Bradley C Gill, Dan Li Lin, Brian M Balog, Charuspong Dissaranan, Hai-Hong Jiang, Margot S Damaser
Bradley Gill, MD, MS, Molecular Assessment of Neuroregenerative Response in the Pudendal Nerve: A Useful Tool in Regenerative Urology(2016)SDRP Journal Of Biomedical Engineering 1(1)
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 ?II-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 ?II-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.
Thor KB, de Groat WC. Neural control of the female urethral and anal rhabdosphincters and pelvic floor muscles. Am J Physiol Regul Integr Comp Physiol. 2010;299(2):R416-38. PMid:20484700 PMCid:PMC2928615View Article PubMed/NCBI
Gill BC, Damaser MS, Vasavada SP, Goldman HB. Stress incontinence in the era of regenerative medicine: Reviewing the importance of the pudendal nerve. J Urol. 2013;190(1):22-28. PMid:23376143 PMCid:PMC4158918View Article PubMed/NCBI
Carr LK, Steele D, Steele S, et al. 1-year follow-up of autologous muscle-derived stem cell injection pilot study to treat stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct. 2008;19(6):881-883. PMid:18204978View Article PubMed/NCBI
Dissaranan C, Cruz MA, Kiedrowski MJ, et al. Rat mesenchymal stem cell secretome promotes elastogenesis and facilitates recovery from simulated childbirth injury. Cell Transplant. 2014;23(11):1395-1406. PMid:23866688 PMCid:PMC4464671View Article PubMed/NCBI
Takahashi S, Chen Q, Ogushi T, et al. Periurethral injection of sustained release basic fibroblast growth factor improves sphincteric contractility of the rat urethra denervated by botulinum-a toxin. J Urol. 2006;176(2):819-823. PMid:16813954View Article PubMed/NCBI
Deng K, Lin DL, Hanzlicek B, et al. Mesenchymal stem cells and their secretome partially restore nerve and urethral function in a dual muscle and nerve injury stress urinary incontinence model. Am J Physiol Renal Physiol. 2015;308(2):F92-F100. PMid:25377914View Article PubMed/NCBI
Gill B, Balog B, Disaranan C, et al. Neurotrophin therapy improves recovery of the neuromuscular continence mechanism following simulated birth injury in rats. Neurourology and Urodynamics. 2013;32(1):82-87. PMid:22581583 PMCid:PMC3419785View Article PubMed/NCBI
Jiang HH, Gill BC, Dissaranan C, et al. Effects of acute selective pudendal nerve electrical stimulation after simulated childbirth injury. Am J Physiol Renal Physiol. 2013;304(3):F239-47. P Mid:23152293 PMCid:PMC3566519View Article
Jiang H, Gustilo-Ashby A, Salcedo L, et al. Electrophysiological function during voiding after simulated childbirth injuries. Exp Neurol. 2009;215(2):342. PMid:19056383 PMCid:PMC2721825View Article PubMed/NCBI
Gill BC, Moore C, Damaser MS. Postpartum stress urinary incontinence: Lessons from animal models. Expert Rev Obstet Gynecol. 2010;5(5):567-580. PMid:21113428 PMCid:PMC2989536View Article PubMed/NCBI
DeLucia TA, Alexander TD, Fargo KN, Jones KJ. Effects of single versus combinatorial treatment strategies on beta II-tubulin gene expression in axotomized hamster rubrospinal motoneurons. Restor Neurol Neurosci. 2007;25(5-6):573-584. PMid:18334774
Kane D, Kerns J, Lin D, Damaser M. Early structural effects of oestrogen on pudendal nerve regeneration in the rat. BJU Int. 2004;93(6):870. PMid:15050008View Article PubMed/NCBI
Willis D, Li KW, Zheng JQ, et al. Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons. J Neurosci. 2005;25(4):778-791. PMid:15673657View Article PubMed/NCBI
Kerns JM, Damaser MS, Kane JM, et al. Effects of pudendal nerve injury in the female rat. Neurourol Urodyn. 2000;19(1):53-69. 1520-6777(2000)19:1<53::AID-NAU7>3.0.CO;2-8View Article
Sakamoto K, Smith GM, Storer PD, Jones KJ, Damaser MS. Neuroregeneration and voiding behavior patterns after pudendal nerve crush in female rats. Neurourol Urodyn. 2000;19(3):311-321. 1520-6777(2000)19:3<311::AID-NAU11>3.0.CO;2-6View Article
Lee VH, Lee LT, Chu JY, et al. An indispensable role of secretin in mediating the osmoregulatory functions of angiotensin II. FASEB J. 2010;24(12):5024-5032. PMid:20739612 PMCid:PMC2992369View Article PubMed/NCBI
Hoffman P, Cleveland D. Neurofilament and tubulin expression recapitulates the developmental program during axonal regeneration: Induction of a specific beta-tubulin isotype. Proc Natl Acad Sci. 1988;85:4530. PMid:3132717 PMCid:PMC280464View Article PubMed/NCBI
Jiang Y, Pickett J, Oblinger M. Comparison of changes in beta-tubulin and NF gene expression in rat DRG neurons under regeneration-permissive and regeneration-prohibitive conditions. Brain Res. 1994;637:233. 91238-6View Article
Erickson HS, Albert PS, Gillespie JW, et al. Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples. Lab Invest. 2007;87(9):951-962. PMid:17643124View Article PubMed/NCBI
Santos AR, Duarte CB. Validation of internal control genes for expression studies: Effects of the neurotrophin BDNF on hippocampal neurons. J Neurosci Res. 2008;86(16):3684-3692. PMid:18655199View Article PubMed/NCBI
Kleinschnitz C, Brinkhoff J, Sommer C, Stoll G. Contralateral cytokine gene induction after peripheral nerve lesions: Dependence on the mode of injury and NMDA receptor signaling. Brain Res Mol Brain Res. 2005;136(1-2):23-28. PMid:15893583View Article PubMed/NCBI
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods. 2001;25(4):402-408. PMid:11846609View Article PubMed/NCBI
Bhatia NN, Ho MH. Stem cell therapy for urinary incontinence and pelvic floor disorders: A novel approach. Current Opinion in Obstetrics and Gynecology. 2004;16(5):397-398.View Article
Ho MH, Heydarkhan S, Vernet D, et al. Stimulating vaginal repair in rats through skeletal muscle-derived stem cells seeded on small intestinal submucosal scaffolds. Obstet Gynecol. 2009;114(2 Pt 1):300-309. PMid:19622991 PMCid:PMC3217318View Article PubMed/NCBI
Lee JY, Cannon TW, Pruchnic R, Fraser MO, Huard J, Chancellor MB. The effects of periurethral muscle-derived stem cell injection on leak point pressure in a rat model of stress urinary incontinence. Int Urogynecol J Pelvic Floor Dysfunct. 2003;14(1):31-7; discussion 37. PMid:12601514View Article PubMed/NCBI
Khorramirouz R, Mozafarpour S, Kameli SM, et al. A novel method of urinary sphincter deficiency: Serial histopathology evaluation in a rat model of urinary incontinence. Anat Rec (Hoboken). 2016;299(2):173-180. PMid:26574901View Article PubMed/NCBI
Sumino Y, Yoshikawa S, Mori KI, Mimata H, Yoshimura N. Insulin-like growth factor-1 as an important endogenous growth factor for the recovery from impaired urethral continence function in rats with simulated childbirth injury. J Urol. 2016. PMid:26767520View Article PubMed/NCBI
Jiang YQ, Oblinger MM. Differential regulation of beta III and other tubulin genes during peripheral and central neuron development. J Cell Sci. 1992;103 ( Pt 3)(Pt 3):643-651.
Jiang YQ, Pickett J, Oblinger MM. Long-term effects of axotomy on beta-tubulin and NF gene expression in rat DRG neurons. J Neural Transplant Plast. 1994;5(2):103-114. PMid:7703290 PMCid:PMC2565286View Article PubMed/NCBI
Erickson HS, Albert PS, Gillespie JW, et al. Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples. Nat Protoc. 2009;4(6):902-922. PMid:19478806 PMCid:PMC2760821View Article PubMed/NCBI
Huang T, Krimm RF. BDNF and NT4 play interchangeable roles in gustatory development. Dev Biol. 2014;386(2):308-320. PMid:24378336 PMCid:PMC3950349View Article PubMed/NCBI
Mesnard NA, Alexander TD, Sanders VM, Jones KJ. Use of laser microdissection in the investigation of facial motoneuron and neuropil molecular phenotypes after peripheral axotomy. Exp Neurol. 2010;225(1):94-103. PMid:20570589 PMCid:PMC2922448View Article PubMed/NCBI
Chen P, Song J, Luo LH, Xiao HJ, Gong SS. Isolating RNAs from rat facial motor neurons with laser capture microdissection after facial-facial anastomosis. Neurosci Lett. 2010;468(3):316-319. PMid:19914333View Article PubMed/NCBI
Ketterer K, Rao S, Friess H, Weiss J, Buchler MW, Korc M. Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer. Clin Cancer Res. 2003;9(14):5127-5136. PMid:14613990
Chang HY, Havton LA. Anatomical tracer injections into the lower urinary tract may compromise cystometry and external urethral sphincter electromyography in female rats. Neuroscience. 2010;166(1):212-219. PMid:20004710 PMCid:PMC2833211View Article PubMed/NCBI