Molecular Assessment of Neuroregenerative Response in the Pudendal Nerve: A Useful Tool in Regenerative Urology

Abstract

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 β_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 β_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.

Keywords: Beta-Tubulin; Nerve Regeneration; Neurogenic; Onuf’s Nucleus; Pudendal Nerve.

Figure 1

Overview of the PCR method for assessing neuroregenerative response in the pudendal nerve.

Figure 2

Schematic showing the rat L4–L6 spinal levels (A) near Onuf’s nucleus and a photographic overlay (B) of an L5 spinal cord section, depicting the separation of Onuf’s nucleus into the two distinct regions for the anal sphincter (AS) and urethral sphincter (US), which is the dorsolateral region. Neuronal cell bodies, stained blue-purple with thionin, are visualized as darkened spots on the grey-brown background staining.

Figure 3

Neuronal cell bodies, stained blue-purple with thionin, are visualized as darkened spots (A) on the grey-brown background staining. Photographic sequence, progressing clockwise from the top left panel that depicts isolation (B and C) and dissection (D) of the urethral sphincter region of Onuf’s nucleus using laser microdissection. All images are 20× magnification.

Figure 4

Comparison of a PCR-based method to the use of in-situ hybridization to assess the neuroregenerative response of the pudendal nerve 7 days after nerve crush injury, as signified by an increase in expression of the cytoskeletal protein β_II-Tubulin relative to the expression levels in uninjured pudendal nerve cell bodies. No statistically significant difference in measured upregulation was detected. In situ hybridization data reprinted from Sakamoto, et al. 2000 with permission.