新生鼠缺氧后脑髓鞘形成障碍及P2X4可能的调控机制研究

Hypoxic and ischemic encephalopathy (HIE)can cause severe long-term behavioral disorder. The designing of an efficient therapeutic method to meliorate it has therefore been the focus of many studies. The defining feature of neuropathology in HIE is the injury susceptibility of the periventricular white matter (PWM).Some studies reported recently that activation of amoeboid microglial cells (AMC) and its related inflammatory response is followed by inflammatory response affecting the axons and oligodendrocytes. Injuries of oligodendurocytes may result in myelin degeneration which contributes to the long-term behavior disorder. However, the underlying mechanism leading to AMC activation has remained obscure. Magnetic resonance imaging (MRI)has been routinely used in clinic to assess the extent of injury in the brain. It allows for detection of subtle structural changes or alteration in PWM which may be linked to the long-term motor and cognitive abmormal in school age patients suffering from HIE. 7 Tesla magnetic resonance imaging can provide us with a more precise change of myelin by measuring the apparent diffusion coef?cient and fractional anisotropy in the white matter.In a previous study, we showed that AMC express ATP receptors P2X4 notably the first-named receptor in hypoxia in neonatal brain. It was suggested that P2X4 may be linked to regulation of AMC activation for production of proinflammatory cytokines especially in altered environment such as hypoxia. However, the putative roles of P2X4 in regulate PWM damage in hypoxic neonatal brain remains to be fully investigate. Here, we propose to investigate the underlying mechanism of P2X4 in regulating the myelin formation and long-term behavior disorder.P2X4 will be blocked by pyridoxal phosphate-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate (a slective blockade for P2X1-3,5-7) and 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate (a slective blokcade for P2X1-7) by intraperitoneal injection at three different concentrations with 4 hours after hypoxic exposure in neonatal rats. To confirm the differences between the hypoxic and P2X4 blockade group, behavior test for understanding the influence of P2X4 on long-term behavior will be conducted. In parallel to this, 7 Tesla magnetic resonance imaging for detection of myelin forming in vivo,HE staining of the PWM and hippocampus for study of the histological changes, immunohistochemistry and western blotting for evaluation of the myelin based protein known to be linked to myelin maturation will be carried out. Additionally, ultrastructural studies of the PWM and hippocampus will be performed. It is envisaged that the information obtained in this study would provide a useful structural, biochemical and molecular basis for designing of new and effective therapeutic strategies for treatement of hypoxia induced injuries in the postnatal brain.

新生儿缺血缺氧性脑病（HIE）可产生严重的远期行为学异常，研究其有效的干预措施已成为国内外关注的焦点。目前的研究发现，HIE发生时脑室周围白质(PWM)呈选择性易损，且该区域阿米巴样小胶质细胞（AMC）明显活化，继而导致髓鞘形成障碍，最终引起远期行为学异常。本项目的前期研究首次报道，AMC表达ATP受体P2X4，它与缺氧后AMC释放炎症介质的过程相关。然而，经体内抑制P2X4对新生大鼠缺氧后髓鞘形成的调控作用，迄今未见报道。本课题以远期行为学评价P2X4选择性抑制的效果，采用目前唯一能进行小动物白质纤维束成像的7T磁共振，监测新生鼠缺氧后白质髓鞘形成异常的改变。重点从整体水平，经器官、组织学、MBP蛋白表达、髓鞘超微结构评价髓鞘形成情况，从而揭示P2X4的调控效能。阐明P2X4对缺氧诱导PWM损伤后，髓鞘形成障碍、异常远期行为学的调控机制，为临床早期干预HIE的病理进程提供理论及实验依据。

项目背景：新生儿缺血缺氧性脑损伤（HIBD）因其严重的远期行为学异常，研究其有效的干预措施已成为国内外关注的焦点。目前的研究发现，HIE 发生时脑室周围白质(PWM)呈选择性易损，且导致髓鞘形成障碍，最终引起远期行为学异常。本项目的前期研究首次报道，AMC 表达ATP 受体P2X4，它与缺氧后AMC释放炎症介质的过程相关。然而，经体内抑制P2X4 对新生大鼠缺氧后髓鞘形成的调控作用，迄今未见报道。本课题以远期行为学评价P2X4 选择性抑制的效果，采用目前唯一能进行小动物白质纤维束成像的7T 磁共振，监测新生鼠缺氧后白质髓鞘形成异常的改变。重点从整体水平，经器官、组织学、MBP 蛋白表达、髓鞘超微结构评价髓鞘形成情况，从而揭示P2X4的调控效能。阐明P2X4 对缺氧诱导远期行为学的调控机制，为临床早期干预HIE 提供理论及实验依据。研究内容：1. P2X4受体对大鼠HIBD后行为学可能的作用；2. 经7-TMRI、电子显微镜、髓鞘化蛋白（MBP）的表达多角度揭示HIBD后髓鞘化的改变；3.研究P2X4受体调控对HIBD后髓鞘化障碍可能存在的作用；4.在研究发现系统性缺氧诱导HIBD模型后脑内炎症反应的情况后，根据结果进一步研究外周血细胞改变及与脑内炎症反应之间可能存在的关系；5.为深入研究新生儿缺氧缺血诱导脑损伤后可能的分子机制，探索了Gal3在该过程中可能的作用。重要结果及意义：1. P2X4受体抑制能够部分修复HIBD 后行为学异常；2. 经7-TMRI 发现HIBD后髓鞘方向性降低、电镜结果揭示HIBD后髓鞘出现疏松分层溶解、但HIBD后早期髓鞘化蛋白（MBP）的表达出现增多，以上结果支持HIBD后髓鞘化发生障碍且与髓鞘的方向性降低关系更为密切；3.P2X4阻断后髓鞘方向性好转、髓鞘超微结构变得紧密、但对MBP表达影响不显著。 4. HIBD后脑内出现炎症反应，同时外周血细胞活跃、外周血炎症介质表达升高，流式结果进一步揭示外周单核细胞入侵脑内。通过调控外周血细胞活跃可能成为减轻脑内炎症反应的有效途径。5. Gal3可导致 新生小鼠缺血再灌注损伤后72h脑内CD11b+/CD45 med-hi数量减少。成果产出：发表8篇文章，其中SCI收录3篇，PUBMED检索1篇，北大核心3篇。授权专利3项。培养研究生6人，毕业研究生2人。