| Cerebral infarction is a serious manifestation of ischemic cerebrovascular disease(ICVD), which is one of the most common diseases of the central nervous system and the high morbidity, mortality and disability rate of this kind of disease are seriously threatening humen’s health and quality of life. So which begins to be the focus of more and more reseachers is how to cure it and make the prognosis better. Recent studies have found that supratentorial focal brain damage can cause functional and pathophysiologic changes in the cerebral infarction focus as well as the distant areas(cerebellum). This phenomenon plays a key role in the recovery of nervous function, whose cause is related to the diaschisis.The definition of diaschisis is that the changes of the function of the remote areas may be result of the injury of cerebral infarction focus which is connected by the fibers. Recently, with the program of the radiologic technique, haemodynamics and metabolism measurement technology, we make a great progress to research the mechanism of diaschisis, and provide the diagnosis and tharepy to the clinic doctors to cure the cerebral infarction.Magnetic resonance diffusion tensor imaging(DTI) characterizes the three-dimensional(3D) distribution of water diffusion, which is highly anisotropic in white matter fibers owing to axonal length. By measuring the degree and direction of water diffusion, it can indirectly evalue the integrity of the brain white matter fibers. The changes of apparent diffusion coefficient and fractional anisotropy after the cerebral infarction could be shown. It is the only way to discrebe the white matter fiber in vivo noninvasively.Repulsive guidance molecule a(RGMa) is a kind of axonal guidance molecule,which plays a key role in the CNS. It is a novel axonal growth inhibitor controlling the growth, proliferation and differentiation of nerve cells and repair and regeneration of the injured cells. In this study, we will treat MCAO rat model with RGMa-specific RNAi and evaluate the effect of treatment on white matter reorganization using DTI. The impact to the express of RGMa, the regeneration of the axon and the recovery of nervous function are also be observed. To certicify the importance of RGMa in the CNS injury and the mechanism of crossed cerebellum diaschisis. To find an effective target to the treatment of cerebral ischemia and CCD.Part 1 The express of RGMa in the rat brain and the relationship to axonObjective Observe the the express of RGMa, the regeneration of the axon and the recovery of nervous function. To evaluate the CCD using DTI.Methods Builting MCAO rat modle, every sixteen rats were randomly divided into one of the six groups as following: Control group, 12 h group, 24 h group, 48 h group, 7d group, 10 d group. The brain were scanned over the time points above and the rats were sacrificed for immunohistochemistry staining, and RT-PCR after MRI scanning over the time points above to assay the RGMa expression in the cortex, basal ganglia and cerebellum respectively.Results The ADC and FA value of MCAO rats declined and the peak was at 12 h. The contralateral cerebellum had the lower parameter than the other side. The express of RGMa was keeping climbing and achieved the maximum at 48h(P <0.05). The value of the protein in cerebellum was higher(P <0.05) compared with controls especiallythe right one. The express of RGMa was negative correlation to the value of MRI and the regeneration of axon.Conclusion The parameters of MRI and pathology of the cerebellum after MACO had significant difference compared to the controls, which provide evidence to CCD. RGMa was related to the regeneration of axon in the injured brain.Part 2 Construction of RGMa RNAi PlasmidObjective Construction of RGMa RNAi Plasmid for the next part.Methods Designed 3 sh RNA at frist for collection. The designed sh RNA will be taken into the target plasmid of pegensil-1 to silence the m RNA of RGMa. Sequencing appraisal and Enzyme identification will be carried out. The PC12 cell will be transfected with the plasmid with sh RNA. The effect of gene silence will be tested later by RT-PCR using the PC12 cell and to choose the best one.Results In the dsigned 3 sh RNA, we choosed the first one for the next part which had the highest transfection transfection efficiency and its concentration was 2.5×109 Pfu/ml. The concentration of Adv(-)was 2.1×109 Pfu/ml for the blank control.Conclusion Construction of RGMa RNAi Plasmid for the next step and the effect is stably and effective.Part 3 Treatment wi th RGMa-specific RNAi i n MCAO rat model and evaluation of effect using DTIObjective Remove the express of RGMa using RNA interference technique with RGMa-special-RNAi. Observe the the impact to the regeneration of the axon and the recovery of nervous function of the remote area. To evaluate the CCD using DTI.Methods Builting MCAO rat modle, every twelve rats were randomly divided into one of the four groups as following: model 2d group, RNAi intervention 2d group, blank control 2d group and negative control 2d group. The brain were scanned over the time points above and the rats were sacrificed for immunohistochemistry staining, and western blotting after MRI scanning over the time points above to assay the RGMa expression in the cortex, basal ganglia and cerebellum respectively.Results The ADC and FA value of RNAi intervention 2d group creased and the express of RGMa declined compared to the model 2d group(P <0.05). The blank control 2d group and negative control 2d group had no change to the model. The contralateral cerebellum had no change too.Conclusion The parameters of MRI and pathology of the focus in supratentorial focal brain after RNAi intervention in MACO rat model improved, which had proved that RNAi was a effective way to treat the cerebral infarction, and DTI can be a new and noninvasive technique for evaluate ICVD and CCD. |
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