Neuroprotective Effect Of Resveratrol In Permanent Focal Cerebral Ischemic Model In Mice

Resveratrol , a polyphenolic compound which is abundantly present in the seeds and skin of grapes , has various biologic activities such as anti-platelet activity, anti-inflammatory activity, anticarcinogenic activity etc. It also acts as free radical scavenger and antioxidant and has cardiovascular protective effect. In recent years, the research that aims at the neuroprotective effect of reveratrol causes more and more attention in the field of neuroscience. A series of pathophysiologic changes occur in cerebral ischemia injury. Nitric oxide synthase(NOS) is activated. NOS can make use of levoarginine to synthesize nitric oxide (NO), which has various toxic effects on the neuron . Free radicals are produced excessively. They can act on protein, nucleic acid ,lipid, hyaluronic acid in extracellular matrix(ECM) etc, which will damage the normal structure and result in death of neuron. Inflammatory mediators also increased after the injury and they can produce subsequent damage to neuron. Being free radical scavenger and inhibitor of NOS andinflammatory mediators, resveratrol can alleviate the damage after ischemia injury.In addition, resveratrol can restrain some stimulative factors of matrixmetalloproteinases-9(MMP-9) such as c-Fos, c-Jun, lipopolysaccharide( LPS ), interleukin-1 (IL-1) etc. MMPs-9 is a kind of zinc-containingprotease that can degrade a variety of molecules in ECM. It is activated afteronset of cerebral ischemia and damages the normal structure of blood-brainbarrier(BBB).Inhibiting the activity of MMP-9 can reduce brain infarct. Soresveratrol has highly likely neuroprotective effect owing to it's inhibiting thethe activity of MMP-9 and alleviating the damage of BBB. Our experimentswill focus in the neuroprotective effect of resveratrol and its mechanism.I. Establishment of the animal model:Aim: To discuss factors which influence the stability of permanent focal cerebral ischemia models in mice. Methods: Ninety mice were divided into three groups by the kind and the weight. An 8-0 segment of monofilament was inserted into proximal position of left middle cerebral artery through carotid artery. 24 hours after the surgery ,brains were cut into thick slices and stained with triphenyl tetrazolium chloride (TTC) and the infarct volume was calculated. Results: The infarct volume and the scale of the neurofunction in Kunming mice(18~22g) are much less than these in ICR mice ,but the rate of success has no difference between two groups. With 8-0 segment of monofilament, the rate of success in Kunming mice(18~22g) is much higher than that in Kunming mice(30~35g ). Conclusion: Through the experiment above, we found the key points to determine the stability of models. 1. The selection of animal species. Due to the difference of the development in Williscircle between the different species, it makes a different selectivity for each species to meet the requirement of different models. 2.The selection of monofilament. Because the disparity of blood vessels in diameter was positively correlated with their body weight, the filament should be selected flexibly according to the body weight. 3. The preparation of monofilament. It requires quite uniform coating of silicon gel resin and the head of segment should be round and blunt, avoiding the formation of curve and hook, which would perforate the blood vessel. In a word, when establishing permanent cerebral focal ischemic model in mice, it is essential to control factors mentioned above strictly. This focal cerebral ischemic model by intraluminal occlusion in mice has a high reproducibility, better comparability and reliability. It minimizes the extra injury and especially has a great benefit on mass experiment, so this model can completely meet our next experiments. II. Study of the neuroprotective effects of resveratrolAim: To study the neuroprotective effects of resveratrol with different dosage on focal cerebral ischemia. Methods: Forty healthy male Kunming mice, weighing 18~22g, were randomly assigned into four groups: groups of resvertrol(10mg/kg, 30mg/kg, 60mg/kg) and group of ischemia. Ischemia was induced by intraluminal middle cerebral artery occlusion(MCAO). The infarct zone was showed by triphenyltertrazolium chloride (TTC) staining. Brain edema was calculated indirectly. Results: The infarct volumes and the degree of edema in group of resveratrol 30mg/kg are obviously less than these in group of resveratrol 10mg/kg, while compared with those in group of resveratrol 60mg/kg, there are no significantly difference (p>0.05). And two indications in three groups of resveratrol are all less than those in groupischemia. Conclusion: Resveratrol has neuroprotective effect on the ischemic brain and the effect is related to the dosage of resveratrol in some degree.III. Influence of resveratrol on the expression of MMP-9 in focal cerebral ischemic injuryAim: To discuss the expression of MMP-9 in permanent focal cerebral ischemic injury and the intervention caused by resveratrol. Methods: Ischemic brain tissue was acquired in different time (0h, 1 h, 3 h, 6h, 12h, 24h) after the cerebral ischemia injury and the expression of MMP-9 was examined by RT-PCR and zymography at the levels of transcription and enzymatic activation. At the same time, we have also detected the effect of resveratrol on the expression of MMP-9 in 24h. Results: The expression of MMP- 9 was augmented after cerebral ischemia at the transcriptional level and enzymatic activities of MMP-9 were also increased. Resveratrol can inhibit the expression of MMP-9 at transcription after ischemia and suppress the enzymatic activity of MMP-9. Conclusion: Resveratrol can suppress the expression of MMP-9 in focal cerebral ischemic injury. So resveratrol has neuroprotective effect owing to it's inhibiting the the activity of MMP-9 .IV. Correlation of resveratrol and the damage of BBB.Aim: To discuss the influence of resveratrol on the damage of BBB in permanent focal cerebral ischemic injury. Methods: Twenty healthy male Kunming mice were randomly assigned into two groups: group of resvertrol(30mg/kg) and group of ischemia. Ischemia was induced by intraluminal middle cerebral artery occlusion(MCAO) and Evans blue was injected. The disruption of BBB was implicated by measuring Evans blue extravasation. Results: Resveratrol can significantly diminish the extent and...