Dynamic Circadian Regulation of the Blood-Brain Interface in a Human Brain-mimicking Microfluid Chip

脑微出血（CMB）和微出血（CMH）是由血液渗漏引起的。 脑接口（BBI）。随后毫米大小的血块导致炎症、细胞损伤和神经系统损伤。 退化这种CMB与BBI完整性随衰老、疾病、创伤性脑损伤而恶化有关。 损伤和中风后遗症，每年影响美国> 795，000人。值得注意的是， CMB和出血性/缺血性卒中的发生不是随机的，而是聚集在白天或晚上。 对BBI的昼夜节律动力学与血液渗漏的脆弱性的理解是有限的。它 很难在体内或通过芯片模型进行研究，并且没有药物治疗。为了弥补这一差距， 本提案响应FOA RFA-HL-20-021。本FOA的目的是支持高风险/高回报 研究血脑屏障的血液成分及其相关界面，以促进脑缺血再灌注损伤的发生。 开发更完整的神经血管-血液模型，用于直接相关的转化应用 对人类这是一个R61/R33探索/发展阶段奖。因为对生物钟的了解 由于BBI对血液渗漏的脆弱性的动力学是有限的，我们的目标是创造一种新的仿生大脑运输 模拟凝血系统和昼夜节律的模型。我们将开发一种创新的微流体 检查凝血因子和血液/血管昼夜节律振荡相互作用的平台1）血液/血管 成分和2）在昼夜节律周期内跨BBI的动态血管压力。我们建议 通过培养含有时钟基因报告基因的人内皮细胞来再现BBI的昼夜节律动力学 在“血管”侧，极化星形胶质细胞、神经元和小胶质细胞在“脑”隔室中。这个项目 汇集了促进创建增强平台所需的专业知识， 人BBI。团队成员的贡献将是：Han-微流体和生物运输分析; Kong- BBI工程; Gillette-组装/验证人类iPSC昼夜节律芯片并评估节律 和通量，以及Flick对凝血因子和Obrietan对昼夜节律报告的咨询 转基因。该补助金将分为两个阶段：第1-2年（R61）的重点将是建立工具， 在YR 3-5（R33）利用这些工具来实现我们的研究目标。这将使我们能够复制动力学 的BBI在人脑中，并探讨它的背景下，振荡昼夜周期，驱动整合 生理和行为，包括睡眠和觉醒。通过针对BBI的双方及其 交叉点，我们将深入了解新兴的观点，即BBI是塑料的，随着时间的推移而变化， 睡眠、感染和衰老的压力。这对生物钟在以下方面的作用具有重要意义： 脑内血液凝固和神经血管功能。成果将有助于发展 针对不良脑血管事件暂时发生的治疗机会，包括 高血压、认知障碍和痴呆以及步态综合征。

Cerebral microbleeds (CMBs) and microhemorrhages (CMHs) result from blood leakage across the blood- brain interface (BBI). Subsequent millimeter-sized blood clots lead to inflammation, cellular injury, and neuro- degeneration. Such CMBs are associated with deterioration of BBI integrity with aging, disease, traumatic brain injury, and the sequelae of strokes, which impact >795,000 people in the United States every year. Notably, CMBs and hemorrhagic/ischemic stroke occurrence is not random, but rather clusters in early day or evening. Understanding of the circadian dynamics of the BBI with respect to vulnerability to blood leakage is limited. It has been difficult to study in vivo or via on-chip models and there is no drug treatment. In addressing this gap, this proposal responds to FOA RFA-HL-20-021. The purpose of this FOA is to support high risk/high reward research on the blood component of the Blood-Brain Barrier and the associated Interface to facilitate the development of a more complete neurovascular-blood model for translational applications with direct relevance to humans. It is an R61/R33 Exploratory /Developmental Phased Award. Because knowledge of the circadian dynamics in BBI vulnerability to blood leakage is limited, we aim to create a new biomimetic brain transport model with mimicry of the coagulation system and circadian rhythm. We will develop an innovative microfluidic platform to examine interactions of coagulation factors and circadian oscillations of both 1) the blood/vascular components and 2) dynamic vascular pressure across the BBI over the circadian cycle. We propose to reproduce circadian dynamics of the BBI by culturing human endothelial cells containing a clock-gene reporter on the ‘vascular’ side with polarized astrocytes, neurons, and microglia in the ‘brain’ compartment. This project assembles the expertise needed to facilitate the creation of enhanced platforms that more closely model the human BBI. Contributions of team members will be: Han–Microfluidics and biotransport analysis; Kong– engineering of BBI; Gillette–Assemble/validate a human iPSC circadian reporter-in-chip and assess rhythms and fluxes, and consultation from Flick on blood coagulation factors and Obrietan on the circadian reporter transgene. This grant will be separated into 2 phases: Focus in YR 1-2 (R61) will be on establishing tools and in YR 3-5 (R33) on utilizing those tools to achieve our research goals. This will enable us to replicate dynamics of the BBI in human brain and to probe it in the context of the oscillatory circadian cycle that drives integrative physiology and behavior, including sleep and wakefulness. By targeting both sides of the BBI and their intersection, we will gain insights into the emerging view that the BBI is plastic, changing with time-of-day, loss of sleep, the stress of infection, and aging. This has significant implications for the role of the circadian clock in blood coagulation in the brain and neurovascular function. The outcome will contribute to developing therapeutic opportunities that target the temporal occurrence of adverse cerebrovascular events, including hypertension, cognitive disorders and dementias, and gait syndromes.