Microdevices to develop ex vivo brain slice models of neurodegenerative diseases: enabling longitudinal, high-resolution, live-tissue imaging

背景：神经退行性疾病如阿尔茨海默病和帕金森病在我们日益老龄化的人口中越来越普遍。目前的治疗方案只能延缓疾病进展，迫切需要确定针对这些破坏性疾病的新药和策略。完整的大脑是极其复杂的，这使得对与神经变性相关的主要改变的清晰理解难以确定。这个项目将开发新的方法来维持完好的脑组织切片离体，在尽可能生理正常的条件下。将开发具有定制机械和表面化学特性的微图案衬底，以支持高分辨率组织，亚细胞成像，集成微流体通道以控制溶液交换和局部药物输送。在这些切片微系统中，组织将浸泡在两侧的介质中，然后将用于再现慢性神经变性。微系统将提供一种独特的能力来观察和量化疾病发病期间的初级细胞事件，并描述潜在治疗方法的功效，以预防、治疗和逆转疾病相关的功能障碍和变性。帕金森病（PD）和阿尔茨海默病（AD）是由于广泛的神经元损伤和蛋白质聚集导致神经网络活动异常而发展起来的。帕金森病、阿尔茨海默病和许多其他神经退行性疾病的一个共同因素是线粒体功能障碍。这些细胞器作为产生atp的代谢中枢至关重要，它们影响信号级联、氧化还原控制和活性氧的处理。线粒体是高度运动性的，由运动蛋白沿着细胞骨架运输，它们经历融合和裂变以保持线粒体质量。脑细胞的复杂结构需要协调远距离传递细胞器、能量和底物。作为有效的钙缓冲剂，线粒体对适当的生理钙信号和病理性钙超载的保护至关重要。有趣的是，线粒体运输蛋白在高钙位点脱离，将细胞器运送到需要钙缓冲和ATP生产的位点。因为线粒体参与了这些“前线”事件，它们也容易受到损伤，如果超过细胞的修复能力，就会引起一系列有害影响：钙超载导致ATP产生的损失，ROS增加和细胞凋亡诱导因子的释放。然而，这些变化往往是紧密相关的，需要进一步的工作来确定哪个是先发生的。研究目的：本项目将利用微工程方法来适应器官型脑切片技术来研究慢性神经退行性疾病。将探索两种平行的策略，每种策略都独立提供信息，然后结合起来，为疾病的发展、进展和治疗提供强有力的见解。目的1：在微流控系统中，在透明微柱支架或超薄多孔膜上维持器官型培养中的脑切片，以便使用倒置显微镜从下方进行活细胞荧光成像，以优化分辨率。目的2：建立PD和AD的长期慢性器官型脑切片模型。目的3：在切片退变过程中多个时间点不同细胞类型和亚细胞位置的线粒体功能、运动和结构的高分辨率成像，并结合钙成像。

Background: Neurodegenerative diseases such as Alzheimer's and Parkinson's Diseases are increasingly prevalent in our increasingly aging population. Current treatment options merely delay disease progression and there is a pressing need to identify new drugs and strategies targeting these devastating disorders. The intact brain is extremely complex, making a clear understanding of the primary alterations associated with neurodegeneration difficult to determine. This project will develop new methods of maintaining intact slices of brain tissue ex-vivo, in as physiologically-normal conditions as possible. Micropatterned substrates, with tailored mechanical and surface chemistry properties, will be developed to support tissue for high-resolution, subcellular imaging, integrating microfluidic channels for controlled solution exchange and localised drug delivery. These slice microsystems, in which the tissue will be bathed in media on both sides, will then be used to recapitulate chronic neurodegeneration. The microsystems will provide a unique ability to observe and quantify primary cellular events during disease onset and characterise the efficacy of potential therapeutics to prevent, treat and reverse disease-associated dysfunction and degeneration.Parkinson's Disease (PD) and Alzheimer's Disease (AD) develop due to extensive neuronal damage and protein aggregation leading to aberrant neural network activity. A common factor in PD, AD and many other neurodegenerative diseases is the dysfunction of mitochondria. These organelles are vital as an ATP-producing metabolic hub and they influence signalling cascades, redox control and the processing of reactive oxygen species. Mitochondria are highly-motile, being trafficked along the cytoskeleton by motor proteins, and they undergo fusion and fission to maintain mitochondrial quality. The complex architectures of metabolically-demanding brain cells necessitate co-ordinated long-distance delivery of organelles, energy and substrates. As efficient calcium buffers, mitochondria are vital for appropriate physiological calcium signals and protection during pathological calcium overload. Fascinatingly, mitochondrial transport proteins disengage at sites of high calcium - delivering the organelles to sites of need for calcium buffering and ATP production. Because mitochondria are involved in these "front-line" events, they are also susceptible to damage which, if it exceeds the cell's capacity to repair, can cause a cascade of deleterious effects: spreading calcium overload that leads to a loss of ATP production, increased ROS and release of apoptosis-inducing factors. These alterations are often tightly inter-related however and further work is needed to ascertain which come first. Research Aims: This project will exploit microengineering approaches to adapt organotypic brain slice techniques to study chronic neurodegenerative diseases. Two parallel strategies will be explored, each independently informative, and then combined to provide powerful insights into disease development, progression and therapies.Objective 1: maintain brain slices in organotypic culture on transparent micropillar supports or ultra-thin porous membranes within a microfluidic system to allow live-cell fluorescence imaging from below using an inverted microscope to optimise resolution. Objective 2: develop long-term chronic organotypic brain slice models of PD and AD. Objective 3: high-resolution imaging of mitochondrial function, motility and structure, combined with calcium imaging, in various cell types and sub-cellular locations, at multiple time-points during slice degeneration.