高频rTMS对全脑缺血大鼠学习记忆的影响及机制探讨
发布时间：2018-07-20 11:11
【摘要】：目的:研究高频rTMS对全脑缺血大鼠学习记忆能力、海马CA1区组织形态学、局部场电位以及海马PKA、p-CREB蛋白表达变化的影响,探讨高频rTMS改善全脑缺血大鼠学习记忆障碍的机制。方法:(1)采用完全随机分组方法将雄性SD大鼠分为假手术组(SO组),模型组(MC组),假刺激组(SrTMS组),真刺激组(rTMS组)和真刺激+H89组(rTMS+H89组),rTMS+H89组大鼠造模前给予腹腔注射蛋白激酶A(protein kinase A,PKA)阻滞剂H89(5mg/kg)。(2)除SO组外,其余各组采用改良的四血管阻断法(four vessels occlusion,4-VO)建立全脑缺血大鼠模型。(3)rTMS组以及rTMS+H89组造模成功后给予连续2周的10Hz rTMS刺激,SrTMS组造模成功后给予假刺激,MC组造模成功后不给予磁刺激。(4)各组大鼠于4-VO术前和rTMS刺激2周后,行Morris水迷宫试验检测大鼠空间学习能力的变化。(5)采用HE染色观察5组大鼠海马CA1区组织形态学改变,在体多通道记录技术记录5组大鼠海马CA1区局部场电位(local field potentials,LFPs),观察θ和γ振荡的变化,免疫组化和Western blot检测5组大鼠海马PKA、p-CREB蛋白表达的变化。结果:1.Morris水迷宫结果:4-VO术前各组大鼠平均逃避潜伏期、跨越平台次数及原平台象限游泳时间差异无统计学意义(p0.05)。rTMS刺激后,与SO组对比,MC组大鼠平均逃避潜伏期延长、跨跃平台次数减少、原平台象限游泳时间缩短(p0.05);rTMS组较SrTMS组、MC组和rTMS+H89组大鼠平均逃避潜伏期缩短、跨跃平台次数增加、原平台象限游泳时间延长(p0.05);MC组和SrTMS组大鼠平均逃避潜伏期、跨越平台次数、原平台象限游泳时间差异无统计学意义(p0.05)。2.海马CA1区LFPs结果:与SO组对比,MC组大鼠θ和γ功率谱密度均值降低(p0.05);rTMS组大鼠θ和γ功率谱密度均值较MC组、SrTMS组和rTMS+H89组增加(p0.05);MC组和SrTMS组大鼠θ和γ功率谱密度均值差异无统计学意义(p0.05)。3.海马CA1区HE染色结果:SO组大鼠海马CA1区细胞排列较整齐,细胞核清晰可见,无细胞凋亡和坏死;MC组和SrTMS组大鼠海马CA1区细胞出现不同程度的细胞凋亡和坏死,表现为细胞数目减少、排列紊乱、胞质嗜伊红、胞核形态不规则;rTMS+H89组海马CA1区细胞坏死程度较MC组轻;rTMS组海马CA1区细胞排列较整齐,仅有少量的变性和坏死。4.海马免疫组化结果:5组大鼠海马CA1区均有PKA、p-CREB蛋白表达(IOD值表示)。MC组PKA、p-CREB蛋白阳性细胞表达较SO组和rTMS组减少,差异有统计学意义(p0.05);rTMS+H89组PKA、p-CREB蛋白阳性细胞表达较rTMS组减少,差异有统计学意义(p0.05);MC组与SrTMS组PKA、p-CREB蛋白阳性细胞表达差异无统计学意义(p0.05)。5.海马Western blot结果:5组大鼠海马均表达一定量的PKA、p-CREB,MC组PKA、p-CREB表达较SO组减少,差异有统计学意义(p0.05);rTMS组PKA、p-CREB表达较MC组和rTMS+H89组增加,差异有统计学意义(p0.05);MC组与SrTMS组PKA、p-CREB表达差异无统计学意义(p0.05)。结论:1.高频rTMS可增强全脑缺血大鼠海马CA1区LFPsθ和γ振荡,改善大鼠的学习记忆障碍。2.高频rTMS可能通过激活海马PKA-CREB信号通路,提高PKA、p-CREB蛋白表达,促进组织细胞的修复与再生,增强神经元的突触可塑性,改善全脑缺血大鼠的学习记忆障碍。
[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.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.1
本文编号：2133332
[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.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.1
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