The microtubule cytoskeleton as a novel target for cavernous nerve regeneration after prostatectomy

摘要 根治性前列腺癌(RP)是治疗局限性前列腺癌的常用方法。 不幸的是，该手术存在勃起功能障碍(ED)的高风险，这对 男性手术后的健康状况。ED的主要病理生理机制是对海绵体的损伤 神经(CN)；RP后CN损伤的再生或修复机制可减轻或治疗这些患者的ED 病人。这项提案中概述的研究将探索一种通过利用神经来修复CN的新方法 新发现的微管调节剂fidgetin-like 2(Fl2)的再生能力。体外研究表明 大鼠外周神经系统原代培养中Fl2缺失对轴突生长的促进作用 神经元。参与这一提议的研究人员最近的工作表明，Fl2的靶向耗尽 通过局部应用包裹在纳米粒中的Fl2-siRNA(Fl2-siRNA-NP)促进闭合和 小鼠皮肤创伤的再生和大鼠勃起功能的改善 在CN损伤时应用RP。值得注意的是，在RP的CN横断模型中，在两周后 使用Fl2-siRNA治疗可以改善勃起功能和明显的神经再生。这些 通过观察，我们得出了这个假设，即在CN损伤后，Fl2的耗竭 激活促进海绵体神经再生的机制，导致海绵体神经损伤加速恢复 勃起功能“。这一假设将在两个具体目标上得到检验。 在第一个具体目标中，我们将证实我们的初步结果，即Fl2的耗尽改善了 CN损伤后勃起功能恢复作为RP动物模型的研究我们将优化配方 以及ED的治疗方案。Fl2-siRNA分别于伤后即刻和一周内应用于CN损伤部位 通过测量冠脉内压/血判断损伤后勃起功能恢复情况 CN刺激后、治疗后不同时间点的压力比(ICP/BP)逐渐升高。跟随 ，我们将进行病理学研究，以提供CN再生和安全性的证据。 不同的配方。 在第二个具体目标中，我们将进行实验，以阐明 缺失Fl2可促进CN损伤后的功能恢复。我们将确定Fl2-siRNA对 轴突的体外生长和再生。我们将使用对照组和经Fl2-siRNA处理的动物的组织进行 广泛的比较组织病理学和基因表达分析，在体部组织和从部位 治疗组与对照组大鼠在CN损伤后不同时间点的损伤情况。 总体而言，在完成这项提议后，我们将确立Fl2作为新目标的潜力 在修复RP后的CN损伤时，确定的治疗方案、配方和安全性考虑因素 优化潜在的临床翻译，并深入了解Fl2介导的CN修复的机制。

Abstract Radical prostatectomy (RP) is a commonly used treatment option for localized prostate cancer. Unfortunately, the procedure carries a high risk of erectile dysfunction (ED) which is highly detrimental to the post-surgical well-being of men. The main pathophysiological mechanism behind ED is damage to the cavernous nerves (CN); mechanisms that regenerate or repair CN injury following RP could alleviate or treat ED in these patients. The studies outlined in this proposal will pursue a novel approach to CN repair by harnessing the nerve regenerative powers of Fidgetin-like 2 (FL2), a newly discovered microtubule regulator. In vitro studies indicate that the depletion of FL2 strongly enhances axonal growth in primary cultures of rat peripheral nervous system neurons. Recent work by the investigators involved in this proposal have shown that targeted depletion of FL2 via topical application of FL2-siRNA encapsulated in nanoparticles (FL2-siRNA-np) promotes the closure and regeneration of cutaneous wounds in mouse models and improved erectile function outcomes in a rat model of RP when applied at the time of CN injury. Remarkably, in a CN transection model of RP, at two weeks post treatment with FL2-siRNA there is both improved erectile function outcomes and visible nerve regrowth. These observations lead us to the hypothesis to be tested in this proposal that “following CN injury, depletion of FL2 activates mechanisms that promote cavernous nerve regeneration, leading to accelerated recovery of erectile function”. This hypothesis will be tested in two specific aims. In the first Specific Aim we will substantiate our preliminary results that depletion of FL2 improves recovery of erectile function following CN injury used as an animal model of RP. We will optimize formulation and treatment regimens for ED. FL2-siRNA will be applied at the site of CN injury both at the time, and one week following injury and recovery of erectile function determined by measuring the intracorporal pressure/blood pressure ratio (ICP/BP) developed after CN stimulation, at various time points after treatment. Following cavernosometry, we will perform pathology studies to provide evidence of CN regeneration and safety of the different formulations. In the second Specific Aim we will conduct experiments to elucidate the mechanism by which the depletion of FL2 promotes functional recovery after CN injury. We will determine the effects of FL2-siRNA on axon growth and regeneration in vitro. Using tissues of controls and FL2-siRNA treated animals we will perform extensive comparative histopathology and gene expression analyses on corporal tissue and from the site of injury of treated versus control rats at various time-points after CN injury. Overall, upon completion of this proposal we will have established the potential of FL2 as a novel target in repairing CN damage following RP, determined treatment regimens, formulation and safety considerations to optimize potential clinical translation and have gained insight into the mechanism of FL2 mediated CN repair.