The Protective Role And Mechanism Of Preconditioning With Resveratrol On Focal Cerebral Ischemia/Reperfusion In Rats

ObjectiveIschemic stroke is one of the common diseases in nervous system and leads to neuronapoptosis after ischemia reperfusion, which may be a major role in causing secondarybrain injury and severe nerve dysfunction. Ischemic preconditioning may attenuatecerebral ischemia/reperfusion injury, in part, by decreasing cell apoptosis. Resveratrolmay be a potential agent for treating neuronal injury associated with stroke, but itsmechanism is unclear. Middle cerebral artery occlusion （MCAO） was employed toestablish focal cerebral ischemia model, and resveratrol （30mg/kg） was given beforeischemia by intraperitoneal injection. Infarction volume was detected by TTC staining.The neuron apoptosis of hippocampus CA1area was determined with TUNEL staining.The expression of apoptosis associated proteins and p-ERK were measured byimmunohistochemical method after ischemia-reperfusion. The aim of this study was toexplore the functional mechanism of resveratrol against neuron apoptosis in ischemiccerebral injury, and provide experimental basis for new drugs to prevent cerebrovasculardiseases in clinic.Methods1Animal groupingSixty Sprague-Dawley rats of clean grade, weighing200-250g, were used. The ratswere randomly divided into sham group, ischemic-reperfusion group （I/R） andresveratrol preconditioning ischemic-reperfusion group （Res+I/R）. Each group was20rats.2Focal cerebral ischemia-reperfusion model preparatingAnesthesia was induced by10%chloral hydrate. The rat was fixed on an operating table in a supine position. After the midline skin incised, the right common carotid artery,external carotid artery and internal carotid artery was isolated. The vagus nerve wasgently isolated by glass needle. A nylon thread was inserted into the external carotidartery and manipulated to enter the internal carotid artery. The thread was advanceduntil felling resistance （approximately20mm） at the point where the filament blocksthe middle cerebral artery. About90min after MCAO, the thread was withdrawn topermit the middle cerebral artery reperfused. The surgical incision was sutured and theanimal was returned to its cage. The rectal temperature of rat was controlled at37±0.5°C with a homoeothermic blanket. The focal cerebral ischemia-reperfusion model couldbe made reproducible.3TTC staining the infarction volumeThe animals were euthanized under chloral hydrate anesthesia followed bydecapitation at24h after reperfusion. The brains were rapidly dissected out and theforebrains were cut into five coronal sections about2mm thick, using a rat brain matrix.The sections were stained by a solution of2%of2,3,5-triphenyltetrazolium chloride（TTC） at37°C for30min to show the infarct volume.4Detecting apoptosis in situ （TUNEL method） to detect the apoptosis ofhippocampus neuron cell5days after ischemia-reperfusion, the rats were perfused with4%paraformaldehyde byheart after chloral hydrate anesthesia until the limbs were stiff. Remove the ischemicside of brain fixed in the corresponding liquid in4°C refrigerator overnight. Interceptfrom optic chiasma to transverse crack part of the brain, paraffin embedding, serialcoronal slices （4μm）. The neurons apoptosis of the hippocampus CA1area wasdetected by TUNEL detection kit.5Detecting apoptosis protein expression by immunohistochemistry.Three sections of specimens of each animal to detect protein apoptosis, such as p-Akt,PI₃K, p-ERK1/2and Caspase-3, were taken by immunohistochemical staining withstreptavidin–peroxidase （SP） and DAB chromogenic method. 6Statistical analysesThe measurement data are expressed in x±s. All statistical analysis was performedusing SPSS13.0software. The data among the groups were compared by t test.Results1TTC staining in sham brains did not show any visualized tissue damage. Conversely,brain sections in the I/R group showed dramatic lesions as pale in the areas that weresupplied by the middle cerebral artery. Resveratrol pretreatment significantly reducedsuch brain damage and improve the obstacle of rat behavior.2Compared with I/R group, preconditioning with resveratrol significantly reduced thenumber of TUNEL-positive neurons in the hippocampal CA1pyramidal layer inducedby cerebral ischemia （P <0.05） with TUNEL staining.3The expressions of PI₃K, p-Akt and p-ERK1/2protein in the hippocampus CA1region of Res+I/R group were significantly higher than that of I/R group （P <0.05）with immunohistochemical method. However, resveratrol pretreatment significantlyreduced the expression of Caspase-3protein （P <0.05）.ConclusionThose results suggest that preconditioning with resveratrol1h before ischemiapossesses has neuroprotective effects on focal cerebral ischemia-reperfusion inducedneuronal apoptosis. The neuroprotective mechanism of resveratrol against neuronalapotosis may attribute to activating PI₃K-Akt and ERK signal pathway and inhibitingCaspase-3proteins expression.