Fabrício Simão

Protective effect of resveratrol against oxygen-glucose deprivation in organotypic hippocampal slice cultures : Involvement of PI3-K pathway

Here we investigated the neuroprotective effect of resveratrol in an in vitro model of ischemia. We used organotypic hippocampal cultures exposed to oxygen-glucose deprivation (OGD). In OGD-vehicle exposed cultures, about 46% of the hippocampus was labeled with PI, indicating a robust percentage of cell death. When cultures were treated with resveratrol 10, 25 and 50 microM, the cell death was reduced to 22, 20 and 13% respectively. To elucidate a possible mechanism by which resveratrol exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase (PI3-k) pathway using LY294002 (5 microM) and mitogen-activated protein kinase (MAPK) using PD98059 (20 microM). The resveratrol (50 microM) neuroprotection was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that resveratrol 50 microM induced the phosphorylation/activation of Akt and extracellular signal-regulated kinase-1 and -2 (ERK1/2) and the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that PI3-k/Akt pathway are involved in the neuroprotective effect of resveratrol.

Resveratrol prevents oxidative stress and inhibition of Na+K+-ATPase activity induced by transient global cerebral ischemia in rats

Increased oxidative stress and energy metabolism deficit have been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In this study, we investigated the oxidative mechanisms underlying the neuroprotective effects of resveratrol, a potent polyphenol antioxidant found in grapes, on structural and biochemical abnormalities in rats subjected to global cerebral ischemia. Experimental model of transient global cerebral ischemia was induced in Wistar rats by the four vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl and fluoro jade C stained indicated extensive neuronal death at 7 days after I/R. These findings were preceded by a rapid increase in the generation of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation, as well as by a decrease in Na(+)K(+)-ATPase activity and disrupted antioxidant defenses (enzymatic and non-enzymatic) in hippocampus and cortex. Administrating resveratrol 7 days prior to ischemia by intraperitoneal injections (30 mg/kg) significantly attenuated neuronal death in both studied structures, as well as decreased the generation of ROS, lipid peroxidation and NO content. Furthermore, resveratrol brought antioxidant and Na(+)K(+)-ATPase activity in cortex and hippocampus back to normal levels. These results support that resveratrol could be used as a preventive, or therapeutic, agent in global cerebral ischemia and suggest that scavenging of ROS contributes, at least in part, to resveratrol-induced neuroprotection.

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.

Effect of treadmill exercise on cell damage in rat hippocampal slices submitted to oxygen and glucose deprivation.

We have recently demonstrated that high intensity training exercise exacerbates brain damage, while a moderate intensity (2 weeks of 20 min/day of treadmill training) reduces the injury caused by in vitro ischemia, oxygen and glucose deprivation (OGD), to hippocampal slices from Wistar rats. In the present paper, the effect of different running programs on severity of ischemic OGD lesion was examined, by the evaluation of three protocols designed to simulate exercise conditions common to humans: one or three 20-min sessions per week, during 12 weeks (moderate intensity), and two 20-min daily sessions for 3 weeks. OGD caused an increase of lactate dehydrogenase (LDH) release into the incubation media, a marker of tissue necrosis, and a decline of cell viability, as assessed by the decrease of mitochondrial dehydrogenase activity (MTT method). Moderate exercise, three times a week during 12-week treadmill training, decreased LDH release after OGD, while one weekly session and 3 weeks of two daily sessions did not affect OGD-induced LDH released. No exercise protocol evaluated altered MTT reduction. Our data support the hypothesis that moderate intensity exercise reduces hippocampal susceptibility to in vitro ischemia.

Pro-angiogenic effects of resveratrol in brain endothelial cells: nitric oxide-mediated regulation of vascular endothelial growth factor and metalloproteinases

Resveratrol may be a powerful way of protecting the brain against a wide variety of stress and injury. Recently, it has been proposed that resveratrol not only reduces brain injury but also promotes recovery after stroke. But the underlying mechanisms are unclear. Here, we tested the hypothesis that resveratrol promotes angiogenesis in cerebral endothelial cells and dissected the signaling pathways involved. Treatment of cerebral endothelial cells with resveratrol promoted proliferation, migration, and tube formation in Matrigel assays. Consistent with these pro-angiogenic responses, resveratrol altered endothelial morphology resulting in cytoskeletal rearrangements of β-catenin and VE-cadherin. These effects of resveratrol were accompanied by activation of phosphoinositide 3 kinase (PI3-K)/Akt and Mitogen-Activated Protein Kinase (MAPK)/ERK signaling pathways that led to endothelial nitric oxide synthase upregulation and increased nitric oxide (NO) levels. Subsequently, elevated NO signaling increased vascular endothelial growth factor and matrix metalloproteinase levels. Sequential blockade of these signaling steps prevented resveratrol-induced angiogenesis in cerebral endothelial cells. These findings provide a mechanistic basis for the potential use of resveratrol as a candidate therapy to promote angiogenesis and neurovascular recovery after stroke.

Resveratrol preconditioning modulates inflammatory response in the rat hippocampus following global cerebral ischemia.

Considerable evidence has been accumulated to suggests that blocking the inflammatory reaction promotes neuroprotection and shows therapeutic potential for clinical treatment of ischemic brain injury. Consequently, anti-inflammatory therapies are being explored for prevention and treatment of these diseases. Induction of brain tolerance against ischemia by pretreatment with resveratrol has been found to influence expression of different molecules. It remains unclear, however, whether and how resveratrol preconditioning changes expression of inflammatory mediators after subsequent global cerebral ischemia/reperfusion (I/R). Therefore, we investigated the effect of resveratrol pretreatment on NF-κB inflammatory cascade, COX-2, iNOS and JNK levels in experimental I/R. Adult male rats were subjected to 10 min of four-vessel occlusion and sacrificed at selected post-ischemic time points. Resveratrol (30 mg/kg) pretreatment was injected intraperitoneally 7 days prior to I/R induction. We found that resveratrol treatment before insult remarkably reduced astroglial and microglial activation at 7 days after I/R. It greatly attenuated I/R-induced NF-κB and JNK activation with decreased COX-2 and iNOS production. In conclusion, the neuroprotection of resveratrol preconditioning may be due in part to the suppression of the inflammatory response via regulation of NF-κB, COX-2 and iNOS induced by I/R. JNK was also suggested to play a protective role through in neuroprotection of resveratrol, which may also be contributing to reduction in neuroinflammation. The study adds to a growing literature that resveratrol can have important anti-inflammatory actions in the brain.

Dietary omega-3 fatty acids attenuate cellular damage after a hippocampal ischemic insult in adult rats ☆

The role of omega-3 polyunsaturated fatty acids (3PUFAs) on brain function is increasingly demonstrated. Here, the effect of dietary deprivation of essential 3PUFAs on some parameters related to neuroprotection was investigated. Rats were fed with two different diets: omega-3 diet and omega-3-deprived diet. To assess the influence of 3PUFAs on brain responses to ischemic insult, hippocampal slices were subjected to an oxygen and glucose deprivation (OGD) model of in vitro ischemia. The omega-3-deprived group showed higher cell damage and stronger decrease in the [(3)H]glutamate uptake after OGD. Moreover, omega-3 deprivation influenced antiapoptotic cell response after OGD, affecting GSK-3beta and ERK1/2, but not Akt, phosphorylation. Taken together, these results suggest that 3PUFAs are important for cell protection after ischemia and also seem to play an important role in the activation of antiapoptotic signaling pathways.

Plasma kallikrein mediates brain hemorrhage and edema caused by tissue plasminogen activator therapy in mice after stroke

Thrombolytic therapy using tissue plasminogen activator (tPA) in acute stroke is associated with increased risks of cerebral hemorrhagic transformation and angioedema. Although plasma kallikrein (PKal) has been implicated in contributing to both hematoma expansion and thrombosis in stroke, its role in the complications associated with the therapeutic use of tPA in stroke is not yet available. We investigated the effects of tPA on plasma prekallikrein (PPK) activation and the role of PKal on cerebral outcomes in a murine thrombotic stroke model treated with tPA. We show that tPA increases PKal activity in vitro in both murine and human plasma, via a factor XII (FXII)-dependent mechanism. Intravenous administration of tPA increased circulating PKal activity in mice. In mice with thrombotic occlusion of the middle cerebral artery, tPA administration increased brain hemorrhage transformation, infarct volume, and edema. These adverse effects of tPA were ameliorated in PPK (Klkb1)-deficient and FXII-deficient mice and in wild-type (WT) mice pretreated with a PKal inhibitor prior to tPA. tPA-induced brain hemisphere reperfusion after photothrombolic middle cerebral artery occlusion was increased in Klkb1-/- mice compared with WT mice. In addition, PKal inhibition reduced matrix metalloproteinase-9 activity in brain following stroke and tPA therapy. These data demonstrate that tPA activates PPK in plasma and PKal inhibition reduces cerebral complications associated with tPA-mediated thrombolysis in stroke.

Effects of skilled and unskilled training on functional recovery and brain plasticity after focal ischemia in adult rats.

Stroke is a leading cause of morbidity and mortality worldwide. Recovery of motor function after stroke can be modified by post-injury experience, but most of surviving patients exhibit persistence of the motor dysfunctions even after rehabilitative therapy. In this study we investigated if skilled and unskilled training induce different motor recovery and brain plasticity after experimental focal ischemia. We tested this hypothesis by evaluating the motor skill relearning and the immunocontent of Synapsin-I, PSD-95 and GFAP (pre and post-synaptic elements, as well as surrounding astroglia) in sensorimotor cortex of both hemispheres 6 weeks after endothelin-1-induced focal brain ischemia in rats. Synapsin-I and PSD-95 levels were increased by skilled training in ischemic sensorimotor cortex. The content of GFAP was augmented as a result of focal brain ischemia in ischemic sensorimotor cortex and that was not modified by rehabilitation training. Unexpectedly, animals remained permanently impaired at the end of motor/functional evaluations. Significant modifications in protein expression were not observed in undamaged sensorimotor cortex. We conclude that skilled motor activity can positively affect brain plasticity after focal ischemia despite of no functional improvement in conditions here tested.

Hyperhomocysteinemia reduces glutamate uptake in parietal cortex of rats.

In the present study we evaluated the effect of acute and chronic homocysteine administrations on glutamate uptake in parietal cortex of rats. The immunocontent of glial glutamate transporter (GLAST) and sodium‐dependent glutamate/aspartate transporter (GLT‐1) in the same cerebral structure was also investigated. For acute treatment, neonate or young rats received a single injection of homocysteine or saline (control) and were sacrificed 1, 8, 12 h, 7 or 30 days later. For chronic treatment, homocysteine was administered to rats twice a day at 8 h interval from their 6th to their 28th days old; controls and treated rats were sacrificed 12 h, 1, 7 or 30 days after the last injection. Results show that acute hyperhomocysteinemia caused a reduction on glutamate uptake in parietal cortex of neonate and young rats, and that 12 h after homocysteine administration the glutamate uptake returned to normal levels in young rats, but not in neonate. Chronic hyperhomocysteinemia reduced glutamate uptake, and GLAST and GLT‐1 immunocontent. According to our results, it seems reasonable to postulate that the reduction on glutamate uptake in cerebral cortex of rats caused by homocysteine may be mediated by the reduction of GLAST and GLT‐1 immunocontent, leading to increased extracellular glutamate concentrations, promoting excitotoxicity.