The interplay between active and passive mechanics in the aging bladder

下尿路症状(LUT)的发生率，如收缩功能障碍和尿潴留， 随着年龄的增长显著增加，全球估计有26亿人至少患有一种LUT 在2008年。人体膀胱的老化改变了机械性能，增加了剩余尿量。这个 逼尿肌是负责膀胱收缩的平滑肌肉，嵌入并被 波浪状、富含胶原的细胞外基质(ECM)。许多与年龄相关的人类LUT的病因与 平滑肌细胞(SMC)收缩和信号通路的改变，这也已经在 衰老膀胱的小鼠模型。然而，在SMCS和ECM之间发生的物理交互作用是 未知，并可能是了解膀胱功能障碍的主动和被动机制的关键因素 随着年龄的增长。这项提案中描述的工作将使用光遗传学、多光子显微镜和一种 测试系统开发一种将向心性收缩结合到膀胱中的体外方法学 机械测试。拟议的研究将由一个具有互补专业知识的团队进行 蛋白质组学、细胞外基质生物学和软组织生物力学(Pi Calve)、膀胱力学和机械学 测试(co-i Tuttle)、本构模型开发和有限元(FE)分析(co-i RocCabianca)。 在目标1中，逼尿肌收缩将膀胱ECM恢复到天然的空组织的能力 将会受到考验。两种诱导SMC收缩的方法(即光生和胆碱能)将与 利用SM22确定光刺激在复制胆碱能逼尿肌收缩中的有效性 ChR2(H134R)-EYFP小鼠。形态和机械数据将在有限元模型中实现， 这将被用来评估ECM组织对机械响应的重要性。验证我们的 方法将增加体内和体外测试条件之间的一致性，并揭示 ECM组织上的逼尿肌收缩。在目标2中，我们将检验这一假设，即 老化的膀胱抑制了逼尿肌充分收缩组织以达到健康休息的能力 配置。老年SM22-Cretg；ChR2(H134R)-EYFP小鼠的膀胱将进行体外测试，以研究如何 老化影响ECM和SMC组织。蛋白质组学将被用来量化ECM和毒扁豆碱 受体丰度随着年龄的增长而变化。此数据将用于通知有限元建模，以帮助解决影响 衰老对组织成分、细胞外基质组织和力学的影响。 这个项目的成功完成将增加对膀胱功能的基本了解 了解这种收缩-重组行为在老化的膀胱中是如何减少的，我们 假设有助于LUTS的发展和进步。开发的实验方法论将 有助于评估各种疾病的膀胱功能障碍，并为新的研究打开大门 下尿路综合征药物治疗的功能效应。

The prevalence of lower urinary tract symptoms (LUTS), such as contractile dysfunction and urinary retention, increases significantly with age and an estimated 2.6 billion people worldwide suffered from at least one LUTS in 2008. The aging human bladder has altered mechanical properties and increased residual urine volume. The detrusor is the smooth muscle that responsible for bladder contraction and is embedded in and surrounded by a wavy, collagen-rich extracellular matrix (ECM). The etiologies of many age-related LUTS in humans are tied to alterations in smooth muscle cell (SMC) contractility and signaling pathways, which has also been shown in mouse models of the aging bladder. However, the physical interactions that occur between SMCs and ECM are unknown and are likely a key factor in understanding the active and passive mechanics of bladder dysfunction with aging. The work described in this proposal will use optogenetics, multiphoton microscopy, and a custom testing system to develop an ex vivo methodology that incorporates concentric contraction into bladder mechanical testing. The proposed studies will be conducted by a team with complementary expertise in proteomics, ECM biology and soft tissue biomechanics (PI Calve), urinary bladder mechanics and mechanical testing (co-I Tuttle), and constitutive model development and finite element (FE) analysis (co-I Roccabianca). In Aim 1, the ability of detrusor contraction to restore the bladder ECM to the native empty organization will be tested. Two methods for inducing SMC contraction will be compared (i.e. optogenetic and cholinergic) to determine how effective light-mediated stimulation is in replicating cholinergic detrusor contraction using SM22- Cretg;ChR2(H134R)-EYFP mice. The morphological and mechanical data will be implemented in an FE model, that will be used to evaluate the importance of ECM organization on the mechanical response. Validation of our approach will increase agreement between in vivo and ex vivo testing conditions and uncover the effect of detrusor contraction on ECM organization. In Aim 2 we will test the hypothesis that remodeling of the ECM in the aging bladder inhibits the ability of the detrusor to adequately contract the tissue to the healthy resting configuration. Bladders from aged SM22-Cretg;ChR2(H134R)-EYFP mice will be tested ex vivo to study how aging affects ECM and SMC organization. Proteomics will be used to quantify how the ECM and muscarinic receptor abundance changes with aging. This data will be used to inform FE modeling to help resolve the effect of aging on tissue composition, ECM organization, and mechanics. Successful completion of this project will increase the fundamental understanding of bladder function gain insight into how this contraction-reorganization behavior is diminished in the aging bladder, which we hypothesize contributes to LUTS development and progression. The experimental methodology developed will be useful in evaluating bladder dysfunction in various diseases and open doors to new research into the functional effects of pharmacological treatments for LUTS.