Designing Neural Tissue Constructs that Mimic Brain-Specific Architecture

设计模拟大脑特定结构的神经元组织结构 目的：该提案开发了一种用于修复受损大脑皮层回路的体内方法， 工程神经组织具体地说，所提出的研究试图产生可移植的结构， 灰色和白色物质结构的属性，以优化它们与正常大脑结构的匹配。后 移植后，将评估这些构建体的存活和整合到宿主脑中。重点 将放在获得移植动物功能恢复的证据上。此外，该提案 将获得原理证明数据，即功能性神经组织可以从人类干细胞来源产生， 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：该建议建立在组织工程的最新进展， 产生可移植的神经组织，其复制灰质和白色物质结构的各个方面。我们 假设重建大脑特定的结构对正确修复大脑回路和恢复大脑功能至关重要， 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分化为 从人类干细胞来源。分化的神经元的表型和电生理学将是 评估，这些细胞的三维培养物将生长。同时，两种类型的构造将 从大鼠胚胎和人类干细胞衍生的神经元中改造而成。第一个是多层次的 反映皮层结构的构造。两个支架层将相互堆叠，其中一个带有神经元 另一个具有来自层5-6的神经元。来自每一层的神经元将被转导 不同的视蛋白用于层特异性刺激。第二个结构是可移植的3D轴突束 使用先前开发的机械伸长方法。每一个的生存和网络活动 将认真分析。多层结构将被移植到初级运动皮层， 未受伤和受伤（侧向液压冲击）的大鼠脑。结构存活率将通过以下方式确定： 移植后2周和1、2和6个月的免疫组化。功能整合将是 在这些时间点通过光学刺激该结构并记录肌肉活动和视觉刺激来评估。 四肢活动的迹象将使用旋转棒和平衡木行走评价运动功能 任务，以及运动速度和距离的评估。轴突组织将被移植到 具有完整或断开的胼胝体的大鼠大脑的两个半球。构装体的生存将是 评价如上。功能整合将通过刺激宿主大脑的一个半球来确定 并从对侧进行记录。运动协调性将通过跑步表现进行评估， 梯级不均匀的轮子。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。 答：这是一个新的建议。 临床关系：创伤性脑损伤（TBI）是美国军人的标志性损伤。 在最近的伊拉克和阿富汗冲突中，导致了广泛的致残后遗症。更换丢失 神经组织对于恢复神经功能和增强互补的效果是重要的。 神经调节和可塑性治疗。

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.