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

摘要 超过70%的脓毒症幸存者会受到长期认知障碍的影响，但潜在的机制 仍然不为人所知。尽管存在广泛的假设，但有证据表明脓毒症患者血脑屏障(BBB)功能障碍 在危重病人的诊断研究中，患者受到实际障碍的限制。而血脑屏障的故障和 认知障碍见于脓毒症的动物模型、体内脓毒症的复杂性和差异 动物和人类之间的反应意味着动物模型不能明确地识别 导致人类脓毒症脑损伤的循环因素。因此，我们建议开发µSIM-hNVU 作为一个以人类IPSC衍生神经血管单位(NVU；脑微血管)为特色的芯片上平台 内皮细胞、周细胞和星形胶质细胞)。“血液方面”将允许以流动为基础的血液输入- 已知或假想在脓毒症相关脑损伤中起作用的携带细胞和分子，以及“脑侧” 将以IPSC来源的小胶质细胞为特色，作为脑部炎症状态的报告。人类 NVU将构建在设备平台-µSIM-上，采用超薄硅纳米膜，提供 血液和大脑之间畅通无阻的溶质交换和活细胞的玻璃状光学性质 成像和高分辨率显微镜。在R61阶段，设备平台将进行升级，以便于- 使用包括用于流动和屏障测量(TEER、扩散)和兼容性的即插即用模块 用一种小体积的数字酶联免疫吸附试验检测分泌的蛋白质。？SIM-hNVU将通过验证 血脑屏障(BBB)功能分析、蛋白质表达研究和转录分析。 我们还将构建一个IPSC NVU，其中NVU的每个细胞组件都携带ApoE4等位基因。这个 ApoE4脂蛋白的表达通过一条已知的途径驱动血脑屏障功能障碍，并增加 经历脑部炎症的人类和动物的认知障碍。我们将使用ApoE4-NVU作为 “芯片上有病的BBB”，我们推测它将对候选机制显示出更强的脆弱性 我们团队和其他人确认的脑损伤。具体地说，我们将测试1)预激活的假设 单核细胞侵入大脑并驱动小胶质细胞激活；2)损伤相关的分子模式 (湿)S100A8/A9复合体导致血脑屏障破坏，促进白细胞浸润和神经炎症； 3)降解内皮细胞糖蛋白的循环因子(如肝素酶)或促进全身性 炎症(无细胞血红蛋白)促进中枢神经系统白细胞的渗透和随后的神经炎症。

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.