Cellular basis of PVL in autopsied brains

该项目的一个关键概念是，心室周围白质软化症（PVL）是由缺血/再灌注引起的，缺血/再灌注会引发自由基对围产儿白质中脆弱的发育中的少突胶质细胞的损伤：结果，发育中的少突胶质细胞死亡，部分原因是细胞凋亡，从而导致成熟少突胶质细胞减少和髓鞘形成受损。该研究的总体假设是双重的：1)在PVL风险最大的时期（24-32周），发育中的少突胶质细胞对脑白质中的氧化应激具有特定的脆弱性，这至少部分与抗氧化酶表达的发展和促氧化途径的发展之间的不匹配有关，例如少突胶质细胞分化所需的铁的获取；2)氧化应激在PVL的发病机制中起主要作用。在基线研究中，我们将使用免疫细胞化学方法和标记物来确定人类大脑白质中少突胶质细胞的时空成熟。我们还将利用组织化学、免疫细胞化学和免疫印迹技术确定人类大脑白质中选定的抗氧化酶和促氧化途径标记物（即铁、一氧化氮合酶定位和12-脂氧合酶表达）的发育特征。在此，我们将验证以下假设：在PVL风险最高的时期，抗氧化酶在未成熟白质中表达水平较低，并且促氧化途径标记物的出现先于抗氧化酶的表达，从而表明自由基的潜在来源与清除它们的系统之间存在发育不匹配。在PVL本身，我们将确定发育中的少突胶质细胞的密度，指示这些细胞的靶向死亡和损失。我们还将确定氧化应激标志物是否在PVL中表达。最后，我们将确定选定的细胞因子在PVL中的作用。提出的研究应提供重要的见解，以发展少突胶质细胞和相关因素的氧化应激在PVL直接在人脑中的作用。

A key concept of the program project is that periventricular leukomalacia (PVL) results from ischemia/reperfusion that triggers free radical injury to vulnerable developing oligodendrocytes in perinatal white matter: as a result, developing oligodendrocytes die, in part by apoptosis, thus resulting in decreased mature oligodendrocytes and impaired myelination. The overall hypothesis of the proposed study is two-fold: 1) there is a specific vulnerability of developing oligodendrocytes to oxidative stress in the cerebral white matter in the period of greatest risk for PVL (24-32 weeks) which relates, at least in part, to a mismatch between the development of the expression of antioxidant enzymes and the development of pro-oxidant pathways, such as the acquisition of iron necessary for oligodendrocyte differentiation; and 2) oxidative stress plays a major role in the pathogenesis of PVL. In baseline studies, we will determine the temporospatial maturation of oligodendrocytes in human cerebral white matter using immunocytochemical methods and markers to stage-specific oligodendrocytes. We will also determine the developmental profile of selected antioxidant enzymes and markers of pro-oxidant pathways (i.e., iron, nitric oxide synthase localization, and 12-lipoxygenase expression) in human cerebral white matter using histochemistry, immunocytochemistry, and immunoblotting. Here we will test the hypothesis that antioxidant enzymes are expressed at low levels in immature white matter during the period of greatest risk for PVL, and that the appearance of markers of pro-oxidant pathways precede the expression of antioxidant enzymes, thus denoting a developmental mismatch between potential sources of free radicals and the systems to clear them. In PVL itself, we will determine the densities of developing oligodendrocytes, indicative of a targeted death and loss of these cells. We will also determine if markers of oxidative stress are expressed in PVL. Finally, we will determine the involvement of selected cytokines in PVL. The proposed studies should provide important insight into the roles of developing oligodendrocytes and factors related to oxidative stress in PVL directly within the human brain.