You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research II1 Apr 2012116 TARGETED MOLECULAR ASSESSMENT OF PUDENDAL NERVE NEUROREGENERATIVE RESPONSE - A MEANS OF ANALYZING INJURY, RECOVERY, AND REGENERATIVE THERAPIES Bradley Gill, Danli Lin, Brian Balog, Charuspong Dissaranan, Hai-Hong Jiang, and Margot Damaser Bradley GillBradley Gill Cleveland, OH More articles by this author , Danli LinDanli Lin Cleveland, OH More articles by this author , Brian BalogBrian Balog Cleveland, OH More articles by this author , Charuspong DissarananCharuspong Dissaranan Cleveland, OH More articles by this author , Hai-Hong JiangHai-Hong Jiang Cleveland, OH More articles by this author , and Margot DamaserMargot Damaser Cleveland, OH More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.164AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES This project aimed to validate an improved method of quantifying mRNA upregulation in small, isolated tissue samples using laser capture microdissection (LMD) and quantitative real-time PCR (Q-PCR). Upregulation of βII-tubulin, a cytoskeletal protein, provides a measure of the neuroregenerative response to peripheral nerve injury. This was investigated in the context of childbirth-related pudendal nerve (PN) injury by studying the PN cell bodies in Onuf's nucleus and validating the new method through comparison to results obtained using radioisotopes and in situ hybridization. METHODS Virgin Sprague-Dawley rats (N=4, 225-250g) received unilateral PN crush injury. Intracardiac perfusion of heparinized saline and lumbo-sacral laminectomy were performed 7-days later, after which the spinal cord was frozen in situ with liquid nitrogen and the L3-S2 segment removed en bloc and stored inside a cryotube in liquid nitrogen. The PN cell bodies, located in Onuf's nucleus at approximately the L6 spinal cord level in female rats, were identified anatomically via serial 10μm cryostat sections. Samples were collected on LMD slides, stored at −80°C, and then stained with thionin and fixed with ethanol and xylene. Using LMD, a total of 5 Onuf's nuclei from the injured side were isolated from serial sections in each rat, collected in cell lysis solution, and then frozen on dry ice. This was repeated on the uninjured contralateral side. Samples then separately underwent RNA isolation and DNA digestion prior to reverse transcription and subsequent non-biasing PCR cDNA pre-amplification that targeted βII-tubulin (gene: TUBB2C). Samples underwent Q-PCR using ribosomal 18s RNA (gene: 18s) as an endogenous control and expression after injury relative to no injury was determined. RESULTS The LMD-PCR method showed βII-tubulin was expressed 2.36 ± 0.46 times higher in injured compared to uninjured PN cell bodies. Prior in situ hybridization methods (N=19) showed a 2.49 ± 0.38 fold greater expression after the same injury done bilaterally and compared to uninjured rats. CONCLUSIONS The LMD-PCR method reproduced previous in situ hybridization findings without radioactive reagents and in 8-9 hours rather than 4 weeks, as with in situ hybridization. This new technique may be useful in preclinical studies assessing the neuroregenerative response to therapeutic agents. It has clinical implications not only in childbirth injury but also prostatectomy-related nerve injury. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e47 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bradley Gill Cleveland, OH More articles by this author Danli Lin Cleveland, OH More articles by this author Brian Balog Cleveland, OH More articles by this author Charuspong Dissaranan Cleveland, OH More articles by this author Hai-Hong Jiang Cleveland, OH More articles by this author Margot Damaser Cleveland, OH More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...