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Molecular Assessment of Neuroregenerative Response in the Pudendal Nerve: A Useful Tool in Regenerative Urology. 阴部神经再生反应的分子评估:再生泌尿外科的一个有用工具。
Pub Date : 2016-02-01 Epub Date: 2016-02-05
Bradley C Gill, Dan Li Lin, Brian M Balog, Charuspong Dissaranan, Hai-Hong Jiang, Margot S Damaser

Aims: Assessing pudendal nerve neuroregenerative response provides valuable insight into injuries and regenerative treatments related to urinary incontinence. This project developed and validated a cost-effective, expedient, and adoptable method of assessing pudendal nerve neuroregenerative response.

Methods: Sprague Dawley rats underwent unilateral pudendal nerve crush prior to spinal cord harvest and laser microdissection for separate collection of the injured and uninjured Onuf's nuclei (pudendal motor neuron cell bodies). Commercially available kits were used to extract and isolate RNA, as well as reverse transcribe and amplify cDNA from cells. Utilizing standard quantitative polymerase chain reaction (Q-PCR), expression of βII-Tubulin, a cytoskeletal protein indicative of nerve growth and neuroregenerative response, was determined in the injured side relative to the uninjured side 1 week after injury.

Results: Injury upregulated βII-Tubulin 2.36±0.46 times via Q-PCR, which was not significantly (p=0.508) different from the 2.49±0.38 times increase noted with in-situ hybridization previously. Starting with tissue collection, results are available within 1 day using PCR, while in-situ hybridization requires 4-weeks.

Conclusions: An easily adoptable PCR-based method of assessing the neuroregenerative response of the pudendal nerve successfully reproduced results obtained with a previous radioisotope-based in-situ hybridization technique.

目的:评估阴部神经再生反应为与尿失禁相关的损伤和再生治疗提供了有价值的见解。本项目开发并验证了一种经济、方便、可采用的评估阴部神经再生反应的方法。方法:Sprague Dawley大鼠行单侧阴部神经挤压术,取脊髓,激光显微解剖,分别收集损伤和未损伤的Onuf核(阴部运动神经元胞体)。市售试剂盒用于提取和分离RNA,以及从细胞中逆转录和扩增cDNA。利用标准定量聚合酶链反应(Q-PCR),在损伤后1周,测定损伤侧相对于未损伤侧β ii -微管蛋白(一种指示神经生长和神经再生反应的细胞骨架蛋白)的表达。结果:损伤后β ii -微管蛋白上调2.36±0.46倍(Q-PCR),与原位杂交法(2.49±0.38倍)差异无统计学意义(p=0.508)。从组织收集开始,使用PCR在1天内可获得结果,而原位杂交需要4周。结论:一种易于采用的基于pcr的评估阴部神经再生反应的方法成功地复制了以前基于放射性同位素的原位杂交技术获得的结果。
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SDRP journal of biomedical engineering
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