected by a significant decrease in HbA1c levels by about 1.2%. Treatment with sitagliptin prevented the changes in the endothelial subcellular distribution of the TJ proteins induced by diabetes. Sitagliptin also decreased the nitrosative stress, the inflammatory state and cell death by apoptosis in diabetic retinas. Diabetic animals presented decreased levels of CD34+ cells in the peripheral circulation and decreased adhesion ability of EPC to the retinal vessels. Sitagliptin allowed a recovery of the number of CD34+ cells present in the bloodstream to levels similar to their number in controls and increased the adhesion ability of EPC to the retinal vessels. CONCLUSIONS: Sitagliptin prevented nitrosative stress, inflammation and apoptosis in retinal cells and exerted beneficial effects on the blood-retinal barrier integrity in ZDF rat retinas.

Evaluation of the impact of diabetes on retinal metabolites by NMR spectroscopy.

PURPOSE/AIM OF THE STUDY: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults in developed countries. Changes in metabolites and in metabolic pathways of the retina caused by hyperglycemia may compromise the physiology of the retina. Using nuclear magnetic resonance (NMR) spectroscopy, we aimed to investigate the effect of diabetes on the levels of intermediate metabolites in rat retinas and the metabolic pathways that could be affected. MATERIALS AND METHODS: Diabetes was induced in male Wistar rats with a single injection of streptozotocin (65 mg/Kg, i.p.). Metabolic alterations were analyzed in streptozotocin-induced diabetic rat retinas by (1)H NMR spectroscopy. Glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose. Lactate production was evaluated by (1)H NMR spectroscopy using [U-(13)C]glucose. RESULTS: Tissue levels of several metabolic intermediates were quantified, but no significant changes in the levels of most metabolites were detected, with the exceptions of glucose, significantly increased, and lactate, significantly reduced in diabetic rat retinas, as compared to age-matched controls. The cytosolic redox ratio, indirectly evaluated by lactate-to-pyruvate ratio, was significantly reduced in diabetic rat retinas, as well as glucose uptake. Parallel studies demonstrated that lactate production rates were significantly diminished, suggesting a reduction in the glycolytic flux. CONCLUSIONS: These results suggest that diabetes may significantly decrease glycolysis in the retina since higher intracellular glucose levels do not translate into higher intracellular lactate levels or into higher rates of lactate production. These changes may alter the normal functioning of the retina during diabetes and may contribute for vision loss in DR.

Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons.

A few studies have reported the existence of depletion of synaptic vesicles, and changes in neurotransmitter release and in the content of exocytotic proteins in the hippocampus of diabetic rats. Recently, we found that diabetes alters the levels of synaptic proteins in hippocampal nerve terminals. Hyperglycemia is considered the main trigger of diabetic complications, although other factors, such as low insulin levels, also contribute to diabetes-induced changes. Thus, the aim of this work was to evaluate whether long-term elevated glucose per se, which mimics prolonged hyperglycemia, induces significant changes in the content and localization of synaptic proteins involved in exocytosis in hippocampal neurons. Hippocampal cell cultures were cultured for 14 days and were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM plus 25 mM glucose), for 7 days. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. The protein levels of vesicle-associated membrane protein-2 (VAMP-2), synaptosomal-associated protein-25 (SNAP-25), syntaxin-1, synapsin-1, synaptophysin, synaptotagmin-1, rabphilin 3a, and also of vesicular glutamate and GABA transporters (VGluT-1 and VGAT), were evaluated by immunoblotting, and its localization was analyzed by immunocytochemistry. The majority of the proteins were not affected. However, elevated glucose decreased the content of SNAP-25 and increased the content of synaptotagmin-1 and VGluT-1. Moreover, there was an accumulation of syntaxin-1, synaptotagmin-1 and VGluT-1 in the cell body of some hippocampal neurons exposed to high glucose. No changes were detected in mannitol-treated cells. In conclusion, elevated glucose per se did not induce significant changes in the content of the majority of the synaptic proteins studied in hippocampal cultures, with the exception of SNAP-25, synaptotagmin-1 and VGluT-1. However, there was an accumulation of some proteins in cell bodies of hippocampal neurons exposed to elevated glucose, suggesting that the trafficking of these proteins to the synapse may be compromised. Moreover, these results also suggest that other factors, in addition to hyperglycemia, certainly contribute to alterations detected in synaptic proteins in diabetic animals.

Diabetes differentially affects the content of exocytotic proteins in hippocampal and retinal nerve terminals.

Diabetes has been associated with cognitive and memory impairments, and with alterations in color and contrast perception, suggesting that hippocampus and retina are particularly affected by this disease. A few studies have shown that diabetes differentially affects neurotransmitter release in different brain regions and in retina, and induces structural and molecular changes in nerve terminals in both hippocampus and retina. We now detailed the impact over time of diabetes (2, 4 and 8 weeks of diabetes) on a large array of exocytotic proteins in hippocampus and retina.The exocytotic proteins density was evaluated by immunoblotting in purified synaptosomes and in total extracts of hippocampus and retina from streptozotocin-induced diabetic and age-matched control animals. Diabetes affected differentially the content of synaptic proteins (VAMP-2, SNAP-25, syntaxin-1, synapsin-1 and synaptophysin) in hippocampal and retinal nerve terminals. Changes were more pronounced and persistent in hippocampal nerve terminals. In general, the alterations in retina occurred earlier, but were transitory, with the exception of synapsin-1, since its content decreased at all time points studied. The content of synaptotagmin-1 and rabphilin 3a in nerve terminals of both tissues was not affected. In total extracts, no changes were detected in the retina, whereas in hippocampus SNAP-25 and syntaxin-1 content was decreased, particularly when more drastic changes were also detected in nerve terminals. These results show that diabetes affects the content of several exocytotic proteins in hippocampus and retina, mainly at the presynaptic level, but hippocampus appears to be more severely affected. These changes might influence neurotransmission in both tissues and may underlie, at least partially, previously detected physiological changes in diabetic humans and animal models. Since diabetes differentially affects exocytotic proteins, according to tissue and insult duration, functional studies will be required to assess the physiological impairment induced by diabetes on the exocytosis in central synapses.

High glucose changes extracellular adenosine triphosphate levels in rat retinal cultures.

Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol-treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high-glucose-treated cells. It was also observed that, in retinal cells cultured under high-glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol-treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR.

Neuropeptide Y protects retinal neural cells against cell death induced by ecstasy.

Ecstasy (3,4-methylenedioxymethamphetamine; MDMA) has potent CNS stimulant effects. Besides the acute effects of MDMA, such as psychomotor activation, euphoria, decreased appetite, and hyperthermia, long-term damage of dopaminergic and serotonergic nerve terminals in multiple brain areas have also been reported. Although some studies have demonstrated that considerable amounts of MDMA reach the vitreous humor of the eye, and that serious visual consequences can result from MDMA consumption, the toxic effect of MDMA on the retina has not been completely elucidated. Neuropeptide Y (NPY) is present in the CNS, including the retina. The aim of the present study was to evaluate the effect of MDMA on rat retinal neural cell viability and investigate the involvement of 5-HT 2A-receptor (5-HT(2A)) activation. Moreover, the neuroprotective role of NPY on MDMA-induced toxicity was also investigated. MDMA induced necrosis [MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and propidium iodide assays] and apoptosis (immunoreactivity of cleaved caspase-3) in mixed cultures of retinal neural cells (neurons, macroglia and microglia), in a concentration-dependent manner. MDMA-induced toxicity was enhanced at higher temperature (40 degrees C) and was reduced by the 5HT(2A)-receptor antagonist, ketanserin (1 microM). Interestingly, necrotic and apoptotic cell death induced by MDMA was inhibited by NPY (100 nM).

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons.

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.

High glucose induces caspase-independent cell death in retinal neural cells.

Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL-positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.

Old and new drug targets in diabetic retinopathy: from biochemical changes to inflammation and neurodegeneration.

Diabetic Retinopathy (DR) is a major complication of diabetes and is a leading cause of blindness in western countries. DR has been considered a microvascular disease, and the blood-retinal barrier breakdown is a hallmark of this disease. The available treatments are scarce and not very effective. Despite the attempts to control blood glucose levels and blood pressure, many diabetic patients are affected by DR, which progresses to more severe forms of disease, where laser photocoagulation therapy is needed. DR has a huge psychological impact in patients and tremendous economic and social costs. Taking this into account, the scientific community is committed to find a treatment to DR. Understanding the cellular and molecular mechanisms underlying the pathogenesis of DR will facilitate the development of strategies to prevent, or at least to delay the progression of the disease. The involvement of the polyol pathway, advanced glycation end products, protein kinase C and oxidative stress in the pathogenesis of DR is well-documented, and several clinical trials have been conducted to test the efficacy of various drugs. More recent findings also demonstrate that DR has characteristics of chronic inflammatory disease and neurodegenerative disease, which increases the opportunity of intervention at the pharmacological level. This review presents past and recent evidences demonstrating the involvement of different molecules and processes in DR, and how different approaches and pharmacological tools have been used to prevent retinal cell dysfunction.

Role of kainate receptor activation and desensitization on the [Ca(2+)](i) changes in cultured rat hippocampal neurons.

We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the [Ca(2+)](i) caused by kainate showed cell-to-cell variability. The [Ca(2+)](i) increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe [Ca(2+)](i) changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the [Ca(2+)](i) and toxicity.

Inhibition of glutamate release by BIA 2-093 and BIA 2-024, two novel derivatives of carbamazepine, due to blockade of sodium but not calcium channels.

We investigated the mechanism(s) of action of two new putative antiepileptic drugs (AEDs), (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), by comparing their effects on the release of endogenous glutamate in hippocampal synaptosomes, with those of carbamazepine (CBZ) and oxcarbazepine (OXC). The AEDs inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) or veratridine in a concentration-dependent manner, being CBZ more potent than the other AEDs. Using conditions of stimulation (30 mM KCl), where Na(+) channels are inactivated, the AEDs did not inhibit either the Ca(2+)-dependent or -independent release of glutamate. The results indicate that BIA 2-093 and BIA 2-024 have sodium channel-blocking properties, but CBZ and OXC are more potent than the new AEDs. Moreover, the present data also indicate that Ca(2+) channels coupled to the exocytotic release of glutamate and the activity of the glutamate transporter were not affected by the AEDs.

Neurotoxic/neuroprotective profile of carbamazepine, oxcarbazepine and two new putative antiepileptic drugs, BIA 2-093 and BIA 2-024.

We investigated and compared the toxicity profile, as well as possible neuroprotective effects, of some antiepileptic drugs in cultured rat hippocampal neurons. We used two novel carbamazepine derivatives, (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b, f]azepine-5-carboxamide (BIA 2-093) and 10, 11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), and compared their effects with the established compounds carbamazepine and oxcarbazepine. The assessment of neuronal injury was made by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay, as well as by analysing morphology and nuclear chromatin condensation (propidium iodide staining), after hippocampal neurons were exposed to the drugs for 24 h. The putative antiepileptic drugs, BIA 2-093 or BIA 2-024 (at 300 microM), only slightly decreased MTT reduction, whereas carbamazepine or oxcarbazepine were much more toxic at lower concentrations. Treatment with the antiepileptic drugs caused nuclear chromatin condensation in some neurons, which is characteristic of apoptosis, and increased the activity of caspase-3-like enzymes, mainly in neurons treated with carbamazepine and oxcarbazepine. The toxic effect caused by carbamazepine was not mediated by N-methyl-D-aspartate (NMDA) or by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors. Moreover, the antiepileptic drugs failed to protect hippocampal neurons from the toxicity caused by kainate, veratridine, or ischaemia-like conditions.