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

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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.