The µSiM-hNVU - a human BBB platform for the study of brain injury mechanisms during systemic infection

摘要 超过70%的脓毒症幸存者受到长期认知障碍的影响，但其潜在机制 仍然未知。虽然广泛假设，但脓毒症患者血脑屏障（BBB）功能障碍的证据 在危重病患者中进行诊断研究的实际障碍限制了患者。当血脑屏障破裂， 脓毒症的动物模型中可见认知障碍，脓毒症在体内的复杂性和差异 动物和人类反应之间的差异意味着动物模型无法明确识别 导致人类败血症脑损伤的循环因子。因此，我们建议开发µ SiM-hNVU 作为一种"芯片上"平台，其特征在于人类iPSC衍生的神经血管单元（NVU;脑微血管 内皮细胞、周细胞和星形胶质细胞）。"血液侧"将允许基于流动的血液引入- 在脓毒症相关脑损伤中具有已知或假设作用的细胞和分子，以及"脑侧" 将以iPSC衍生的小胶质细胞作为大脑炎症状态的报告者为特色。人类 NVU将建立在一个设备平台上-µ SiM-具有硅纳米膜，提供 "血"和"脑"间无阻碍的溶质交换和活细胞的玻璃样光学性质 成像和高分辨率显微镜。在R61阶段，设备平台将更先进， 使用包括"即插即用"模块，用于流量和屏障测量（TEER、扩散）和兼容性 用小容量的数字ELISA检测分泌蛋白。µ SiM-hNVU将通过以下方式进行验证： 血脑屏障（BBB）功能的功能测定、蛋白质表达研究和转录分析。 我们还将构建iPSC NVU，其中NVU的每个细胞组分携带ApoE4等位基因。的 ApoE4脂蛋白的表达通过已知的途径驱动BBB功能障碍，并增加 认知障碍的人类和动物经历脑炎症。我们将使用ApoE4-NVU作为 一个"芯片上的病态BBB"，我们假设它将显示出对候选机制的增强的脆弱性 我们的团队和其他人发现了脑损伤。具体来说，我们将检验以下假设：1）预激活 单核细胞侵入大脑并驱动小胶质细胞活化; 2）损伤相关的分子模式 （DAMP）复合物S100A8/A9驱动BBB破坏以促进白细胞浸润和神经炎症; 和3）降解内皮糖链的循环因子（例如，肝素酶）或有助于全身 炎症（无细胞血红蛋白）促进白细胞CNS浸润和随后的神经炎症。

Abstract Long-term cognitive impairment affects more than 70% of sepsis survivors, but the underlying mechanisms remain unknown. Though widely hypothesized, evidence of blood-brain barrier (BBB) dysfunction in septic patients is limited by practical barriers to diagnostic studies in critically ill subjects. While BBB breakdown and cognitive impairment are seen in animal models of sepsis, the complexity of sepsis in vivo and differences between animal and human responses means that animal models cannot unambiguously identify the circulating factors that cause brain injury in human sepsis. Therefore, we propose to develop the µSiM-hNVU as an `on-chip' platform featuring a human iPSC-derived neurovascular unit (NVU; brain microvascular endothelial cells, pericytes and astrocytes). The `blood side' will allow the flow-based introduction of blood- borne cells and molecules with known or hypothesized roles in sepsis related brain injury, and the `brain side' will feature iPSC-derived microglial cells serving as a reporter of the brain inflammatory status. The human NVU will be built on a device platform – the µSiM – featuring ultrathin silicon nanomembranes that provide for unhindered solute exchange between `blood' and `brain' compartments and glass-like optical quality for live cell imaging and high-resolution microscopy. In the R61 phase, the device platform will be advanced for ease-of- use including `plug-and-play' modules for flow and barrier measurements (TEER, diffusion), and compatibility with a small-volume, digital-ELISA assay for secreted proteins. The µSiM-hNVU will be validated with functional assays of blood-brain barrier (BBB) function, protein expression studies, and transcriptional analysis. We will also build a iPSC NVU in which each cellular component of the NVU carries the ApoE4 allele. The expression of the ApoE4 lipoprotein drives BBB dysfunction by a known pathway and increases the risk of cognitive impairment in humans and animals experiencing brain inflammation. We will use the ApoE4-NVU as a `diseased BBB on a chip” which we hypothesize will show enhanced vulnerabilities to candidate mechanisms of brain injury identified by our team and others. Specifically, we will test the hypotheses that 1) pre-activated monocytes invade the brain and drive microglial activation; 2) the damage associated molecular pattern (DAMP) complex S100A8/A9 drive BBB breakdown to promote leukocyte infiltration and neuroinflammation; and 3) circulating factors that degrade endothelial glycocaylx (e.g., heparinase) or contribute to systemic inflammation (cell-free hemoglobin) promote CNS infiltration of leukocytes and subsequent neuroinflammation.