早产儿脑损伤微环境及VEGF-Wnt/β-catenin信号轴重塑NVU的机制

Reconstruction of neuron vessel unit (NVU) is the key to treat preterm neonates with brain injury. And a good microenvironment is the fundamental guarantee of NVU remodeling in damaged brain. We proved previously that gene therapy with vascular endothelial growth factor (VEGF) was effective in the treatment of ischemic stroke. We recently found that VEGF level decreased significantly in preterm rats with white matter damage(WMD) , in vitro cultured hippocampal organotypic ischemia model, we also found that VEGF can significantly increase the expression of β-catenin protein. These results and analysis have led to the formation of our hypothesis of this study that VEGF improves the microenvironment and activate Wnt/β-catenin signaling pathways to promote reconstruction of NVU in preterm rats with brain injury . We have developed the following three specific aims to test our hypothesis on a preterm rat (3d neonatal rat) WMD model: 1) Detecting the molecular events in brain microenvironment, especially VEGF level and newborn vessles, before and after brain damage and treatment with VEGF gene therapy 2) Detecting the number of newborn neurons, vascular endothelial cells, oligodendrocytes and electrophysiological changes in different brain regions with immunofluorescence and patch-clamp techniques 3) Analyzing the ways and rules of VEGF promoting nerve regeneration, angiogenesis and its effect on Wnt / β-catenin signaling pathways with single-cell gene expression profile. The completion of this study will help to uncover the molecular mechanism triggered by VEGF gene therapy that improves the microenvironment to promote neurogenesis and angiogenesis in preterm neonates with brain injury.

神经血管单元（NVU）重塑是治疗早产儿脑损伤的关键，而良好的脑微环境是其根本保障。我们前期研究证实血管内皮生长因子（VEGF）基因治疗可明显改善大鼠缺血性脑损伤，最近发现早产脑白质损伤（WMD）鼠脑组织VEGF水平显著下降；且VEGF可明显增加体外培养海马器官型脑片β-catenin的表达。因此，我们假设：VEGF通过改善脑微环境，激活Wnt/β-catenin信号通路，促进NVU重塑。籍此，本研究拟采用早产鼠WMD模型，检测脑损伤前后及VEGF治疗前后脑微环境中的生物学事件，尤其是VEGF水平及新生血管，全面解析早产WMD鼠脑炎性微环境的时空特征；运用免疫荧光技术及膜片钳技术检测不同脑区域的新生神经元、血管内皮细胞、少突胶质细胞的数量及电生理，结合单细胞基因表达谱，解析VEGF促进神经再生、血管新生的途径和规律及对Wnt/β-catenin信号的依赖性，揭示VEGF重塑NVU的机制。

背景：本课题探索血管内皮生长因子A (Vascular endothelial growth factor A, VEGF)基因治疗在早产儿缺氧缺血性脑损伤(Hypoxic ischemic brain damage, HIBD)HIBD中的疗效，及其与Wnt/β-catenin轴相关的作用机制。方法：通过缺氧及右侧颈总动脉结扎诱导新生3天的SD大鼠缺氧缺血，建立早产HIBD模型，立体定位脑室注射过表达VEGF基因的慢病毒，观察并记录VEGF基因治疗1周及2周后乳鼠的神经行为学变化；分别取治疗7天和14天的乳鼠脑组织作组织切片，进行组织免疫荧光染色，检测皮层及海马中VEGF、Wnt3a、β-catenin、CD34、神经元特异核蛋白（Neuronal nuclei, NeuN）的蛋白表达，TTC染色以观察脑组织梗死体积；腹腔注射Wnt/β-catenin通路抑制剂LGK-974后，对上述指标进一步检测；最后，体外培养原代神经元建立细胞糖氧剥夺（Glucose and oxygen deprivation, OGD）模型，通过PI/Hoechst染色观察VEGF基因治疗的神经保护作用；类似地，使用LGK-974干预，检测VEGF、Wnt3a、β-catenin的蛋白表达。结果：1. VEGF基因治疗可显著降低早产鼠HIBD的行为学评分（p＜0.001）；2. 组织免疫荧光CD34和NeuN结果显示，VEGF基因治疗促进了血管的新生（p＜0.05），改善了神经元的损伤（p＜0.001）；3. 组织TTC染色显示，VEGF基因治疗组脑梗死体积相比于HIBD组减少了2倍（p＜0.01）；4. VEGF基因治疗显著上调了Wnt/β-catenin的mRNA和蛋白表达（p＜0.01）；5. 在原代神经元OGD模型中，通过PI/Hoechst染色发现，VEGF显著减少了神经元的死亡（p＜0.05），并且上调了Wnt/β-catenin的蛋白表达，而使用LGK-974后，VEGF的促血管新生及神经保护作用均显著降低。结论：1.VEGF基因治疗对早产大鼠HIBD具有显著的促血管新生和神经保护作用；2. VEGF基因治疗可以改善早产HIBD大鼠的行为学评分；3. VEGF通过激活Wnt/β-catenin轴对早产HIBD大鼠发挥神经保护作用。