【摘要】：目的:研究高頻rTMS對全腦缺血大鼠學習記憶能力、海馬CA1區(qū)組織形態(tài)學、局部場電位以及海馬PKA、p-CREB蛋白表達變化的影響,探討高頻rTMS改善全腦缺血大鼠學習記憶障礙的機制。方法:(1)采用完全隨機分組方法將雄性SD大鼠分為假手術組(SO組),模型組(MC組),假刺激組(SrTMS組),真刺激組(rTMS組)和真刺激+H89組(rTMS+H89組),rTMS+H89組大鼠造模前給予腹腔注射蛋白激酶A(protein kinase A,PKA)阻滯劑H89(5mg/kg)。(2)除SO組外,其余各組采用改良的四血管阻斷法(four vessels occlusion,4-VO)建立全腦缺血大鼠模型。(3)rTMS組以及rTMS+H89組造模成功后給予連續(xù)2周的10Hz rTMS刺激,SrTMS組造模成功后給予假刺激,MC組造模成功后不給予磁刺激。(4)各組大鼠于4-VO術前和rTMS刺激2周后,行Morris水迷宮試驗檢測大鼠空間學習能力的變化。(5)采用HE染色觀察5組大鼠海馬CA1區(qū)組織形態(tài)學改變,在體多通道記錄技術記錄5組大鼠海馬CA1區(qū)局部場電位(local field potentials,LFPs),觀察θ和γ振蕩的變化,免疫組化和Western blot檢測5組大鼠海馬PKA、p-CREB蛋白表達的變化。結果:1.Morris水迷宮結果:4-VO術前各組大鼠平均逃避潛伏期、跨越平臺次數(shù)及原平臺象限游泳時間差異無統(tǒng)計學意義(p0.05)。rTMS刺激后,與SO組對比,MC組大鼠平均逃避潛伏期延長、跨躍平臺次數(shù)減少、原平臺象限游泳時間縮短(p0.05);rTMS組較SrTMS組、MC組和rTMS+H89組大鼠平均逃避潛伏期縮短、跨躍平臺次數(shù)增加、原平臺象限游泳時間延長(p0.05);MC組和SrTMS組大鼠平均逃避潛伏期、跨越平臺次數(shù)、原平臺象限游泳時間差異無統(tǒng)計學意義(p0.05)。2.海馬CA1區(qū)LFPs結果:與SO組對比,MC組大鼠θ和γ功率譜密度均值降低(p0.05);rTMS組大鼠θ和γ功率譜密度均值較MC組、SrTMS組和rTMS+H89組增加(p0.05);MC組和SrTMS組大鼠θ和γ功率譜密度均值差異無統(tǒng)計學意義(p0.05)。3.海馬CA1區(qū)HE染色結果:SO組大鼠海馬CA1區(qū)細胞排列較整齊,細胞核清晰可見,無細胞凋亡和壞死;MC組和SrTMS組大鼠海馬CA1區(qū)細胞出現(xiàn)不同程度的細胞凋亡和壞死,表現(xiàn)為細胞數(shù)目減少、排列紊亂、胞質嗜伊紅、胞核形態(tài)不規(guī)則;rTMS+H89組海馬CA1區(qū)細胞壞死程度較MC組輕;rTMS組海馬CA1區(qū)細胞排列較整齊,僅有少量的變性和壞死。4.海馬免疫組化結果:5組大鼠海馬CA1區(qū)均有PKA、p-CREB蛋白表達(IOD值表示)。MC組PKA、p-CREB蛋白陽性細胞表達較SO組和rTMS組減少,差異有統(tǒng)計學意義(p0.05);rTMS+H89組PKA、p-CREB蛋白陽性細胞表達較rTMS組減少,差異有統(tǒng)計學意義(p0.05);MC組與SrTMS組PKA、p-CREB蛋白陽性細胞表達差異無統(tǒng)計學意義(p0.05)。5.海馬Western blot結果:5組大鼠海馬均表達一定量的PKA、p-CREB,MC組PKA、p-CREB表達較SO組減少,差異有統(tǒng)計學意義(p0.05);rTMS組PKA、p-CREB表達較MC組和rTMS+H89組增加,差異有統(tǒng)計學意義(p0.05);MC組與SrTMS組PKA、p-CREB表達差異無統(tǒng)計學意義(p0.05)。結論:1.高頻rTMS可增強全腦缺血大鼠海馬CA1區(qū)LFPsθ和γ振蕩,改善大鼠的學習記憶障礙。2.高頻rTMS可能通過激活海馬PKA-CREB信號通路,提高PKA、p-CREB蛋白表達,促進組織細胞的修復與再生,增強神經(jīng)元的突觸可塑性,改善全腦缺血大鼠的學習記憶障礙。
[Abstract]:Objective: To study the effect of high frequency rTMS on learning and memory ability, histomorphology, local field potential and the changes of PKA and p-CREB protein expression in hippocampus in CA1 region of the hippocampus of rats. Methods: (1) the male SD rats were divided into sham operation group by complete random grouping method. (group SO), model group (group MC), pseudo stimulus group (group SrTMS), real stimulation group (group rTMS) and real stimulation +H89 group (group rTMS+H89), and group rTMS+H89 rats were given intraperitoneal injection of protein kinase A (protein kinase A, PKA) blocker before modeling. (2) the other groups were established by improved four vascular blocking method. The model of whole brain ischemia rat. (3) rTMS group and rTMS+H89 group were given 10Hz rTMS stimulation after successful modeling, SrTMS group was given fake stimulation after successful modeling, and group MC did not give magnetic stimulation after successful model building. (4) the rats in each group were tested by Morris water maze test before and 2 weeks after 4-VO and rTMS stimulation. (5) HE staining was used to observe the histomorphological changes in the hippocampal CA1 region of the 5 groups of rats. The local field potential (local field potentials, LFPs) in the hippocampal CA1 region of the 5 groups of rats was recorded in the body multichannel recording technique. The changes of theta and gamma oscillations were observed. The changes of PKA and p-CREB protein in the hippocampus of 5 groups of rats were detected by immunohistochemistry and Western blot. Results: the 1.Morris water maze junction Results: the average escape latency of rats in each group before 4-VO was not significant, and there was no significant difference between the number of cross platform times and the original platform quadrant swimming time (P0.05).RTMS stimulation. Compared with the SO group, the average escape latency of the MC group was prolonged, the frequency of the leap platform decreased and the original platform quadrant swimming time shortened (P0.05); the rTMS group was compared with the SrTMS group, MC group and rTMS+H89. The average escape latency was shortened, the number of leaping platforms increased and the original platform quadrant swimming time was prolonged (P0.05). The average escape latency of the MC and SrTMS group rats, the number of crossing platforms, and the difference in the original platform quadrant swimming time were not statistically significant (P0.05), LFPs results in the CA1 region of the.2. hippocampus: the power spectral density of theta and gamma in the MC group was compared with the SO group. Mean value decreased (P0.05), and the mean value of theta and gamma power spectral density in group rTMS rats was higher than that of group MC, SrTMS and rTMS+H89 (P0.05), and there was no significant difference in the mean value of theta and gamma power spectral density in MC and SrTMS rats (P0.05).3. hippocampus CA1 region HE staining results: the cells in the hippocampus of the hippocampus were neatly arranged, the nuclei were clearly visible, and no cell apoptosis was found. In group MC and group SrTMS, the cells of CA1 area of hippocampus of the rats of group SrTMS were apoptotic and necrotic in varying degrees, which showed that the number of cells decreased, the arrangement of the cells was disorderly, the cytoplasm eosinophil was irregular, and the necrosis degree of the cells in the CA1 area of the hippocampus of the rTMS+H89 group was lighter than that of the MC group, and the cells in the CA1 area of the hippocampus of the rTMS group were neatly arranged, only a small amount of degeneration and necrotic.4. were found. The results of hippocampal immunohistochemical staining: 5 groups of rats in the hippocampus CA1 area were PKA, p-CREB protein expression (IOD value).MC group PKA, p-CREB protein positive cells expressed less than the SO group and rTMS group, the difference was statistically significant (P0.05), rTMS+H89 group PKA, protein positive cells decreased, the difference was statistically significant. The difference in expression of p-CREB protein positive cells was not statistically significant (P0.05).5. hippocampus Western blot results: the hippocampus of the 5 rats expressed a certain amount of PKA, p-CREB, MC group PKA, p-CREB expression was less than SO group, the difference was statistically significant (P0.05). There is no significant difference in the expression of PKA and p-CREB in group rTMS (P0.05). Conclusion: 1. high frequency rTMS can enhance LFPs theta and gamma oscillation in hippocampus CA1 region of rats with whole brain ischemia, and improve the high frequency rTMS of learning and memory impairment.2. in rats by activating hippocampus PKA-CREB signaling pathway, enhancing PKA, p-CREB egg white expression, promoting the repair and regeneration of tissue cells and enhancing the God. The synaptic plasticity of Yuan Yuan can improve learning and memory impairment in rats with global cerebral ischemia.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.1

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