CAREER: Development of a Tissue-Engineered Model of Ischemic Microstroke

死脑组织的小体积区域（微梗死）在神经功能障碍和认知障碍中的作用还不清楚。对痴呆症患者大脑的分析表明，存在大量广泛的微梗死，在某些情况下，单个大脑中有数千个微梗死，这与认知障碍的增加有关。动物研究支持这一发现。虽然已经建立了因果关系，但小血管卒中期间发生的细胞、分子和生理事件及其在神经损伤中的作用仍然未知。在人类或动物大脑中进行高分辨率细胞实验极具挑战性。为了克服这一限制，该项目的重点是开发和验证一个组织工程，微流体，在体外，微中风模型，准确地概括了在体内发生的事件的级联。这种流化的体外微中风模型的开发和验证有可能为微中风引起的基本生物学变化提供重要的见解。据设想，利用该模型实现的生物学发现可能会对人类健康产生重大影响，并可能导致智能设计的治疗方法的发展，以在未来的应用中治疗微中风和痴呆症。为了增加从小学到本科的广泛学生的曝光率，制定了一项教育计划，旨在激发学生的兴趣，通过科学展览中的艺术，科学模块中的实践艺术，实验室研究经验，以及将微生理系统的研究纳入现有的本科课程。该项目的重点是开发和验证一种组织工程，微流控、体外、微卒中模型，可用于研究微卒中诱导的神经损伤闭塞和再灌注诱导的变化期间发生的细胞、分子和生理事件。 该模型克服了在动物大脑中进行高分辨率，时空细胞/分子实验的挑战。 该研究计划是根据3个具体目标组织的：1）量化毛细血管闭塞和再灌注对3D合成，仿生血管网络中脑血管流量的影响，该网络基于整个小鼠大脑血管系统的3D图像堆栈，该图像堆栈通过高分辨率成像，通过刀口扫描显微镜，灌注印度墨水生成; 2）定量毛细血管闭塞和再灌注对人干细胞衍生的脑微血管内皮细胞（BMEC）的影响，和3）通过时空监测毛细血管闭塞的进展来定量毛细血管闭塞和再灌注对微梗塞进展的影响。和再灌注诱导的微梗死在水凝胶包封的人星形胶质细胞和神经元。 实现这些目标需要进一步开发和实施先进的生物制造技术（双光子水凝胶降解和聚合），先进生物材料（酶可降解构建体和半合成血凝块），以及分化的人神经干细胞的掺入（BMEC，星形胶质细胞，神经元）使用所有人体细胞创建微生理学中风模型。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估。

The role that small volume regions of dead brain tissue (microinfarcts) play in neurological dysfunction and cognitive impairment is not well understood. Analysis of dementia patients' brains has linked the presence of numerous and wide-spread microinfarcts, in some cases thousands in a single brain, to increased cognitive impairment. Animal studies support this finding. While a causal link has been established, the cellular, molecular, and physiological events that occur during small blood vessel stroke and their role in neurological impairment remain unknown. Performing high-resolution cellular experiments in humans or animal brains is extremely challenging. To overcome this limitation, this project focuses on the development and validation of a tissue-engineered, microfluidic, in vitro, microstroke model that accurately recapitulates the cascade of events that occur in vivo. Development and validation of this fluidized, in vitro, microstroke model has the potential to provide significant insights into the fundamental biological changes induced by microstrokes. It is envisioned that the biological discoveries achieved with this model could significantly impact human health and potentially lead to the development of intelligently designed therapies to treat microstroke and dementia in future applications. To increase exposure across a broad range of students, from elementary to undergraduate, an educational plan has been developed that aims to spark interest in students to pursue science, technology, engineering and math education through Art in Science Exhibits, hands-on Art in Science Modules, laboratory research experiences, as well as to incorporating research in microphysiological systems into existing undergraduate courses.The project focuses on developing and validating a tissue-engineered, microfluidic, in vitro, microstroke model that can be used to study the cellular, molecular and physiological events that occur during microstroke-induced neurological impairment occlusion- and reperfusion-induced changes. The model overcomes challenges in performing high-resolution, spatiotemporal cellular/molecular experiments in animal brains. The Research Plan is organized under 3 specific aims: 1) Quantify the influence of capillary occlusion and reperfusion on cerebrovascular flow in a 3D synthetic, biomimetic, vascular network based on a 3D image stack of the vascular system of an entire mouse brain that was generated by high-resolution imaging, via knife edge scanning microscopy, of perfused india ink; 2) Quantify the influence of capillary occlusion and reperfusion on human stem cell derived brain microvascular endothelial cells (BMECs) and 3) Quantify the influence of capillary occlusion and reperfusion on microinfarct progression by spatiotemparally monitoring the progression of a capillary occlusion- and reperfusion induced microinfarct in hydrogel encapsulated human astrocytes and neurons. Fulfillment of these aims requires further development and implementation of advanced biofabrication techniques (two-photon hydrogel degradation and polymerization), advanced biomaterials (enzymatically-degradable constructs and semi-synthetic blood clots), and incorporation of differentiated human neural stem cells (BMECs, astrocytes, and neurons) to create a microphysiological ìstroke model using all human cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.