AB288. SPR-15 Motor unit number estimation of the external anal sphincter in rats

Objective: Reflexive and active control of the external anal sphincter (EAS) is essential for maintaining regulated colonic functions. Anorectal dysfunction, often related to compromised function of the EAS, is one of the major complaints in patients. Currently, the clinical diagnosis of anorectal dysfunction includes manometry, motor latency test, and single fiber electromyography (EMG). However, none of these techniques can be used to document the global innervation of the EAS in vivo. In this study, a novel approach was developed to non-invasively estimate the number of functioning motor units in the EAS. We further validated results with immunostaining of EAS in rats.

Methods: Compound muscle action potential (CMAP) responses were elicited by series of preset stimuli delivered intra-vaginally to the pudendal nerve and the variation of the CMAPs was used to estimate the single motor unit potential (SMUP). The motor unit number estimation (MUNE) was implemented by calculating the ratio between the maximal CMAP and mean SMUP. Immunostaining of nicotinic acetylcholine receptors (AChRs) was used as a marker for identifying myofibers in the EAS. Linear regression analysis was performed to determine the relationship between MUNE and corresponding number of myofibers, with a significance level set at 0.05.

Results: The MUNE was successfully performed in 7 female rats, while immunofluorescence was implemented in 5 of them. The averaged SMUP was estimated as 45.00±17.24 µV, and the mean MUNE was calculated as 41±12 among the 7 rats tested. The results show good consistency with previous findings that the number of motoneurons innervating the EAS varies between 31.7±8.5 to 73±9.96 using retrograde tracing. The mean number of successfully identified myofibers was 580±45 myofiber/EAS, where a linear relationship between the MUNE and number of myofibers was observed (P<0.05).

Conclusions: This study represents the first effort to non-invasively assess the innervation of EAS in vivo using the rat as a pre-clinical model. The performance of the proposed MUNE approach was validated by comparison with immunostaining results, suggesting a consistent innervation ratio in rat EAS. This approach can potentially enable future clinical applications for advanced diagnosis and treatment of neurogenic EAS disorders.

Funding Source(s): NIH DK082644, University of Houston, the Brown foundation, and Houston Methodist foundation

Keywords: External anal sphincter (EAS); motor unit estimation; electromyography (EMG); immunostaining