低频功能性电刺激对痴呆大鼠认知功能影响的cAMP/PKA-MAPK-CREB-BDNF-LTP通路的机制研究

Exercise can improve the cognition of the healty and the patient ,such as the stroke, the Alzhiher disease, the TBI and the Parkinson disease.Our previous study on the stroke rats found that low-frenquency functional electrical stimulations (LFFES) could increase the brain-derived neurotrophic factor (BDNF) at the area of hippocampus, which indicated that LFFES facilitates synaptic plasticity through the related pathway. The randomized clinical trial on patients with stroke at either acute or chronic stage conducted by our team have demonstrated that LFFES improved the functional recovery of patients with stroke through strengthening the activity-dependent plasticity. Therefore, we hypothesis that LFFES strengthen the cortical plasticity of rat with ischemic stroke via the pathway of BDNF-LTP,and improve the cogniton. The LFFES maybe can increase the BDNF level, active the pathway of cAMP/PKA-MAPK-CREB-BDNF, and facilatate the LTP at the hippocampus, and then improve the cognition of the demetia rat. In this study, we will first establish the dementia model induced by four-vessel occlusion(4-VO) in Wistar rat. And then usethe low-frenquency functional electrical stimulation. Moreover, after the treatment with LFFES, the following was measured, including the structure of the synaptic junction, amplitude of LTP in vivo and in the hippocampus area and the changes of protein expression associated cAMP/ PKA-CREB-of BDNF-LTP signaling pathways in the maintain phase of LTP. The results of this study are expected to identify whether LFFES on the limb of rats with 4-VO can improve the cogniton of the dementia rat,the treatment of LFFES can be used to the early stage stroke and the patient without the initiative to do exercise ,enhence the effectcy of the neurorehabilitation.

运动可以通过BDNF介导的信号机制改善认知。我们的基础研究发现低频功能性电刺激（LFFES）能增强脑卒中大鼠的运动功能，提高海马BDNF水平，提示其通过BDNF相关通路易化突触可塑性；临床研究显示LFFES通过增强使用依赖性可塑性改善脑卒中肢体运动功能。因此我们假设LFFES诱导产生的运动也可以通过BDNF-LTP通路提高脑梗死大鼠皮层可塑性，并通过cAMP/PKA-MAPK-CREB通路改善认知功能，为临床提供新的认知康复手段。本研究采用大鼠四血管阻断痴呆模型，给予低频电刺激，诱导肢体运动，采用电镜和体视学方法研究突触结构可塑性变化，分子生物学技术检测cAMP/PKA-CREB-BDNF -LTP通路相关核酸/蛋白的表达，电生理技术检测在体和脑片海马区LTP变化，morris水迷宫、新事物认知实验和巴恩斯迷宫检测学习记忆功能，以阐明痴呆大鼠应用此方法提高认知的效果和机制，便于临床推广应用

我们的既往研究表明低频功能性电刺激(LFFES)能增强脑卒中大鼠的运动功能，提高海马BDNF水平，提示其通过 BDNF相关通路易化突触可塑性；已有的临床研究显示功能性电刺激可以通过依赖性神经可塑性改善脑卒中肢体运动功能。本课题假设 LFFES 诱导产生的运动也可以通过 BDNF-LTP通路提高脑梗死大鼠皮层可塑性，为临床提供新的认知康复手段。本研究采用双侧颈总动脉永久性结扎法制作血管性痴呆模型。用随机数字法分为假手术组、模型组、自主运动、强迫运动和功能性电刺激组，采用新奇事物识别实验、巴恩斯试验和水迷宫试验测试大鼠学习记忆能力，取大鼠海马及前额叶皮层采用Western blot技术检测上述各组SYN、SYP、PSD-95及MAP-2、TAU蛋白表达，采用免疫组织化学染色方法检测上述各组MAP-2表达。结果显示：1.自主运动、强迫运动和功能性电刺激诱导的运动均可促进VD大鼠学习记忆能力的恢复，其可能机制为运动训练可改善VD大鼠海马及前额叶皮层区突触结构可塑性；2. 功能性电刺激诱导的运动可通过促进海马及前额叶皮层区SYN、PSD-95、MAP-2、TAU蛋白表达，以改善VD大鼠学习记忆能力。3.低频电刺激组可显著上调可塑性经典通路AMPAR，pAMPAR，NMDARR1表达，明显上调BDNF，pERK1/2，pCaMKII，pCREB蛋白的表达，增加新奇事物鉴别指数。