Cytokines and the blood-brain barrier in the ovine fetus

描述（由申请人提供）：缺氧缺血性脑损伤是围产期最重要的神经系统问题。子宫内血流动力学异常可能与促炎性细胞因子（细胞因子）（如IL-12、IL-6和TNF-1）升高相关，易导致脑损伤，特别是在早产儿中。在母体感染期间产生的全身性细胞因子和/或缺血后细胞因子的增加可能加重对胎儿脑的损伤。神经血管单位是由脑微血管内皮、神经胶质和神经元组成的特殊部位。虽然已知细胞因子可穿过成人血脑屏障（BBB），但支持全身性细胞因子可穿过胎儿和早产儿血脑屏障这一假设的证据很少。我们的具体目标是测试特定细胞因子穿过胎儿中完整和受损的血脑屏障以损害大脑的假设。该假设的结果是，阻断这些细胞因子将减弱对神经血管单元（BBB）和可能的脑的缺血相关损伤。一个多学科的方法将被用来解决我们的假设，将包括生理，生化，病理，免疫组织化学和分子方法。目的1检验细胞因子如IL-12和IL-6在绵羊胎儿中以成熟依赖性方式穿过血脑屏障的假设，以及细胞因子屏障通透性的成熟相关变化主要与紧密连接组成的变化有关。将通过积分技术用1-氨基异丁酸和放射性标记的细胞因子定量BBB渗透性。将通过蛋白质印迹、免疫组织化学和北方印迹测量紧密连接（TJ）蛋白和mRNA。目的2确定缺血性损伤是否增加血脑屏障对细胞因子的通透性，作为胎龄和紧密连接成熟的函数。脑缺血是由颈动脉闭塞引起的。将如目标1中测量BBB渗透性和TJ组分，并通过病理学、免疫组织化学和分子方法评估脑损伤。目的3确定是否阻断细胞因子与中和抗体的全身输注的影响减弱缺血性损伤的胎儿神经血管单位（BBB），并可能在早产儿比近足月胎儿的大脑。在缺血前输注IL-6和IL-12阻断抗体。将诱导脑缺血，并如目的2中所述测量BBB渗透性、TJ组分和脑损伤。这些研究将提供第一个直接的证据，是否系统性细胞因子穿过完整或受损的胎儿血脑屏障，以及是否用中和抗体阻断细胞因子的作用保护胎儿神经血管单位（BBB）和大脑。该项目可能为预防人类胎儿和/或早产儿脑损伤的新策略提供新的见解。 围产期缺氧缺血性脑损伤常导致脑性瘫痪和智力低下。早产儿CP的发病率为40 - 148/1，000，足月儿为1 - 2/1，000。该项目可以为预防人类胎儿和/或早产儿脑损伤的新策略提供新的见解。

DESCRIPTION (provided by applicant): Hypoxic-ischemic brain injury is the single most important neurologic problem in the perinatal period. In utero hemodynamic abnormalities possibly in association with elevated pro-inflammatory cytokines (cytokines) such as IL-12, IL-6, and TNF-1 predispose to brain injury, particularly in premature neonates. Systemic cytokines produced during maternal infection and/or increases in cytokines after ischemia may accentuate damage to the fetal brain. The neurovascular unit is a privileged site that consists of brain microvascular endothelium, glia, and neurons. Although cytokines are known to cross the adult blood-brain barrier (BBB), evidence to support the hypothesis that systemic cytokines cross the BBB of the fetus and premature neonate is sparse. Our specific aims test the hypothesis that specific cytokines cross both the intact and injured BBB in the fetus to damage the brain. A consequence of this hypothesis is that blockade of these cytokines would attenuate the ischemia related damage to the neurovascular unit (BBB) and possibly the brain. A multidisciplinary approach will be used to address our hypothesis and will include physiological, biochemical, pathological, immunohistochemical, and molecular methods. Aim 1 tests the hypothesis that cytokines such as IL-12 & IL-6 cross the BBB in a maturation-dependent manner in ovine fetuses, and that maturation-related changes in barrier permeability to cytokines are primarily related to changes in the composition of the tight junction. BBB permeability will be quantified by the integral technique with 1-aminoisobutyric acid and radiolabeled cytokines. Tight junction (TJ) proteins and mRNA will be measured by Western blot, immunohistochemistry, and Northern blot. Aim 2 determines whether ischemic injury increases the permeability of the BBB to cytokines as a function of gestational age and tight junction maturation. Brain ischemia is induced by carotid occlusion. BBB permeability and TJ components will be measured as in Aim 1 and brain injury assessed by pathological, immunohistochemical, and molecular methods. Aim 3 determines whether blocking the effects of cytokines with systemic infusions of neutralizing antibodies attenuates ischemic injury to the fetal neurovascular unit (BBB) and possibly the brain more in preterm than near term fetuses. IL-6 and IL-1 2 blocking antibodies will be infused before ischemia. Brain ischemia will be induced and BBB permeability, TJ components, and brain injury measured as in Aim 2. These studies will provide the first direct evidence whether systemic cytokines cross the intact or injured fetal BBB and whether blocking the effects of cytokines with neutralizing antibodies protect the fetal neurovascular unit (BBB) and brain. This project may provide new insights into novel strategies to prevent brain injury in the human fetus and/or premature infant. Perinatal hypoxic/ischemic brain injury often results in cerebral palsy (CP) and mental retardation. The incidence of CP is 40-148/1,000 in premature and 1-2/1,000 in full term infants. This project could provide new insights into novel strategies to prevent brain injury in the human fetus and/or premature infant.