早期多感觉刺激对缺氧缺血性脑损伤新生大鼠纹状体突触结构重塑及p-ERK/BDNF信号反馈环路的影响

关键词：新生缺氧缺血性脑损伤  早期多感觉刺激  突触重塑  磷酸化的细胞外信号调节激酶  脑源性神经营养因子

Keywords:neonatal hypoxic-ischemic brain damage  early multi-sensory stimulation  synaptic remodeling  phosphorylated extracellular signal-regulated kinase  brain-derived neurotrophic factor

摘要：

      摘要 目的：观察早期多感觉刺激对缺氧缺血性脑损伤（hypoxic-ischemic brain damage， HIBD）新生大鼠的治疗作用及对磷酸化激活的细胞外调节蛋白激酶（phosphorylated extracellular regulated protein kinases，p-ERK）、磷酸化激活的环磷腺苷效应元件结合蛋白（phosphorylated cAMP-response element binding protein，p-CREB）、脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）、BDNF的特异性受体（tyrosinekinase receptor B，TrkB）、突触蛋白1（synaptophysin 1，SYN1）、突触囊泡蛋白（synapsin，SYP）蛋白表达的影响，从p-ERK/p-CREB/BDNF/TrkB信号反馈环路探索早期多感觉刺激对HIBD新生大鼠纹状体突触重塑的影响。 方法：64只7日龄健康新生大鼠以随机数字表法分为空白组、假手术组、模型组、早期多感觉刺激组，每组16只。空白组大鼠不做任何处理，模型组和早期多感觉刺激组大鼠采用经典Rice法制备HIBD模型，假手术组仅暴露颈总动脉，不进行电凝。早期多感觉刺激组造模后24h予持续28d的多感觉刺激干预。采用悬吊试验检测各组大鼠随意运动功能肢体的肌力；平衡木试验检测各组大鼠平衡及运动控制能力；HE染色观察纹状体组织细胞形态学变化；电镜观察纹状体突触数量、突触活性区长度、厚度以及间隙宽度改变；免疫组织化学法观察纹状体TrkB、Syn1表达水平； Western Blot法检测各组大鼠纹状体中p-ERK、p-CREB、BDNF、TrkB、Syn1、Syp的蛋白表达水平。 结果：①与空白组和假手术组比较，造模后第14、21、28天模型组新生大鼠悬吊时间明显减少（P<0.01），造模后第21、28天模型组新生大鼠平衡木试验评分显著升高（P<0.01）；缺血侧纹状体突触密度降低（P<0.01），突触后致密区变薄（P<0.01）；缺血侧纹状体p-ERK（P<0.05）、p-CREB（P<0.01）、BDNF（P<0.01）、TrkB（P<0.01）、Syn1（P<0.05）、Syp（P<0.05）蛋白表达水平显著下调。②与模型组比较，造模后第28天早期多感觉刺激组新生大鼠悬吊时间明显增加（P<0.01），造模后第21、28天早期多感觉刺激组新生大鼠平衡木试验评分显著降低（P<0.01）；纹状体突触密度增加（P<0.05），突触后致密区增厚（P<0.01）；缺血侧纹状体p-ERK（P<0.05）、p-CREB（P<0.05）、BDNF（P<0.05）、TrkB（P<0.01）、Syn1（P<0.01）、Syp（P<0.01）蛋白表达水平显著上调。 结论：早期多感觉刺激可有效促进缺氧缺血性脑损伤新生大鼠神经运动功能恢复和突触重塑，其作用机制与激活p-ERK/p-CREB/BDNF/TrkB正反馈环路有关。

Abstract:

      Abstract Objective： To investigate the therapeutic effects of early multi-sensory stimulation on neonatal rats with hypoxic-ischemic brain damage （HIBD） and its influence on the protein expression of phosphorylated extracellular signal-regulated kinase （p-ERK）， phosphorylated cAMP-response element-binding protein （p-CREB）， brain-derived neurotrophic factor （BDNF）， BDNF's specific receptor （tyrosine kinase receptor B， TrkB）， synaptophysin 1 （Syn1）， and synapsin （SYP）. The study aims to explore the impact of early multi-sensory stimulation on synaptic remodeling in the striatum of neonatal HIBD rats， with a focus on the p-ERK/p-CREB/BDNF/TrkB signaling feedback loop. Method： Sixty-four healthy 7-day-old neonatal rats were randomly assigned to four groups using a random number table： the blank group， sham-operated group， model group， and early multi-sensory stimulation group， with 16 rats per group. The blank group received no treatment. In the model and early multi-sensory stimulation groups， the HIBD model was established using the classical Rice method， while the sham-operated group underwent exposure of the common carotid artery without electrocoagulation. In the early multi-sensory stimulation group， multi-sensory stimulation was initiated 24 hours post-modeling and continued for 28 days. The hanging test was utilized to assess limb muscle strength related to voluntary motor function in each group， while the balance beam test evaluated balance and motor control. Electron microscopy was employed to examine changes in the number of synapses in the striatum， including alterations in the length and thickness of the active zone and synaptic cleft width. Immunohistochemistry was used to assess TrkB and Syn1 expression levels in the striatum. Western Blot analysis was conducted to determine the protein expression levels of p-ERK， p-CREB， BDNF， TrkB， Syn1， and Syp in the striatum of each group. Result： ①Compared to the blank and sham-operated groups， the model group exhibited a significant decrease in hanging time on the 14th， 21st， and 28th days post-modeling （P<0.01）. On the 21st and 28th days post-modeling， balance beam test scores were significantly higher in the Model group （P<0.01）； the number of synaptic vesicles in the striatum was significantly reduced， and the length and thickness of the active zone were diminished. Protein expression levels of p-ERK （P<0.05）， p-CREB （P<0.01）， BDNF （P<0.01）， TrkB （P<0.01）， Syn1 （P<0.05）， and Syp （P<0.05） in the striatum were significantly downregulated.②In comparison to the Model group， the early multi-sensory stimulation group showed a significant increase in hanging time on the 28th day post-modeling （P<0.01）. On the 21st and 28th days post-modeling， balance beam test scores were significantly lower in the early multi-sensory stimulation group （P<0.01）； the number of synaptic vesicles in the striatum significantly increased， and the length and thickness of the active zone were enhanced. Protein expression levels of p-ERK （P<0.05）， p-CREB （P<0.05）， BDNF （P<0.05）， TrkB （P<0.01）， Syn1 （P<0.01）， and Syp （P<0.01） in the striatum were significantly upregulated. Conclusion： Early multi-sensory stimulation effectively enhances the recovery of motor function and promotes synaptic remodeling in neonatal rats with hypoxic-ischemic brain damage. This effect may be associated with the activation of the p-ERK/p-CREB/BDNF/TrkB positive feedback loop.