Resveratrol reduces matrix metalloproteinase-2 activity induced by oxygen-glucose deprivation and reoxygenation in human cerebral microvascular endothelial cells.

The main pathophysiology in cerebral ischemia is the structural alteration in the neurovascular unit, coinciding with neurovascular matrix degradation. Among the human matrix metalloproteinases (MMPs), MMP-2 and -9, known as gelatinases, are the key enzymes for degrading type IV collagen, which is the major component of the basal membrane that surrounds the cerebral blood vessel. In the present study, we investigated the effects of resveratrol on cytotoxicity, reactive oxygen species (ROS), and gelatinases (MMP-2 and -9) in human cerebral microvascular endothelial cells exposed to 6 hours of oxygen-glucose deprivation and a subsequent 24 hours of reoxygenation with glucose (OGD/R), to mimic ischemia/reperfusion in vivo. Lactate dehydrogenase increased significantly, in comparison to that in the normoxia group. ROS was markedly increased in the OGD/R group, compared to normoxia. Correspondingly, ROS was significantly reduced with 50 µM of resveratrol. The proMMP-2 activity in the OGD/R group showed a statistically significant increase from the control cells. Resveratrol preconditioning decreased significantly the proMMP-2 in the cells exposed to OGD/R in comparison to that in the OGD/R group. Our results indicate that resveratrol regulates MMP-2 activity induced by OGD/R via its antioxidant effect, implying a possible mechanism related to the neuroprotective effect of resveratrol.

Effect of erythropoietin on oxygen-induced brain injury in the newborn rat.

The developing nervous system is sensitive to supraphysiological oxygen concentrations. Recent studies showed that exposure to hyperoxia in infant rats leads to extensive apoptotic degeneration in the cortex and white matter of the developing brain. A wide variety of experimental studies have shown that erythropoietin exerts a remarkable neuroprotection in both cell cultures and in animal models of nervous system disorders. In the present study, we investigated the effect of erythropoietin against hyperoxia-induced neurodegeneration in the developing brain. Eighteen Wistar rat pups were divided into three groups: control group, hyperoxia+saline-treated group and hyperoxia+erythropoietin-treated group. Hyperoxia groups were exposed to 80% oxygen (n=12) in a plexiglas chamber in which the oxygen concentration was monitored twice daily from birth until postnatal day 5. Hyperoxia exposure was 24h/day for 5 days. The hyperoxia+erythropoietin group received an intraperitoneal injection of recombinant human erythropoietin at a dose of 1000U/(kgday). At postnatal day 5, all animals were sacrificed. Neuronal cell death and apoptosis were evaluated. Histopathological examination showed that erythropoietin significantly diminished apoptosis in the CA1 region and dentate gyrus of hippocampus and parietal cortex in hyperoxia+erythropoietin-treated group. Regarding the safety profile of erythropoietin in premature and mature infants, this agent may be potentially beneficial in preventing hyperoxic brain injury.

Neuroprotective effect of the peptides ADNF-9 and NAP on hypoxic-ischemic brain injury in neonatal rats.

Perinatal asphyxia is an important cause of neonatal mortality and subsequent serious sequelae such as motor and cognitive deficits and seizures. Recent studies have demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. However, the effect of these peptides on the hypoxic-ischemic brain injury model is unknown. The aim of this study is to investigate the effects of the peptides ADNF-9 and NAP on neurodegeneration and cerebral nitric oxide (NO) production in a neonatal rat model of hypoxic-ischemic brain injury. Seven-day-old Wistar Albino rat pups have been used in the study (n=42). Experimental groups in the study were: sham-operated group, ADNF-9-treated hypoxia-ischemia group, NAP-treated hypoxia-ischemia group, ADNF-9+NAP-treated hypoxia-ischemia group, and vehicle-treated group. In hypoxia-ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was applied for 2.5 h. ADNF-9, NAP, and ADNF-9+NAP were injected (intraperitoneally; i.p.) as a single dose immediately after the hypoxia period. Brain nitrite levels, neuronal cell death, and apoptosis were evaluated in both hemispheres (carotid ligated or nonligated) 72 h after the hypoxic-ischemic insult. Histopathological evaluation demonstrated that ADNF-9 and NAP significantly diminished number of "apoptotic cells" in the hippocampal CA1, CA2, CA3, and gyrus dentatus regions in both hemispheres (ligated and nonligated). When compared with vehicle-treated group, combination treatment with ADNF-9+NAP did not significantly reduce "apoptotic cell death" in any of the hemispheres. ADNF-9 and NAP, when administered separately, significantly preserved the number of neurons CA1, CA2, CA3, and dentate gyrus regions of the hippocampus, when compared with vehicle-treated group. The density of the CA1, CA2, and dentate gyrus neurons was significantly higher when combination therapy with ADNF-9+NAP was used in the carotid ligated hemispheres. In the nonligated hemispheres, combination therapy preserved the number of neurons only in the CA1 and dentate gyrus regions. Brain nitrite levels were evaluated by Griess reagent and showed that hypoxic-ischemic injury caused a significant increase in NO production. Brain nitrite levels in ADNF-9+NAP-treated animals were not different in carotid ligated or nonligated hemispheres. The peptides ADNF-9 and NAP significantly decreased NO overproduction in the hypoxic-ischemic hemisphere, whereas no significant change appeared in hypoxia alone and also in the sham-operated group. These results suggest the beneficial neuroprotective effect of ADNF-9 and NAP in this model of neonatal hypoxic-ischemic brain injury. To our knowledge, this is the first study that demonstrates a protective effect of these peptides against hypoxia-ischemia in the developing brain.

Protective effects of erythropoietin against ethanol-induced apoptotic neurodegenaration and oxidative stress in the developing C57BL/6 mouse brain.

The developing central nervous system is extremely sensitive to ethanol, with well-defined temporal periods of vulnerability. Recent studies have shown that administration of ethanol to infant rats during the synaptogenesis period triggers extensive apoptotic neurodegeneration throughout many regions of the developing brain. Furthermore, acute ethanol administration produces lipid peroxidation in the brain as an indicator of oxidative stress. In recent years, it has been shown that erythropoietin (EPO) has a critical role in the development, maintenance, protection, and repair of the nervous system. In the present study, we investigated the effect of EPO against ethanol-induced neurodegeneration and oxidative stress in the developing C57BL/6 mouse brain. Seven-day-old C57BL/6 mice were divided into three groups: control group, saline-treated group, EPO-treated group. Ethanol was administered to mice at a dosage of 2.5 g/kg for two times with a 2-h interval. Recombinant human EPO (rhEPO) was given 1000 U/kg. Twenty-four hours after the first dose of ethanol, all the animals were killed. Neuronal cell death, apoptosis, thiobarbituric acid substance (TBARS) levels, superoxide dismutase (SOD), and glutathione peroxidase (Gpx) enzymes activities were evaluated. Histopathological evaluation demonstrated that EPO significantly diminished apoptosis in the cerebellum, prefrontal cortex, and hippocampus and also spared hippocampal CA1, CA2, and CA3 neurons. Simultaneous administration of EPO along with ethanol attenuated the lipid peroxidation process and restored the levels of antioxidants. Regarding the wide use of erythropoietin in premature newborns, this agent may be potentially beneficial in treating ethanol-induced brain injury in the perinatal period.