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IL-12、IL-6和TNF-1等促炎细胞因子升高有关，易导致脑损伤，尤其是早产儿。母体感染期间产生的全身细胞因子和/或缺血后细胞因子的增加可能加重胎儿脑的损伤。神经血管单元是由脑微血管内皮、神经胶质和神经元组成的特殊部位。虽然已知细胞因子能穿过成人血脑屏障（BBB），但支持全身细胞因子能穿过胎儿和早产新生儿血脑屏障这一假设的证据很少。我们的具体目的是验证一个假设，即特定的细胞因子穿过胎儿完整的和受伤的血脑屏障来损伤大脑。这一假设的结果是，阻断这些细胞因子将减轻缺血相关的神经血管单位（BBB）和可能的大脑损伤。我们将采用多学科方法来解决我们的假设，包括生理、生化、病理、免疫组织化学和分子方法。Aim 1验证了细胞因子如IL-12和IL-6在绵羊胎儿中以成熟依赖的方式穿过血脑屏障的假设，并且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。血脑屏障的通透性将通过1-氨基异丁酸和放射性标记细胞因子的积分技术进行量化。严密连接（TJ）蛋白和mRNA将通过Western blot、免疫组织化学和Northern blot检测。目的2确定缺血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功能。颈动脉闭塞引起脑缺血。与Aim 1一样，测量血脑屏障通透性和TJ成分，并通过病理、免疫组织化学和分子方法评估脑损伤。目的3确定通过全身输注中和抗体阻断细胞因子的作用是否能减轻胎儿神经血管单位（BBB）的缺血性损伤，并可能减轻早产儿比近期胎儿对大脑的损伤。缺血前注射IL-6和IL-1 - 2阻断抗体。将诱导脑缺血，并测量血脑屏障通透性、TJ成分和脑损伤。这些研究将提供第一个直接证据，证明全身性细胞因子是否穿过完整或受损的胎儿血脑屏障，以及用中和抗体阻断细胞因子的作用是否能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瘫（CP）和智力低下。早产儿的CP发病率为40-148/ 1000，足月婴儿的CP发病率为1-2/ 1000。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.