Targeting the Choroid Plexus-Cerebrospinal Fluid System to Treat Post-Hemorrhagic Hydrocephalus

项目摘要 出血后脑积水（PHH）是早产儿发病的主要原因。PHH被触发 脑室内出血（IVH）导致脑脊液（CSF）蓄积， 周围脑组织的脑压迫和永久性神经缺陷。虽然PHH显然 由于CSF产生和清除平衡的改变，其机制知之甚少， 我们引导理性治疗的能力在这里，我们建议检查两个过程，可以操纵 治疗上缓解PHH：（1）脉络丛（ChP）的离子和液体转运，和（2）心室血液 通过巨噬细胞清除。在正常生理条件下的成人中， 细胞通过一组不完全了解的膜蛋白分泌CSF，包括NKCC 1，磷酸化 激活的双向钠钾氯协同转运蛋白。引人注目的是，我们最近发现NKCC 1参与CSF 在大脑发育的早期阶段，脑脊液被清除而不是分泌。CSF-K+水平显著高于 在胚胎中高于成人，可能解释了NKCC 1水运输的相反方向8。实验 在发育过程中，向心室中引入血液似乎进一步提高了CSF-K水平，并驱动 ChP上皮细胞内钙离子活性，即早基因c-fos表达， NKCC 1的表达/磷酸化。我们的研究结果表明，在IVH中存在一种新的反调节反应， 早产儿：ChP通过NKCC 1吸收CSF，由K+驱动。我们将通过确定 如果NKCC 1激活抑制或减轻我们的小鼠IVH模型中的脑积水（目的1;初步数据 建议后者）。我们还发现，IVH后，血液制品滞留在发育中的心室中， 为PHH的持续存在提供了解释。脑室和ChP的顶面是特定的 巨噬细胞称为Kolmer细胞。虽然Kolmer细胞被认为是大脑的反应者， 它们的出血、清除和其他功能仍然难以捉摸。我们的数据表明，在早期 在大脑发育的各个阶段，心室巨噬细胞/Kolmer细胞被激活并被招募到大脑发育的部位。 心室内的血液渗漏（目的2A），这些巨噬细胞是必要的，足以清除 来自心室的血液和/或炎性信号（目标2B，C）。总的来说，我们的数据表明， PHH的严重程度取决于血液制品、离子和蛋白质之间的发育阶段特异性相互作用， 浓度（例如[K]）、免疫和炎症反应以及NKCC 1表达水平。估计有20% 发生脑室内出血的婴儿会发展为PHH。我们怀疑这是由于缺乏足够的内源性 补偿性反应本提案的最终目标是通过奠定基础， 用于开发临床治疗，促进CSF和血液的内源性清除，这些CSF和血液驱动致病性 导致PHH的过程。这一建议还应指导成人IVH和其他疾病的治疗， 破坏细胞外离子平衡

PROJECT SUMMARY Post-hemorrhagic hydrocephalus (PHH) is a leading cause of morbidity in premature infants. PHH is triggered by germinal matrix intraventricular hemorrhage (IVH) that results in accumulation of cerebrospinal fluid (CSF) in the brain compression of surrounding brain tissue, and permanent neurological deficits. While PHH is clearly caused by an altered balance of CSF production and removal, the mechanisms are poorly understood, limiting our ability to guide rational therapies. Here, we propose to examine two processes that could be manipulated therapeutically to alleviate PHH: (1) ion and fluid transport by the choroid plexus (ChP), and (2) ventricular blood clearance by macrophages. In adults under normal physiological conditions, sheets of specialized ChP epithelial cells secrete CSF via an incompletely understood set of membrane proteins including NKCC1, a phosphorylation activated bi-directional Na-K-Cl cotransporter. Strikingly, we recently discovered that NKCC1 participates in CSF removal rather than CSF secretion during early stages of brain development. CSF-K+ levels are significantly higher in embryos than adults, likely explaining this opposite direction of NKCC1 water transport8. Experimental introduction of blood into the ventricles during development appears to further elevate CSF-K levels, and to drive intracellular calcium activity in ChP epithelial cells, expression of the immediate early gene c-fos, and increased expression/phosphorylation of NKCC1. Our findings suggest a novel counter-regulatory response to IVH in premature infants: ChP absorption of CSF via NKCC1, driven by K+. We will test this hypothesis by determining if NKCC1 activation either worsens or mitigates hydrocephalus in our mouse IVH model (Aim 1; preliminary data suggests the latter). We also found that following IVH, blood products linger in the developing ventricles and may account for the persistence of PHH. The brain's ventricles and the apical surface of the ChP are home to specific macrophages known as Kolmer cells. While Kolmer cells have been implicated as responders to brain hemorrhage, their scavenging and other functions have remained elusive. Our data suggest that during early stages of brain development, ventricular macrophages/Kolmer cells are activated and recruited to the site of blood leakage within the ventricle (Aim 2A) and that these macrophages are necessary and sufficient to clear blood and/or inflammatory signals from the ventricles (Aim 2B, C). Collectively, our data suggest that the ultimate severity of PHH depends on a developmental stage-specific interplay between blood products, ion concentrations (e.g. [K]), immune and inflammatory reactions, and NKCC1 expression levels. An estimated 20% of infants that experience intraventricular bleeds develop PHH. We suspect this is due to insufficient endogenous compensatory responses. The ultimate goal of this proposal is to improve outcomes by laying the groundwork for development of clinical treatments that boost endogenous removal of CSF and blood that drive the pathogenic processes that lead to PHH. This proposal should also guide therapies for adult IVH and other conditions with disrupted extracellular ionic homeostasis.