| ObjectiveThe middle cerebral artery was occluded by internal carotid artery suture to cause transient focal cerebral ischemia. Three doses of resveratrol (10, 30, 60 mg/kg, respectively) were intraperitoneally injected after ischemia-reperfusion. Using 2,3,5-triphenyl tetrazolium chloride (TTC) staining to show the infarct volume, dry-wet weight method to calculate the water content was calculated, and HE staining to observe the morphology of neurons aim To explore the dosage dependence relation of resveratrol on brain protection after acute ischemia and reperfusion.Methods1. Animal groupingSeventy-five male Sprague-Dawley rats, weighing 220-250g, were used. The rats were randomly divided into sham group, model group, low dose group, middle dose group and high dose group. Each group was 15 animals.2. Focal cerebral ischemia-reperfusion preparationAnesthetized by 10% 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 was gently isolated by glass needle. A nylon thread was inserted into the external carotid artery and manipulated to enter the internal carotid artery. The thread was advanced until felling resistance (approximately 20mm) at the point where the filament blocks the middle cerebral artery. About 90min after middle cerebral artery occlusion (MCAO), the thread was withdrawn to permit the middle cerebral artery reperfused. The surgical incision was sutured and the animal was returned to its cage. The rectal temperature of rat was controlled at (37±0.5)°C with a homoeothermic blanket. The focal cerebral ischemia-reperfusion model could be made repeatably.3. TTC stainingThe rats were intraperitoneally injected with 3 doses of resveratrol (10, 30, 60 mg/kg) about 1h after ischemia-reperfusion as low, middle, and high dose group, respectively. The animals were euthanized under chloral hydrate anesthesia followed by decapitation at 24h after reperfusion. The brains were rapidly dissected out and the forebrains were cut into five coronal sections about 2mm thick, using a rat brain matrix. The sections were stained by a solution of 2% of 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 30 min to show the infarct volume.4. Evaluation of brain edemaThe water content in ischemia brain was determined by wet weight to dry weight ratio about 24h after ischemia-reperfusion. The ischemia brain was weighed and dried for 24h at 100°C, and re-weighed. The percentage of water was calculated according to the method of dry-wet weight .5. Histological examinationHistologic examinations were made on seventh day after ischemia. The animals were anesthetized by 2% chloral hydrate and transcardially perfused with 200ml of 0.1M phosphate buffer saline at 4°C (pH 7.4), followed by 200 ml of 4% buffered paraformaldehyde phosphate. The brains were removed, postfixed in the same fixative for 24h, and then processed for paraffin embedding. Coronal sections (4mm in thickness) including the dorsal hippocampus were cut for HE staining. The sections were examined under a light microscope at 100 magnifications, and the numbers of abnormal CA1 pyramidal cells per 1mm length along the extent of CA1 pyramidal layer were counted as neuronal density (cells per mm).6. Statistics AnalysisAll statistical analysis was performed using SPSS11.0. Values were expressed as means±SEM. Student's t-test were performed to compare the means in two groups. Statistics significance was defined P < 0.05.Results1. The brains in sham group did not show any detectable lesion or non-viable tissue damage. Conversely, brain sections obtained from model group showed detectable lesions as white patches in the areas that were supplied by the MCAO. The infarct volume and neurological score in each resveratrol groups were lower than that in the model group.2. Ischemic brain edema is one of the major reason of brain mortality. Resveratrol could reduce the edema caused by cerebral ischemia. The brain water content was significantly increased in model group compared with sham group. Whereas, the quantities of water contain in each resveratrol groups were significantly lower than that in the model group.3. The microphotographs of the CA1 hippocampal region were integrated in the sham group e. In the model group, the microphotographs of the CA1 hippocampal region were obvious degeneration, necrosis and inflammation. In the middle dose group of resveratrol, the abnormal pyramidal neurons of hippocampus CA1 area were less than that in the model group.However, neither MCAO nor the administration of 3 doses of resveratrol after ischemia reperfusion did not reverse granule cells loss in the dentate gyrus.ConclusionThe results suggest that resveratrol has a neuroprotective effect on focal cerebral ischemia-reperfusion injury in rats. The middle dose of resveratrol has the best therapeutic efficacy. |
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