| Rationale: Vascular dementia (VaD) refers to the severe cognitive dysfunction syndrome which caused by ischemic stroke, hemorrhagic stroke, or various cerebrovascular diseases which resulting the hypoperfusion of brain areas associated with memory, cognitive and behavior. Its clinical manifestation mainly included the disabilities of language, movement, spatial perceptual and personality disorder. So far,its underlying mechanism has largely remained unknown. Epidemiological data of Europe and the US have shown that the prevalence of VaD is approximately between 0.9% and 3.0%, which account for 15% to 20% of all the dementias,rank only second to Alzheimer's disease. In China, the annual prevalence rate of VaD is among 5/1000 to 9/1000 in the population over 60. Besides, the incidence of VaD has increased every year. It is one of the most important factors which lead to seniledementia and bring the decline of life quality among the aged people. Therefore, VaD not only brings great pain to the patients and affects their life quality, but also brings heavy burden to their family and society. All in all, the way to prevent and treat VaD has become an urgent medical problem.In clinical treatment practices, the strategy of exercise intervention, as a means of rehabilitation treatment of cerebral vascular disease, has been widely used and accepted by the international medical community, due to its effect on the recovery of cognition of VaD. By now there has been no three-stage rehabilitation system for cerebrovascular disease in China. A large amount of the cerebrovascular disease patients had not got systematic rehabilitative treatment after their acute phase of disease. So the study and rational application of the exercise intervention strategy may make a special significance for VaD patients in china.Glu is the highest amount and the most important excitatory amino acid in central nervous system, which widely distributed in cerebral cortex, hippocampus,cerebellum, striatal and basal nucleus. By combining of its corresponding GluR, it participates in various important physiological functions of the brain, including learning, memory, movement, and sensation. The further study have verified that the N-methyl-D-aspartate receptor (NMDAR) in the GluR is associated with learning and memory process described above. Various NMDAR functions largely depend on NR2.NR2 includes NR2A and NR2B. NR2B has a better biological activity than NR2A .Besides, NR2B has a close relationship with LTP and nerve damage after cerebral ischemia. S1480 is an important loci of NR2B, whose phosphorylation can make NR2B activated. Activation of NR2B and instantaneous influx of Ca2+ will activate the associated protein kinase of Ca2+, such as calmodulin dependent protein kinase ? (CaMK ?). The interaction of NR2B and CaMK ? will further activate the extracellular signal regulated protein (ERK1/2) signal pathway, and ERK1/2 signal pathway can maintain a long-term activation to induce LTP. CK2 is the casein/serine protein kinase widely distributed in the body which is considered being related to learning and memory. CK2 can participate in the learning and memory function of hippocampus by phosphorylation of the S1480 loci of the NR2B.In this study, the author choose the VaD rats with bilateral artery ischemia reperfusion following peritoneal injection sodium nitroprusside as animal model, and use treadmill training as a form of exercise. Different experimental methods were applied such as behavioral detection, cell morphology observation,immunohistochemistry, PCR, and western blot. The purpose of the present study is to explore the protective function of the exercise on reducing nerve cell apoptosis of hippocampus, increasing the gene expression and protein regulation of CK2, p-NR2B,NR2B, NR1 and p-ERK1/2 receptors. It has an important significance in providing the experimental data for VaD treatment by using exercise.Part 1: The establishment of the VaD rat model and the influence of exercise training on behavioral and hippocampus pathologic changes in the ratsWith the development of medical technology, plenty of drugs for treating dementia are developed every year, but there is no drug has been approved by the FDA for the treatment of VaD. After stroke, giving patients moderate exercise is a good rehabilitation strategy. The influence of exercise on learning and memory is still at the frontier and means a hot spot in the human movement science. Exercise can exert influence on various factors which associated with learning and memory activities. But its exact biological mechanism still need be further clarifed. In clinical practice, the strategy of exercise intervention has been regarded as one of the rehabilitation methods for cerebrovascular disease, and its value for cognitive functional recovery has been admitted by the world. Currently in China, most researchers only focused on clinical studies, while the study of the animal model is quite few. Therefore, establishing an ideal animal model will greatly promote the study of its pathologic mechanism and even the clinical treatment of VaD.Objectives: The purpose of the present study is to establish a reliable VaD cerebral ischemia-reperfusion rat model and explore whether the exercise training may change the learning and memory disorder which caused by VaD. Furthermore,the study also would provide a view to explore the mechanism for the exercise intervention of VaD.Methods: We adopt the method of recurrent ischemia and reperfusion on the bilateral carotid artery following peritoneal injection sodium nitroprusside to create VaD model causing the rat brain tissue in hypoperfusion state. The learning and memory scores of the rats were detected by applying step-down test and water maze test. Meanwhile, the changes of the hippocampus nerve cell structure of the rats were observed by applying cresyl violet staining and the therapeutic effect of the treadmill training.Results:1. Step-down test: The results showed that there was a statistical difference in error times and staying time within 10 minutes between the exercise group and the model group (P<0.05) in the step-down test, indicating that exercise training can improve the learning performance of VaD rats.2.Morris water maze test: The results showed that there was a statistical difference in the escape latency and the total swim course between the exercise group and the model group (P<0.05),indicating that exercise training can improve VaD rats'learning achievement. Morris space exploration test showed that there was a statistical difference in the staying time in the original platform quadrant between the exercise group and the model group (P<0.05), which indicated that treadmill training can improve the memory scores of VaD rats.3. Cell morphology in the hippocampus CA1 areaRat hippocampus staining showed that the hippocampus cells of the rats that have been exercised for 4 weeks are arranged more neatly than cells of the rats for the 6 weeks. Besides, there is also an obvious reduction of apoptosis in Nissl appoptotic cells , and the number of cells have increased significantly in the rats for the 4 weeks compared with the model group.Conclusion:1. The learning scores of animals in the model group decrease greatly after their bilateral carotid arteries ligated repeatedly, which indicates that the VaD rat model is built successful.2. Exercise can significantly improve the ability of learning and memory function of VaD rats, which indicates that exercise can relieve the damage of nerve cells in the hippocampus CA1 area.Part 2 Influence of exercise training on learning,memory, gene expression and protein expression of CK2, NR1, NR2B, P-NR2B, ERK1/2 and p-ERK in hippocampus area of the VaD ratsExercise training for stroke is an effective rehabilitation therapy. Clinical data show that it can effectively reduce the depressive level of patient, increase motor and sensory control abilities and improve the walking speed and accuracy, thereby significantly improve the patients' quality of life. In animal studies, the author also find that active exercise training after stroke can reduce the cerebral infarction volume and nerve cell apoptosis caused by ischemia, promote motor function recovery of the animals and improve the learning and memory performance. The mechanism may involve not only the increasing of the neurotrophic factor BDNF and CREB but also promoting the hyperplasia of the hippocampus synapses and the neurogenesis.Clinical studies have shown that exercise can increase capillary density in the motor cortex area of the brain and increase the cerebral blood flow for healthy people. These results suggest that angiogenesis plays an important role in brain functional recovery for the stroke. An increasing number of studies suggest that the mechanism of exercise enhancing the plasticity of hippocampus neuronal synapses (such as long term potential) may be an important way to improve the learning and memory ability.During the process, glutamate (Glu) and its receptors (GluR) can play a key role.Objectives: The purpose is to explore the protective effect of exercise training on nerve cells and molecular mechanism, by the way we tried to illustrat the formation mechanism of VaD and provide treatment ideas for exercise intervention of VaD.Methods: In this study, we have built rat model of cerebral ischemia/reperfusion after peritoneal injection sodium nitroprusside to investigate the protective functions of the exercise training by the treadmill training. Then, we detected the expression of NR 1 mRNA,NR2B and the protein expression change of CK2?p-NR2B?NR2B?p-ERK1/2?ERK1/2 in the CA1 area of the VaD rats using PCR and western blot.Results: 1.PCR shows the NR1mRNA and NR2BmRNA expression in hippocampus decreased significantly in the 3d group, which has a statistic difference compared with sham group. The expression level of Group 4w+s and Group 6w+s increase significantly compared with their respective model groups, and there is a statistic significance (P<0.05). After two weeks, the expression of each exercise group NR2BmRNA increases significantly compared with the model group, and there is statistic significance (P<0.05).2. Western blot shows that the density values of CK2 in the hippocampus area of group 3d and 2w Group are significantly lower than those of the sham surgery control groups, and there is statistic significance (p < 0.05). With the time passing, CK2 gradually increases and its expression was the highest in 4w+s group. This expressions in 2w+s group and 4w+s group increased significantly compared with the respective model control group (both p < 0.05); the expression of p-NR2B protein showed an increasing trend with the time, and the density values of p-NR2B/NR2B in Group 4w+s and 6w+s group increase significantly compared with their respective model group (p < 0.05). With the time and exercise intervention, the p-ERK1/2 protein expressions in both exercise group and non-exercise model group increase gradually,while the ERK1/2 level remains unchanged. Density analysis shows that the density values of the p-ERK1/2 protein expressions in 4w+s group and 6w+s group increasing significantly compared with their respective model group (p < 0.05).Conclusion: Exercise training can improve the learning and memory of the VaD rats by increasing the expression of NR2B in the hippocampus and activating the ERK1/2 signaling pathway. |
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