Precision-cut bladder slices: an enabling technology for urologic research

平滑肌的机制评估是必不可少的，以严格了解下尿路功能的健康和疾病，并促进转化泌尿系统疾病的研究。平滑肌评价的现有策略包括来自动物模型的全膀胱制备物或膀胱条以及来自人组织的单细胞。然而，利用这些方法，在捕获天然组织环境的标本中的单个实验内执行收缩、刚度和潜在信号传导的综合分析的能力是有限的。 精密切割组织切片技术已被应用于许多中空器官产生新的见解细胞和组织生理学和病理生理学。Krishnan实验室的研究表明，精密切割肺切片（PCLS）可以与组织牵引显微镜（TTM）相结合，以定量评估远端气道中的生理相关参数，包括组织收缩和硬度。在这个应用中，我们建议开发的精密切割膀胱切片（PCBS）作为一种新的平台，询问收缩，松弛和刚度的膀胱在体外，同时保留天然的细胞微环境。本提案的总体目标是证明PCBS技术用于评价健康小鼠以及健康和患病人类膀胱组织中膀胱收缩和僵硬的效用。我们假设，精确切割膀胱切片的发展将能够在体外定量评估平滑肌功能和信号传导，同时保留天然细胞环境。我们将用以下具体目的来检验这一假设：目的1：评价用于测量平滑肌收缩的精密切割膀胱切片。目的2：评价精密切割膀胱切片用于评估膀胱壁硬度。我们将使用组织牵引显微镜来评估小鼠和人类膀胱组织中诱发的收缩和舒张。此外，我们将进行生化和免疫组织化学分析，以确定PCBS保留活力，表型和功能反应的程度，包括冷冻保存后。我们还将应用一种新型的组织拉伸装置来探索来自患病与健康人类膀胱的PCBS的刚度，使我们能够评估细胞外基质微环境中的平滑肌活性。 在项目期结束时，我们预计将创建一个新的平台，能够测量膀胱组织中的生理相关终点，包括收缩，松弛和僵硬，并确定PCBS可以用药理学试剂调节的程度，以实现机制研究。这些研究的成功完成有望建立一个新的平台，有助于验证膀胱功能障碍的新治疗干预措施。

Mechanistic evaluation of smooth muscle is essential to a rigorous understanding of lower urinary tract function in both health and disease, and to facilitate translational urologic disease research. Existing strategies for smooth muscle evaluation include whole bladder preparations or bladder strips from animal models as well as single cells from human tissues. However, with these approaches the ability to perform integrated analysis of contraction, stiffness and underlying signaling within a single experiment in specimens that capture the native tissue environment is limited. Precision-cut tissue slice technology has been applied to a number of hollow organs yielding novel insights into cell and tissue physiology and pathophysiology. Research from the Krishnan laboratory has demonstrated that precision-cut lung slices (PCLS) can be coupled with tissue traction microscopy (TTM) to enable quantitative assessment of physiologically relevant parameters in distal airways, including tissue contraction and stiffness. In this application we propose to develop the precision-cut bladder slice (PCBS) as a novel platform to interrogate contraction, relaxation and stiffness of the bladder in vitro, while preserving the native cellular microenvironment. The overall objective of this proposal is to demonstrate the utility of PCBS technology for evaluation of bladder contraction and stiffness in healthy murine and healthy and diseased human bladder tissues. We hypothesize that development of precision-cut bladder slices will enable quantitative assessment of smooth muscle function and signaling in vitro while preserving the native cellular environment. We will test this hypothesis with the following Specific Aims: Aim 1: Evaluate precision-cut bladder slices for measurement of smooth muscle contraction. Aim 2: Evaluate precision-cut bladder slices for assessment of bladder wall stiffness. We will use tissue traction microscopy to assess evoked contraction and relaxation in mouse and human bladder tissues. In addition, we will perform biochemical and immunohistochemical analysis to determine the extent to which PCBS retain viability, phenotypic and functional responses, including after cryopreservation. We will also apply a novel tissue stretching device to explore stiffness in PCBS from diseased versus healthy human bladders, enabling us to evaluate smooth muscle activity within the microenvironment of the extracellular matrix. At the end of the project period, we expect to have created a novel platform enabling measurement of physiologically relevant endpoints in bladder tissue, including contraction, relaxation and stiffness, and to have determined the extent to which PCBS can be modulated with pharmacological agents to enable mechanistic studies. Successful completion of these studies is expected to establish a new platform that can facilitate the validation of new therapeutic interventions for bladder dysfunction.