Role of Extracellular Matrix in Hypoxic-Ischemic Perinatal White Matter Injury

描述（由申请人提供）：白色物质损伤（白质损伤）是早产幸存者终身神经系统残疾的主要原因。我们最近在一个大型人体尸检系列中证明了这一点（Buser等人，2012），主要形式的脑梗死包括弥漫性病变，坏死是次要成分。弥漫性脑脊髓炎髓鞘形成失败的机制涉及晚期少突胶质细胞祖细胞（preOL）的成熟停滞，发生时无明显轴突病变。前OL停滞与星形胶质细胞增生的程度显著相关，这支持了来自星形胶质细胞病变的因素有助于前OL停滞的假设。弥漫性人视网膜富含星形胶质细胞相关的透明质酸（HA），其在细胞外基质中弥漫性积聚。我们发现，高分子量（MW）形式的HA被透明质酸酶加工成低分子量形式，其在体外抑制前OL成熟并延迟实验诱导的脱髓鞘后的髓鞘再生。我们鉴定了GPI锚定的透明质酸酶PH 20的新CNS表达，其定位于新生儿脑中的前OL，并且在白色病变中的前OL和星形胶质细胞内显示出升高的表达。我们建议定义新的机制，涉及处理HA的生物活性较低的MW形式，防止前OL在体外和体内成熟。我们将测试的总体假设，即增强表达的PH 20介导的低分子量形式的HA，抑制前OL成熟髓鞘化OL的生成。目的1解决了一系列相关的机制问题，这些问题将定义在新生大鼠慢性脑缺血的两种互补模型中介导前OL停滞的HA分子的特定大小范围：缺氧缺血（H-I）的早产等效模型和显示出稳健的星形胶质细胞增生和前OL停滞的切片培养模型。 目的2与目的1整合以定义正常对照中体内HA的组织水平的调节和从H-I演变的新生儿视网膜病变中HA的组织水平的调节。我们将定量HA酶和透明质酸酶（包括PH 20）的发育表达。目的3将确定PH 20功能的获得或丧失对前OL成熟和髓鞘形成的影响。我们将量化在正常WM发育过程中和体外慢性胰腺炎中以及新生儿H-I后由慢病毒驱动的PH 20过表达介导的preOL停滞的程度。补充研究将量化由PH 20抑制剂递送至H-I引起的慢性白色病变促进的前OL成熟和髓鞘形成的程度。目的4将确定人类慢性脑脊髓炎髓鞘形成失败的时间进程，以及它是否持续或显示部分解决。这些人体研究将与目标1-3中的啮齿动物研究相结合，以确定人前OL停滞与反应性星形胶质细胞增生生物标志物（GFAP和CD 44）、PH 20表达和总HA水平的相关性。我们希望获得新的见解机制，触发失败的正常髓鞘在慢性脑脊髓炎，从而定位于开发新的策略，以促进正常髓鞘的早产幸存者。

DESCRIPTION (provided by applicant): White matter injury (WMI) is the leading cause of life-long neurological disability in survivors of premature birth. We recently demonstrated in a large human autopsy series (Buser et al., 2012) that the major form of WMI comprises diffuse lesions and necrosis is a minor component. The mechanism of myelination failure in diffuse WMI involves arrested maturation of late oligodendrocyte progenitors (preOLs) and occurs without significant axonopathy. PreOL arrest was significantly associated with the magnitude of astrogliosis, which supports the hypothesis that factors derived from the astrogliotic lesion contribute to preOL arrest. Diffuse human WMI was enriched in astrocyte-associated hyaluronic acid (HA) that diffusely accumulated in the extracellular matrix. We found that higher molecular weight (MW) forms of HA are processed by hyaluronidases to lower MW forms that inhibit preOL maturation in vitro and delay re-myelination after experimentally-induced demyelination. We identified novel CNS expression of a GPI-anchored hyaluronidase, PH20, that localizes to preOLs in neonatal brain and which shows elevated expression within preOLs and astrocytes in white matter lesions. We propose to define novel mechanisms that involve processing of HA to bioactive lower MW forms that prevent preOL maturation in vitro and in vivo. We will test the overall hypothesis that the enhanced expression of PH20 in WMI mediates the generation of lower MW forms of HA that inhibit preOL maturation to myelinating OLs. Aim 1 addresses a series of related mechanistic questions that will define the specific size ranges of HA molecules that mediate preOL arrest in two complementary models of chronic WMI in the neonatal rat: a preterm- equivalent model of hypoxia-ischemia (H-I) and a slice culture model that displays robust astrogliosis and preOL arrest. Aim 2 integrates with aim 1 to define the regulation of tissue levels of HA in vivo in normal controls and in evolving neonatal WMI from H-I. We will quantify the developmental expression of both the HA synthases and hyaluronidases, including PH20. Aim 3 will define the effect of PH20 gain or loss of function on preOL maturation and myelination. We will quantify the magnitude of preOL arrest mediated by lentiviral-driven PH20 over-expression during normal WM development and in chronic WMI in vitro and after neonatal H-I. Complementary studies will quantify the magnitude of preOL maturation and myelination promoted by delivery of PH20 inhibitors to chronic white matter lesions arising from H-I. Aim 4 will define the temporal course of myelination failure in chronic human WMI and whether it persists or shows partial resolution. These human studies will integrate with the rodent studies in aims 1-3 to define the association of human preOL arrest with biomarkers of reactive astrogliosis (GFAP and CD44), PH20 expression, and total HA levels. We expect to gain new insights into mechanisms that trigger a failure of normal myelination in chronic WMI, and, thereby, be positioned to develop new strategies to promote normal myelination in survivors of premature birth.