Designing Neural Tissue Constructs that Mimic Brain-Specific Architecture

设计模仿大脑特定结构的神经元组织结构 目的(S)：这项建议为修复受损的大脑皮层回路提供了一种体内方法。 工程化的神经组织。具体地说，拟议的研究试图产生可移植的结构， 灰质和白质结构的属性，以优化它们与正常大脑结构的匹配。vt.在.的基础上 移植后，将评估这些构建物的存活率和与宿主大脑的整合情况。重点 将放在获得移植动物功能恢复的证据上。此外，这项提案 将获得可以从人类干细胞来源产生功能神经组织的原理证明数据， 这是将这项工作转化为临床可行疗法的关键一步。 研究设计和方法学：这项建议建立在组织工程学的最新进展基础上，以 生成可移植的神经组织，复制灰质和白质结构的各个方面。我们 假设重建大脑特有的结构对于正确修复大脑回路和恢复 大脑功能。这项工作将分三个阶段进行。谷氨酸能皮层神经元将被分化 来自人类干细胞来源。分化的神经元的表型和电生理学将是 经过评估，这些细胞的三维培养将被培养出来。同时，两种类型的构造将 从大鼠胚胎，然后是人类干细胞来源的神经元进行工程改造。第一个将是多层的 反映皮质建筑的建筑。两层支架将相互堆叠，其中一层带有神经元 来自第2-4层，另一个含有来自第5-6层的神经元。每一层的神经元都将被转化为 不同的蛋鸡特异性刺激蛋白。第二个结构将是可移植的3D轴突区域 使用先前开发的机械伸长方法。每一个人的生存和网络活动 构造将被仔细分析。多层结构将被移植到初级运动皮质中 未损伤和损伤(侧向流体冲击)大鼠脑。建筑的存活率将由 移植后2周、1、2、6个月免疫组织化学染色。功能集成将是 在这些时间点通过光刺激结构和记录肌肉活动和视觉进行评估 四肢有活动的迹象。运动功能将使用旋转和横梁行走进行评估 任务，以及对运动速度和距离的评估。轴突组织将被移植到 大鼠大脑的两个半球，有完整的或断开连接的胼胝体。构建生存将是 如上所述进行评估。功能整合将通过刺激宿主大脑的一个半球来确定 并从对侧记录。运动协调性将通过运行性能进行评估 有凹凸不平横档的车轮。我们还将评估移植动物的认知表现 确定移植过程是否有任何有害影响。 调查结果：这是一项新的建议。 临床关系：创伤性脑损伤(TBI)是美国军事人员的标志性损伤 在最近的伊拉克和阿富汗冲突中，并导致了广泛的致残后遗症。取而代之 神经组织对于恢复神经功能和增强补体的作用非常重要 神经调节和基于可塑性的治疗。

Designing neuronal tissue constructs that mimic brain-specific architecture Objective(s): This proposal develops an in vivo approach for repairing damaged cerebral cortex circuits with engineered neural tissue. Specifically, the proposed studies seek to generate transplantable constructs with attributes of grey and white matter structure to optimize their match to normal brain architecture. Upon transplantation, these constructs will be evaluated for their survival and integration into host brains. Emphasis will be placed on obtaining evidence of functional recovery in transplanted animals. In addition, this proposal will obtain proof-of-principle data that functional neural tissue can be generated from human stem cell sources, a crucial step toward translating this work into a clinically viable therapy. Research Design & Methodology: This proposal builds upon recent advances in tissue engineering to generate transplantable neural tissue that replicate various aspects of grey and white matter architecture. We hypothesize that recreating brain-specific architecture is vital to properly repairing brain circuits and restoring brain function. There will be three phases to this work. Glutamatergic cortical neurons will be differentiated from human stem cell sources. The phenotype and electrophysiology of the differentiated neurons will be assessed, and three-dimensional cultures of these cells will be grown. In parallel, two types of constructs will be engineered from rat embryonic and then human stem cell-derived neurons. The first will be a multi-layered construct that reflects cortical architecture. Two scaffold layers will be stacked on each other, one with neurons from layers 2-4 and the other with neurons from layers 5-6. Neurons from each layer will be transduced with different opsins for layer-specific stimulation. The second construct will be transplantable 3D tracts of axons using previously developed mechanical elongation methods. The survival and network activity of each construct will be carefully analyzed. Multi-layered constructs will be transplanted into primary motor cortex in uninjured and injured (lateral fluid percussion) rat brains. Construct survival will be determined with immunohistochemistry at 2 weeks and 1, 2, and 6 months after transplantation. Functional integration will be assessed at these time points by optically stimulating the construct and recording muscle activity and visual evidence of movement in the extremities. Motor function will be evaluated using the rotarod and beam walk tasks, as well as assessments of locomotion speed and distance. Axonal tissue will be transplanted across the two hemispheres of the rat brain with an intact or disconnected corpus callosum. Construct survival will be evaluated as above. Functional integration will be ascertained by stimulating one hemisphere of the host brain and recording from the contralateral side. Motor coordination will be assessed through running performance on a wheel with uneven rungs. We will also evaluate the cognitive performance of transplanted animals to determine if the transplantation procedure has any detrimental effects. Findings: This is a new proposal. Clinical Relationships: Traumatic brain injury (TBI) is the signature injury of U.S. military personnel involved in recent conflicts in Iraq and Afghanistan and results in a wide range of disabling sequelae. Replacing lost neural tissue is important for restoring neurological function and enhancing the effects of complementary neuromodulation and plasticity-based therapies.