| Chronic cerebral ischemia is a common pathological status in neural impairment, which always contribute to permanent or progressing cognitive dysfunction. Cognitive dysfunction induced by chronic cerebral ischemia is a disease which is affecting heavily people,s living quality. Its morbidity and mortality of cognitive dysfunction are descending year by year. Nowadays , study on mechanism of cognitive dysfunction due to chronic cerebral ischemia has been a hot spot for the neurologists both native and abroad。Untill today,Its occurence mechanism remain incompletely known and there haven,t been specifically drugs and effective treatment method ,then the treatment of cognitive dysfunction is still a difficult subject in clinic study. It is one of the greatest tasks faced by currently medical science.We adopted the permanent bilateral carotid arteries occlusion (2VO) model, and successfully established cognitive dysfunction model due to chronic cerebral ischemia. Based on the model , target first on HIF-1α, observed changes of the related HIF-1αand its target gene HO-1, TH mRNA and protein , observed the changes of neuron apoptosis and cell cycle control element CyclinD1 mRNA in frontal cortex and hippocampus area. A new anti-plate drug cilostazol was administration to ischemia group, to investigate the mechanism of learning and memory impairment after chronic cerebral ischemia and the protective mechanism of cilostazol on cognitive dysfunction , aim to find out new therapeutic target and strategy for chronic ischemic cerebrovascular Diseases, aim to offer a new idea for chronic ischemic cerebral diseases .Cilostazol, a selective inhibitor of phosphodiesterase3, has the function of inhibiting platelet aggregation and vasodilatation. Cilostazol is a kind of medicine curing acute cerebral infarction, exerts neuroprotective effects on acute focal cerebral ischemia in rats. However, it was regarded as a result of a target of curing and studing diseases ,from the expression of HIF-1αand its target gene HO-1, TH mRNA and protein , the changes of neuron apoptosis and cell cycle control element CyclinD1 mRNA, it has never reported in China and abroard that the mechanisms of learning and memory impairment after chronic cerebral ischemia and the protective mechanism of cilostazol on cognitive dysfunction induced by 2VO chronic cerebral ischemia.Part 1 Establishment and evaluation of cognitive dysfunction model induced by chronic cerebral ischemiaObjective: To establish and evaluate the cognitive dysfunction model induced by chronic cerebral ischemia.Methods: Healthy Male Wistar rats were selected whose body weight was 250-350g , breeding under normal temperature.After the training of Morris water maze place navigation experiment, the rats were divided randomized pseudo-operation group and ischemia group, each group was divided into 3w, 6w and 9w group according to ischemic time .The chronic ischemia model was established with the method of two-vessel occlusion (2VO), ability of learning and memory measured with Morris water maze including escape latency and swimming distance, including spanning platform time by rats. Pathological changes were measured with HE staining.Results: The sham-operation groups had no different change in escape latency and swimming distance at the time of 3w, 6w and 9w after 2VO. The ischemia groups had significant longer escape latency and swimming distance at the time of 3w, 6w and 9w after 2VO, compared with sham-operation groups; The escape latency and swimming distance in 9w-ischemia group were longer than the other two groups . Spanning platform times after 2VO, the ischemia groups had significantly reduced at the time of 3w, 6w and 9w, compared with sham-operation groups; The times at the 9th week ischemia group is the least, compared with the other two groups. There was no difference in sham-operation groups. HE staining of sham-operation groups shew neuoral cells in the cortical neuron of frontal lobe and hippocami line up in order ,uniformity,cellar nucleus were big and globular, nucleolus were clear.In 3w after2VO, there were neuoral cells loss,cytomorphosis and nucleolus were unclear; In 6w after2VO, there were obviously neuoral cells loss,nuclear fragmentation,astrogliosis and proliferation of small branching capillaries;In 9w after2VO, there were more obviously neuoral cells loss,severe cytomorphosis,structural disappearance and astrogliosis knots.Conclusion: cognitive dysfunction rat model after chronic cerebral ischemia was successfully established and evaluated with Morris water maze place navigation and spanning platform experiment, and HE dyeing also shows that it is a good method to investigate neural impairment.Part2 Expression of HIF-1αand its targets in the rat cognitive dysfunction model with chronic cerebral ischemiaObjective: To investigate the changes of HIF-1αand its targets on the cognitive dysfunction in rats after chronic cerebral hypoperfusion. Methods: cortex and hippocampus at different time point was measured with the method of immunohistochemistry, RT-PCR and Western-blot. HIF-1αand its targets HO-1 in frontal lobe cortex and hippocampus at different time point was measured with the method of immunohistochemistry,Western-blot and RT-PCR . TH ,the target of HIF-1α, was measured with the method of immunohistochemistry in frontal lobe cortex and hippocampus at different time point was measured . We have respectively analyzed the relationship between HIF-1αand the scores of learning and remembering with Pearson Correlation correlative analyzing system. So did its targets HO-1.The relationship between HIF-1αand its targets HO-1 was also analyzed with the same method.Result: The results of immunohistochemistry,Western-blot and RT-PCR experiment were showed below: HIF-1αmRNA and protein expression of the ischemia groups were higher than sham-operation groups, HIF-1αexpression of ischemia groups reached the peak at the time of 3w, and then declined progressively in 6w and in 9w but still above the normal level. HO-1expression of sham-operation groups were weaker compared with that of ischemia groups. HO-1expression of ischemia groups raised in 3w and peak in 6w, then declined in 9w. TH expression of ischemia groups were higher than pseudo-operation groups, raised in 3w and peak in 6w, then declined in 9w. In ischemia group, the expression of HIF-1αand HO-1 in immunohistochemistry in each ischemic time point and their corresponding learning-memory ability were in significant positive correlation. There are significant positive correlation between HIF-1αand HO-1,HIF-1αand TH, the related coefficients are respectively r=0.4786and r=0.4305. There is no correlation in sham-operation groups.Conclusion:HIF-1αand HO-1,TH are all highly expressed in the process of the cognitive dysfunction in rats after chronic cerebral ischemia. Peak of HIF-1αadvanced HO-1,TH, HIF-1αand HO-1 participate in the process of the cognitive dysfunction likely as a protective role, yet TH likely as a harming role. HIF-1αpathway may be a new therapeutic target for the cognitive dysfunction after chronic cerebral ischemia.Part3 Protective effects of cilostazol on the rat of the cognitive dysfunction induced by chronic cerebral ischemiaObjective:To observe the effectsof cilostazol on histology and behavioristics,on the effects of HIF-1α,HO-1,TH expression, on the effects of cell apoptosis in cortex and hippocampus and cell cycle control element CyclinD1mRNA and in order to investigate its possible mechanism of cilostazol on cognitive dysfunction in rats induced by chronic cerebral ischemia. Methods: Male Wistar rats were selected whose body weight was 250-300g , healthy. After the training of Morris water maze place navigation experiment, the rats were divided randomly sham-operation group ,ischemia group and cilostazol group . The chronic ischemia model was established with the method of two-vessel occlusion (2VO). After 9w, histology and behavioristics were measured with Morris water maze and HE staining . The expressive changes of HIF-1α,HO-1 in cortex and hippocampus at different time point were measured by the method of immunohistochemistry,Western-blot and RT-PCR . The expressive changes of TH were measured by the method of immunohistochemistry. Cell apoptosis was measured by the method of Flow CytoMeter. Cell cycle control element CyclinD1Mrna was measured by the method of RT-PCR.Result:The ischemia and sham-operation group had significant longer escape latency and swimming distance at the time of 9w after 2vo, compared with sham-operation groups; The escape latency and swimming distance in 9w-cilostazol group were longer than the ischemia group ,but still above the normal level. Spanning platform times after 2vo, the ischemia group and cilostazol group had significantly reduced at the time of 9w, compared with sham-operation group; The times of cilostazol group is more than that of the ischemia group. the investigation of histology shows that compared with ischemia group ,the changes of cilostazol group significantly increased at the time of 9w. HIF-1αand HO-1,TH were all highly expressive in the process of the cognitive dysfunction in rats after 9w-chronic cerebral ischemia, yet cilostazol group significantly decreased their expression at the same time point. The percentage of cell apoptosis of ischemia and cilostazol group was higher than sham-operation group in frontal lobe and hippocampus, yet cilostazol group is lower than ischemia group .cell cycle control element CyclinD1mRNA was expressed by the method of RT-PCR at sham-operation group, ischemia group and cilostazol group in frontal lobe of rats. cilostazol group was lower than ischemia group, but higher than sham-operation group. There is significant difference between ischemia group and cilostazol group in the result of OD.Conclusion:Cilostazol can significantly enhance the scores of learning-memory and reduce neuronal necrosis and apoptosis , improve the cognitive function of rat with chronic cerebral ischemia. HIF-1αand HO-1,TH,cell cycle control element CyclinD1mRNA and the percentage of cell apoptosis are all highly expressed in the process of the cognitive dysfunction in rats after chronic cerebral ischemia. Cilostazol had the neuroprotective function by decreasing the expression of HIF-1αand HO-1,TH , reducing the expression of cell cycle control element CyclinD1mRNA and the percentage of cell apoptosis during the processs of cognitive dysfunction after chronic cerebral ischemia . Cilostazol has not only the function of antiplatelet aggregation, but also the function of neuroprotective function, it can be used to prevent and cure cognitive dysfunction after chronic cerebral ischemia. Cilostazol prospects to be one of new drugs in cognitive dysfunction after chronic cerebral ischemia. |
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