CREB信号调控大鼠脑白质损伤后海马神经再生及对认知功能损伤康复的研究

Perinatal infection is an important cause of neonatal neurologic morbidity. Periventricular white matter damage (periventricular leukomalacia) is the predominant form in premature infants and the most common antecedent of cerebral palsy. Neurogenesis arising from populations of neural progenitors that persist in the sub-ventricular zone (SVZ) and sub-granular zone (SGZ) of the dentate gyrus (DG) after hypoxia-ischemia are now a universally accepted feature of the adult mammalian brain. However, it remains unknown whether neurogenesis is induced by perinatal infection in the developing brain. The cAMP response element binding protein (CREB) is a transcription factor in eukaryote and plays important roles in the regulation of neurogenesis，synapse formation，learning and memory. The phosphorylation of CREB is the important way to regulate transcription, and the transcription of many target genes is activated after the phosphorylation of CREB affected by extracellular signals, showing various physiological functions. The goal of the current study is to examine the effect of intrauterine infection on hippocampal neurogenesis and to examine whether the CREB signaling pathway is involved in inflmmation-induced neurogenesis and the associated molecular mechanism. This hypothesis is supported by further findings that LY294002, the PI3K inhibitor, induced a reduction of hippocampal progenitor cells in DG after intrauterine infection and down-regulated p-Akt and p-CREB expression. Using the intrauterine E. coli infected model, we study the spatial correlation between Akt and CREB, learning and memory. Furthermore, the BDNF and its receptor TrkB, both being downstream gene transcripts of p-CREB, are studied. In this study, we use immunohistochemistry to examine cell proliferation and investigate neuronal survival and differentiation in hippocampal DG. The Morris Water Maze (MWM) test is used to study hippocampal-dependent learning, including acquisition of spatial memory and long-term spatial memory after blocking PI3K/Akt signaling pathway in intrauterine infection model. Here we use patch clamp technique to demonstrate deficits in long-term synaptic plasticity and loss of dendritic spines in the hippocampus of white matter damage model, and study the role of CREB signaling pathway in regulating hippocampal cognitive function and synaptic plasticity. The identified genetic pathways provide insight into the inflmmation-induced neurogenesis that is so central in learning and memory and will direct future clinic studies on the functional rehabilitation of cerebral palsy.

妊娠早期胎儿炎症反应综合征是造成胎儿早产和早产儿脑白质损伤的最常见原因，临床与流行病学资料证实早产儿脑白质损伤是儿童脑瘫的一个重要危险因素。由于早产儿脑白质损伤将影响新生儿后期神经系统发育，所以脑白质损伤后神经再生修复机制的研究将显得尤为重要。cAMP反应序列结合蛋白(CREB)是一种重要的核转录因子，调节启动子中具有环磷酸腺苷反应单元(CRE)的基因转录，在神经元再生、突触形成及学习记忆等方面具有重要调节作用，是细胞内多种信号通路的一种关键成分。前期研究发现宫内炎症脑白质损伤能诱导海马神经再生，但CREB信号通路是否参与脑白质损伤后神经再生及相关分子机制尚未明确。因此，通过建立宫内炎症脑白质损伤模型来研究CREB信号通路与海马神经再生的相互关系，以及通过对CREB信号通路在神经再生中分子机制的研究，阐述CREB信号通路在神经再生及认知功能改善中的重要作用，为脑瘫的康复治疗提供了新的思路。

妊娠早期胎儿炎症反应综合征是造成胎儿早产和早产儿脑白质损伤的最常见原因，早产儿脑白质损伤是儿童脑瘫的一个重要危险因素。由于围生期是大脑发育的关键时期，早产儿脑白质损伤将影响新生儿后期神经系统发育，将造成儿童严重的运动障碍和不可逆的认知功能障碍，所以对脑白质损伤后神经再生修复及认知功能损伤康复机制的研究将显得尤为重要。近年研究发现哺乳动物海马齿状回颗粒细胞下区（SGZ）神经再生能力持续存在，神经前体细胞能在颗粒细胞下层发生增殖，迁移到颗粒细胞层，逐渐成熟、分化为神经元及胶质细胞，对已发生凋亡的神经元起到修复和替代的作用，并能功能性整合到海马神经环路中，这些可塑性改变在海马依赖性空间学习和记忆中发挥重要作用。cAMP反应序列结合蛋白(CREB)是一种重要的核转录因子，在神经元再生、突触形成及学习记忆等方面具有重要的调节作用，是细胞内多种信号通路的一种关键成分。本研究应用免疫组化、激光共聚焦显微镜观察海马神经细胞增殖、神经元和星形胶质细胞分化；采用Morris水迷宫观察宫内感染脑白质损伤后仔鼠空间学习记忆改变；应用膜片钳检测技术观察海马神经突触功能；RT-PCR及Western blot方法检测CREB信号通路上下游基因的表达，进而明确CREB信号通路参与宫内感染脑白质损伤后海马神经再生。分别应用PI3K阻滞剂LY294002阻断PI3K/Akt/CREB信号通路，磷酸二酯酶抑制剂rolipram，增加脑内cAMP浓度从而激活CREB信号通路，来观察海马神经再生、学习记忆功能改变以及CREB信号通路上下游基因的表达变化。研究发现宫内感染后仔鼠海马齿状回BrdU+细胞和BrdU+/Nestin+细胞显著增加（P<0.01），说明宫内炎症脑白质损伤后可能会促进海马齿状回细胞增殖，且增殖细胞可能为神经干细胞来源。BrdU+/GFAP+和BrdU+/NeuN+细胞的比率无统计学差异（P>0.01），说明宫内感染不影响星形胶质细胞和神经元的分化。宫内感染后BDNF、TrkB、Akt、Survivin表达明显增高（均P<0.05），提示BDNF/PI3K/Akt信号通路可能参与调节宫内炎症脑白质损伤后海马的神经发生。Morris水迷宫实验发现宫内感染后仔鼠游泳轨迹杂乱无章，逃避潜伏期延长（P<0.05），象限停留时间和穿越数减少（P<0.05），说明宫内感染可影响仔鼠的学习记忆功能。