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

死亡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尚不清楚。对痴呆症患者大脑的分析表明，大量且广泛存在的微梗死（在某些情况下，单个大脑中有数千个微梗死）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，但在小血管中风期间发生的细胞、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的细胞实验是极具挑战性的。为了克服这一限制，该项目专注于开发和验证一种组织工程、微流体、体外微中风模型，该模型可以准确地概括体内发生的级联事件。这种流态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，通过该模型取得的生物学发现可能会对人类健康产生重大影响，并可能导致未来应用中治疗微中风和痴呆的智能设计疗法的发展。为了增加从小学到本科生的广泛学生的接触，已经制定了一项教育计划，旨在通过科学艺术展览，实践科学艺术模块，实验室研究经验，以及将微生理系统研究纳入现有的本科课程，激发学生对科学，技术，工程和数学教育的兴趣。该项目专注于开发和验证组织工程、微流体、体外微中风模型，该模型可用于研究微中风引起的神经损伤闭塞和再灌注引起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。本研究计划有3个具体目标：1)基于全鼠脑血管系统的三维图像堆栈，定量研究毛细血管闭塞和再灌注对三维合成仿生血管网络中脑血管流量的影响，该网络是通过灌注墨水的刀口扫描显微镜高分辨率成像生成的；2)量化毛细血管闭塞和再灌注对人干细胞来源的脑微血管内皮细胞（BMECs）的影响；3)通过对水凝胶包膜的人星形胶质细胞和神经元的毛细血管闭塞和再灌注诱导的微梗死的时空监测，量化毛细血管闭塞和再灌注对微梗死进展的影响。实现这些目标需要进一步发展和实施先进的生物制造技术（双光子水凝胶降解和聚合），先进的生物材料（酶可降解结构和半合成血凝块），并结合分化的人类神经干细胞（BMECs，星形胶质细胞和神经元）来创建使用所有人类细胞的微生理ìstroke模型。该奖项反映了美国国家科学基金会的法定使命，并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，被认为值得支持。

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.