| Background: Cerebral infarction(CI), also known as ischemic stroke, refers to the local blood supply to the brain caused by cerebral tissue ischemia and hypoxia, which can cause localized necrosis or brain softening. At present, there are 7 million patients with CI in our country, which is one of the most deadly diseases. The average incidence rate is(116-219) /10 million population, and the annual mortality rate is(58~ 142) /10 million people. CI has a high incidence, disability and mortality, in which different levels to lose labor ability can reach 75%, which is the most common cause of disability and also is the leading cause of disability for adults in China. In recent years, with the accelerated process of aging in China, the incidence of CI is increasing year by year. The clinical manifestations of Ci is very complex, and the extent of the disease depends on infarct size and location, such as headache, dizziness, nausea, vomiting, motion and(or) feeling aphasia, hemiplegia, incontinence, etc..CI has become a serious public health problem because of the great economic and mental burden to the family and society. Therefore, it has become an important issue in the study of Neural Department of internal medicine to explore the reasonable and effective methods of CI treatment and reduce the mortality, disability rate and recurrence rate of CI.Aspirin and clopidogrel are the common antiplatelet aggregation drugs in clinical. A large number of studies prove that the use of aspirin or clopidogrel can significance prevent CI recurrence. In clinical practice, the combination of aspirin and clopidogrel in the treatment of intracranial atherosclerotic stenosis has also been reported, and the joint use of the two drugs has been gradually received attention in the Department of internal medicine. However, how to choose the timing of the application, drug measurement and other issues are still to be studied, and there is no experimental report on the combination of two drugs in the treatment of cerebral ischemia reperfusion animal model in rats.Bone marrow stromal cells(BMSCs) deriving from mesoderm have high self-renewal capacity and multilineage differentiation potential of adult stem cells, which is currently the most studied and has representative and differentiation tendency of most adult stem cells. A large number of in vitro experiments show that, under different conditions, the mesenchymal stem cells can differentiate into neuron like cells. At present, the method of separation and extraction of BMSCs is more mature and safe, which can also play a central role in the protection and repair of brain tissue by migration and proliferation.In summary, because of the superiority of stem cell therapy with antiplatelet drug therapy, this study compared aspirin and clopidogrel on cerebral ischemia again perfusion in rats model of curative effect, from the significance of clinical application. At the same time, the combination of aspirin and clopidogrel was used to observe the effect of combination therapy on the expression of nerve function and expression of brain tissue in rats. In addition, this study first put forward the idea that stem cells combined with two kinds of anti platelet aggregation drugs in animal model of cerebral ischemia reperfusion rat model. Comparing the therapeutic effect of stem cell therapy and drug therapy on rat model aim to provide experimental data and theoretical basis for clinical practice.Purpose:(1) To observe and compare the effects of three different treatments of aspirin, clopidogrel and both of them on cerebral ischemia reperfusion model.(2) To observe and compare the efficacy of BMSCs therapy and anti platelet aggregation drugs in the treatment of cerebral ischemia reperfusion model.(3)To propose a new approach for the treatment of CI patients, that is stem cells combined with anti platelet aggregation drugs in the treatment of CI patients, and provide real experimental data and reliable theoretical support for its clinical applications.Methods: Three-four month old SD rats were randomly assigned to nine groups(n=13), and the weight is 250~300 g, which are MCAO model: Sham operation group(group A), model of natural recovering group(group B), aspirin group(Group C), clopidogrel treated group(D group), aspirin combined with clopidogrel treatment group(E group), BMSCs treatment group(F Group), BMSCs in combination with aspirin treatment group(G group), BMSCs combined with clopidogrel in the treatment group(Group H) and BMSCs in combination with aspirin and clopidogrel therapy group(group I). Among them, B group without any intervention; group C and g daily aspirin orally(each only 10.3 mg / kg / D),; D and H group daily clopidogrel orally(each only 7.8 mg / kg / D); E and I was daily given aspirin and clopidogrel orally(each aspirin 10.3 mg / kg / day, clopidogrel 7.8 mg / kg / D); F, G, h and I were after 7d given intravenous injection of BMSCs(each 2 x 106 / ml, 1 ml). In the treatment of fifteenth days, the Morris water maze was used to evaluate the spatial learning and memory ability of rats. The 21 st day of the experimental specimens were drawn, part of the rats with 10% hydration chloric aldehyde anesthesia after blood were collected from heart, and cut off the head for rats hippocampus. In another part of the rat, the brain was prepared for paraffin embedding and tissue sections by using the method of cardiac perfusion. The difference of serum ELISA kit were used for detecting the contents of inflammatory factors. Slices were stained by HE staining and Nissl staining to observe the changes of brain tissue. TUNEL staining was used to observe the neuronal apoptosis in the rat brain. The expression of different neurotrophic factors was observed by immunohistochemistry staining. The apoptosis pathway genes(Bcl-2, Bax, Caspase-3 and Surivivin) expression were detected by Western Blott.Results:(1) The MCAO rat model was successfully established in this study.(2) BMSCs was successfully isolated and cultured, which was successfully transplanted through the rat tail vein.(3) Effects of antiplatelet aggregation drugs combined with BMSCs on learning and memory function in MCAO rats. The neurological function score results show: compared with group B, other treatment group rats neural function scores were increased and I group was the highest(p<0.05); within the drug treatment group, compared with each other, the C group was the lowest, the highest in the group E(p<0.05); BMSCs treatment group of neural function score higher than drug treatment group(p<0.05). Morris water maze results show that: at the same time, the escape latency gradually shortened in different treatment group rats; compared with group B, in the treatment group, the escape latency of rats were shortened and combined treatment group escape latency shorter than the single drug group; the time of BMSCs group was less than that of medication group, among which BMSCs combined with two kinds of drug group was the shortest escape latency; at different time points, the escape latency was also gradually shortened in the same treatment group with the extension of time(p<0.05). It showed that anti platelet aggregation drugs could effectively improve the learning and memory function of MCAO rats, and the effect of combination was more effective. In addition, the effect of BMSCs combined with drug therapy was the best.(4) Effects of antiplatelet aggregation drugs combined with BMSCs in the treatment of inflammatory injury in MCAO rats. ELISA results showed that: compared with B group, in the treatment group, the serum inflammation factor of TNF-a, IL-1, IL-6, HSP-70 and ACTH values were decreased(p<0.05), and the serum concentrations of inflammatory factor decreases gradually in simple drug group,combined treatment group, BMSCs group and BMSCs combined with drug therapy group, which BMSCs combined with two kinds of anti platelet aggregation drug group rats serum inflammatory factor level was the lowest(p<0.05). The results showed that the anti platelet aggregation drugs could effectively reduce the inflammatory response in MCAO rats, and the effect of BMSCs combined with drug treatment was the best.(5) Effects of antiplatelet aggregation drugs combined with BMSCs on the pathological changes of brain tissue in MCAO rats. Slides stained with hematoxylin and eosin results can be seen that:performance of the B group of brain ischemic area for brain tissue present sparse form, neural cell deformation, and nuclear condensation or fragmentation, massive necrosis of nerve cells in the brain tissue of hippocampal CA1 and CA4 region, and a group of nerve cells in the normal; Nerve cell necrosis in the cerebral cortex of C and D treated rats, but less than the number of necrosis in B group; the number of nerve cell necrosis in the cerebral cortex of E treatment group was less than that in the group; the number of cell necrosis in the BMSCs treatment group was less than that in the drug treatment group, and the lesion was the lightest after combined drug treatment. Nissl staining results: under the microscope, in the rats brain tissue of A group Nissl bodies is blue; while B group staining sections showed that Nissl bodies and ischemic necrosis of nerve cells were rare and the amount of necrotic were reduced. Nissl bodies in a few neurons also disappeared, and cell nuclear pyknosis; the number of the Nissl bodies in the different drug treatment group was increased, and the number of the combined drug group was more than that of the simple drug group; BMSCs transplantation group was significantly increased in the number of the Nissl bodies, and the number of I group was largest.(6) Effects of antiplatelet aggregation drugs combined with BMSCs on the apoptosis of hippocampal neurons in the brain of MCAO rats. The results of TUNEL staining showed that B group had obvious degeneration and death in the hippocampal neurons, and the injury site was mainly located in the cerebral cortex, subcortical and hippocampus area of the ischemic side(the apoptosis rate was 51.33%); compared with the B group, the apoptosis rate of different treatment group was significantly lower(p<0.05); the apoptosis rate of D group was lower than that of group C(p<0.05); the apoptosis rate of E group was lower than that of C、D drug group; the apoptosis rate of BMSCs in treatment group was 36.72%,which was lower than that in drug therapy group; the apoptosis rate of group I was the lowest.(7) Effects of antiplatelet aggregation drugs combined with BMSCs on the secretion of neurotrophic factors in the brain of MCAO rats: the results showed that neurotrophic factors GFAP, GDNF and NSE rare in the B group; the number of positive cells increased gradually with the different treatment methods. Under the microscope, the cytoplasm of cells appeared to see a lot of brown or tan; comparison of different treatment methods, combining with two kinds of antiplatelet drugs group the number of positive cells were more than a single drug treatment group; but the number of positive cells increased after BMSCs combined with drug treatment.(8) Effects of antiplatelet aggregation drugs combined with BMSCs in the treatment of brain tissue protein in MCAO rats. Western blot results showed that: A group does not express the apoptosis related proteins; compared with the B group, the expression of surivivin in other treatment group increased gradually, but the expression of Caspase-3 decreased gradually; among them, the expression of surivivin was higher than that of single drug therapy(p>0.05) in combination with two drug treatment groups; there was no significant difference in protein expression between C and D group(p>0.05); the expression level of surivivin in the BMSCs treatment group was higher than that in the drug treatment group, but the expression level of Caspase-3 was lower than that of the drug therapy group(p<0.05); the expression level of surivivin in the combined drug treatment group was higher than that in the other groups, but the expression of surivivin was highest in I group, and the expression of Caspase-3 in BMSCs group was the lowest(p<0.05).Conclusion:(1) The efficacy of aspirin and clopidogrel in the treatment of rat MCAO model is better than that of the single use;(2) BMSCs combined with aspirin or clopidogrel treatment on rat model of MCAO therapy was better than the single using with antiplatelet drug therapy;(3) The three treatment methods commonly used are the best efficacy in the rat MCAO model, including BMSCs, aspirin and clopidogrel. |
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