| Cerebral ischemic stroke is one of the most common diseases affecting human health.It has a high mortality rate and many clinically difficult sequelae such as hemiplegia,numbness,dysphasia and cognitive deficits.Hypoxic-ischemic brain injury(HIBI)is a syndrome of acute global brain injury resulting from critical reduction or loss of blood flow and supply of oxygen and nutrients.HIBI occurs frequently in infancy and childhood.Events such as perinatal asphyxia,near drowing,respiratory arrest,and near sudden infant death syndrome cause significant mortality and morbidity.Despite current critical care practices,the outcomes from HIBI may be life-long neurologic deficits.For adults,HIBI can result from insufficient cerebral blood flow,reduced oxygen availability,reduced oxygen carriage by blood,or metabolic interference with the use of available oxygen.The enormous burden of human suffering and financial cost caused by HIBI make neuroprotection a major health care priority and potential targets for neuroprotective interventions has been studied over time.Ischemia-reperfusion brain damage(IRBI)is also a leading cause of morbidity and mortality,and its incidence increases with age.IRBI is a leading global cause of mortality,responsible for more deaths than cancer.Even in patients presenting comparable degrees of ischemia,a huge variability in the recovery of brain function is observed,indicating a complex pathological process which,to date,still lacks a specific therapy for its effective treatment.Indeed,even reopening of the stroke-related artery with either thrombolysis or catheter intervention is still far from providing a safe and effective therapy for most patients.HIBI and IRBI are associated with varying degrees of neurologic sequelae,depending upon the severity and length of HI.It triggers a series of cellular and biochemical pathways that lead to neuronal injury.One of the key cellular pathways of neuronal injury is inflammation and oxidative stress,leading to secondary neuronal injury.The inflammation is characterized by activation of microglia,the innate immune cells of brain,migration of peripheral macrophages;release of pro-inflammatory cytokines and chemokines,and phagocytosis of injured and uninjured neurons.Together at the cell level,HIBI and IRBI set in motion a cascade of biochemical events commencing with a shift from oxidative to anaerobic metabolism,intracellular build-up of calcium,mitochondrial dysfunction,and free radical[mainly reactive oxide species(ROS)]generation,which lead to extensive and prolonged cell death.The role of free radicals in HI induced brain injury appear to be crucial because the developing brain is particularly vulnerable to oxidative damage due to the limited capacity of antioxidant enzymes.Part 1:Deletion of the p66Shc Gene is Neuroprotective for Hypoxic-Ischemic Brain Injury in MiceBackgroundP66Shc is a splice variant of p52Shc/p46Shc,two cytoplasmic adaptor proteins involved in the propagation of intracellular signals from activated tyrosine kinases to Ras.p66Shc has the same modular structure of p52Shc/p46Shc(SH2-CH1-PTB)and contains a unique N-terminal region(CH2);however,it is not involved in Ras regulation,but rather functions in the intracellular pathway(s)that regulates ROS metabolism and apoptosis.Genetic deletion of p66Shc(p66Shc-/-)in mice extends lifespan by 30%and protects mice from IRBI,myocardial IRBI injury,hind limb ischemia and hyperglycemia-induced endothelial dysfunction through a blunted production of free radicals.ObjectivesIt’s crucial to elucidate the molecular mechanisms underlying the pathogenesis of HIBI to set the basis for the design of novel effective therapeutical strategies.In the present study,we thus analyzed the effects of genetic deletion of p66Shc in HIBI.We investigated whether p66Shc knockout mice can be protected from hypoxia-ischemia induced and ROS-mediated brain injury and neurological deficits.Methods and resultsA common carotid artery ligation plus hypoxia exposure model was performed to induce HIBI in wild-type(Wt)and p66Shc knockout mice(p66Shc-/-).Cerebral blood flow and blood pressure measurements revealed comparable hemodynamics in both experimental groups.Magnetic Resonance Imaging(MRI)of the brain and neuronal nuclear antigen(NeuN)immunohistochemical staining showed a significantly reduced infarct volume in p66Shc-/-when compared with Wt mice(P<0.05,n = 20).8-OHdG immunoactivity was not as prominent in p66Shc-/-mice as in the WT mice,indicating the oxidative stress intensity decreased by p66Shc knockout.TNF-a immunoactivity levels were all lower in p66Shc-/-mice than those in WT mice,indicating the inflammation degree,intertwined with oxidative stress,decreased by p66Shc knockout.CD34 staining showing the microvessel density angiogenesis level in p66Shc-/-mice was higher than in WT mice.Bax,bcl-2 and TUNEL staining demonstrating that p66Shc knockout may inhibit the apoptosis induced by HIBI.In line with these,p66Shc-/-mice exhibited a less impaired neurological function and a decreased production of free radicals systemically(P<0.05,n = 15).Following HIBI,protein levels of gp91phox nicotinamide adenine dinucleotide phosphate oxidase subunit were increased in brain homogenates of Wt(P<0.05,n = 15),but not of p66Shc-/-mice.The expression levels of malondialdehyde(MDA)and superoxide anion free radicals in plasma,reflecting systematic oxidative stress degrees,were higher in wide type mice than in p66shc knockout mice.Furthermore,p66shc knockout also significantly increased the activities of endogenous antioxidant enzymes,including superoxide dismutase(SOD)(P<0.05,n = 15).ConclusionIn the present study,we show that the deletion of the gene p66Shc protects mice from HIBI through a blunted production of free radicals,inflammation cells and inflammatory cytokines;enhancing angiogenesis in the peripheries of the infarct focus;and decreasing apoptosis intensity.The ROS mediator p66Shc may represent a novel therapeutical target for the treatment of HIBI.Part 2:Neuroprotective Effects of Grape Seed Procyanidin Extract on Ischemia-Reperfusion Brain InjuryBackgroundIschemia-reperfusion brain damage is an important cause for mortality and morbidity,and the incidence increases with age.Studies on humans and animals showed that oxidative stress(OS)plays a crucial role in ischemic stroke with or without reperfusion.Grape seed polyphenol extract(GSPE)has powerful antioxidant properties and protects neurons and glia during ischemic injury.In a study by Prior et al.,a dramatically increased post-prandial antioxidant capacity was observed following the consumption of mixed grape powder.Nevertheless,the exact effects of GSPE on ischemia-reperfusion brain injury remains unclear.AimsWe hypothesize that GSPE might also be protective in ischemia-reperfusion brain injury.Toward this reason,we explored whether GSPE administration can protect from ischemia-reperfusion brain injury.GSPE’s effects on mitochondrial signaling pathways which are crucially involve in oxidative-stress-processes are also investigated.Methods and resultsTransient middle cerebral artery occlusion(MCAO)was conducted to induce ischemia-reperfusion brain injury in mice received GSPE(MCAOG)and normal saline(MCAONS),followed by reperfusion for 24 hours.Blood pressure and cerebral blood flow measurements revealed comparable hemodynamics in the two groups.Via brain MRI and neuronal nuclear antigen(NeuN)immune-histochemical staining,MCAOG mice had a significantly reduced stroke size than MCAONS mice(P<0.05,n=20).GSPE administration seemed to be able to attenuate the OS,protect the neurological function,and attenuate the apoptosis(all P<0.05).Furthermore,GSPE also significantly increased the activities of endogenous antioxidant enzymes,including glutathione peroxidation(GSH-Px,P<0.05,n = 15)and improved the angiogenesis in the peri-infarct zone(P<0.05,n = 20).ConclusionWe showed that GSPE administration protects mice from ischemia-reperfusion brain injury via blunted free radicals,inflammation,apoptosis and a promotion of angiogenesis and activation of antioxidant enzymes.GSPE represents a new therapeutical direction for treatments of ischemia-reperfusion brain injury. |
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