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

脑微出血(CMBS)和微出血(CMHS)是由血液渗漏引起的- 脑接口(BBI)。随后毫米大小的血栓会导致炎症、细胞损伤和神经- 退化。这种CMBS与BBI完整性随着年龄、疾病、创伤性脑损伤而恶化有关 伤害和中风的后遗症，每年影响着美国795,000人。值得注意的是， CMBS和出血性/缺血性卒中的发生不是随机的，而是在白天或晚上聚集在一起。 关于血液渗漏的脆弱性，对BBI昼夜节律动态的了解有限。它 在体内或通过芯片模型进行研究一直很困难，也没有药物治疗。在解决这一差距方面， 本提案是对FOA RFA-HL-20-021的回应。这个FOA的目的是支持高风险/高回报 促进血脑屏障功能的血液成分及相关界面研究 开发更完整的神经血管-血液模型，用于直接相关的翻译应用 对人类来说。它是R61/R33探索/开发阶段奖。因为对昼夜节律的了解 在BBI动态对血液泄漏的脆弱性是有限的，我们的目标是创造一种新的仿生脑运输 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控技术 检查凝血因子和昼夜节律波动相互作用的平台：1)血液/血管 2)昼夜循环中BBI上的动态血管压。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复制BBI的昼夜动态 在“血管”侧，在“脑”隔区内有极化的星形胶质细胞、神经元和小胶质细胞。这个项目 汇集了促进创建增强平台所需的专业知识，以便更紧密地模拟 人类BBI。团队成员的贡献将是：HAN-微流体和生物传输分析；KONG- BBI的工程学；吉列-组装/验证人类IPSC昼夜节律报告芯片并评估节律 和通量，以及Flick关于凝血因子和Obrietan对昼夜节律记者的咨询 转基因。这笔赠款将分为两个阶段：年第1-2年(版本61)的重点将是建立工具和 在YR 3-5(R33)中关于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态 BBI在人脑中的作用，并在推动一体化的振荡昼夜循环的背景下探索它 生理和行为，包括睡眠和清醒。通过瞄准BBI的两边和他们的 交集，我们将深入了解一种新兴的观点，即BBI是塑料的，随着时间的推移而变化，损失 睡眠、感染和衰老的压力。这对生物钟在体内的作用有着重要的影响。 脑部的血液凝结和神经血管功能。其结果将有助于发展 针对不良脑血管事件的暂时发生的治疗机会，包括 高血压、认知障碍和痴呆症以及步态综合症。

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.