The male urethra performs urinary and reproductive functions. In contrast to other pelvic viscera, normal urethral functions require the activation of striated musculature. In rats, the external urethral sphincter (EUS) is a complex musculature with circular and oblique fibers that surround the prostatic and membranous urethra, and contribute to maintaining urinary continence and prevent retrograde ejaculation. The ischiocavernosus (IC) muscles are localized lateral to the base of the penis, and their contraction have been related to the control of penile erection. Both, EUS and IC muscles present evoked response to urethral stimulation [1]. Anatomically, the EUS do not present clear division between left and right muscles, however, the innervation is bilateral and arises from two, left and right, motor branches of the sacral plexus (MBSP) [2]. This pattern of innervation suggest the possibility of ipsilateral and contralateral innervation. The aim of the present study was to determine the pattern of innervation of the EUS and IC fibers as well as its reinnervation after unilateral surgical nerve injury.

The experimental protocol was approved by the University Committee on Laboratory Animals, according to the guidelines of the Mexican Council on Laboratory Animals Care (NOM-062-ZOO-1999) and NIH Guide for the Care and Use of Laboratory Animals. We employed adult Wistar male rats (250–300 g). The animals were randomized to undergo IC and EUS electromyography (EMG) before and 30 min after unilateral axotomy of the MBSP (Experiment 1, n=6), or after two, three, five and 20 weeks after the axotomy (Experiment 2, n=6 per group). In the Experiment 1 the animals were anesthetized with urethane (1.2 g/kg) and a laparotomy was performed to localize the IC and EUS muscles. Bipolar stainless steel electrodes (0.05 mm) were placed in the left and right sides of the EUS and IC muscles, and connected to an electrophysiological recorded system. Periurethral muscles EMG activity was evoked by mechanostimulation of the penile urethra using a catheter with a 0.8 mm in diameter. The catheter was introduced through the urinary meatus to the diverticulum (bulbar urethra). EMGs were simultaneously recorded before, during and after urethral stimulation in intact condition and after the axotomy of the right MBSP. In the Experiment 2 the animals were anesthetized with ketamine (60 mg/kg) and xylazine (7.5 mg/kg) and the right MBSP was localized at the level of the ischiorectal fossa and transected (5 mm of nerve was removed). The animals were anesthetized (urethane 1.2g/kg) two, three, five and twenty weeks after surgery and EMGs recorded as described above. The pattern of the EMG of periurethral muscles was characterized and one second sample during mechanical stimulation was analyzed to determine mean amplitude (µV) and frequency (Hz). Statistical analysis was performed using Sigma Plot Software (version 12, Systat Software, Inc.). Paired Student´s t-test (Experiment 1) or ANOVA (Experiment 2) were used to analyze the data. P values of <0.05 indicated a statistically significant differences for statistical comparisons.

In Experiment 1, both sides of the EUS discharged synchronically during stimulation of penile urethra and diverticulum. The EMG activity was characterized by tonic activity (amplitude, 140 ± 21 µV; frequency, 201 ± 8 Hz) and a long afterdischarge (~10 sec). Left and right IC muscles only discharged during stimulation of the diverticulum. EMG responses were in burst (amplitude, 78 ± 12 µV; frequency, 120 ± 34 Hz) and rarely presented afterdischarge (1-2 sec). Right MBSP axotomy reduced the amplitude of the ipsilateral to the transected nerve EUS reflex response to 35% and eliminated the response of the right IC, but did not affect neither the frequency nor amplitude of the left IC and EUS fibers (intact side) (p>0.05). In the Experiment 2, the EUS and IC responses to urethral stimulation recovered gradually across time, and three and five weeks after the axotomy no statistical differences were observed neither in amplitude nor frequency between the denervated side versus the non denervated side (p>0.05). However, after 20 weeks of axotomy the IC EMG response of the ipsilateral side to the sectioned MBSP was exacerbated and the EMG amplitude was larger than the value of the non-denervated side (p<0.05). In addition, the pattern of the IC muscle discharge in response to the urethral stimulation was atypical responding with tonic long after-discharges, a characteristic pattern of the EUS.

The present findings show that the pattern of discharge of the EUS differs from the IC response, which indicates that motoneurons innervating each periurethral muscle have differential electrophysiological properties. It despite IC and EUS motoneurons are hosted in the same spinal nucleus, the dorsolateral nucleus of the lumbosacral spinal cord. The fact that the EUS responded to mechanostimulation of the penile and bulbar urethra but the IC muscles only to the diverticulum indicates that regional urethral afferents converge on IC and EUS motoneurons and may explain the fact that IC muscles discharge during micturition [3], even though its contraction seems no to be crucial for the control of micturition in male rats. Our data also demonstrate that while the EUS fibers have 35% of contralateral innervation, IC muscles of male rats are only innervated by the ipsilateral MBSP. However, after unilateral denervation both muscles seems to be reinnervated by contralateral fibers of the MBSP. The denervated fibers of the EUS get reinnervation probably through the sprouting of the contiguous contralateral nerve fibers, which allow a correct function. In contrast, the denervated IC muscle gets aberrant innervation, which may affect IC function, such as penile erection. The non-specific reinnervation seems to arise from an extension of the EUS motor-units from contiguous muscle. Nerve lesion is common during pelvic surgeries, such as prostatectomies, and for this reason is important to clarify the regenerative mechanisms and reinnervation of striated muscles of the genitourinary tract, in order to develop innovative techniques to facilitate functional reinnervation of these structures.

Periurethral striated muscles are innervated by motoneuron pools with different electrophysiological properties. After unilateral nerve lesion, functional contralateral reinnervation appears for contiguous fibers, however, aberrant reinnervation may occur for not contiguous musculature. Further studies are necessary to facilitate specific reinnervation of bilateral, non-contiguous muscles of the lower urinary tract.

Juarez, R., et al., Activity of the external urethral sphincter evoked by genital stimulation in male rats. Neurourology and Urodynamics, 2016. 35(8): p. 914-919.
Juarez, R. and Y. Cruz, Urinary and ejaculatory dysfunction induced by denervation of specific striated muscles anatomically related to the urethra in male rats. Neurourology and Urodynamics, 2014. 33(4): p. 437-42.
Cruz, Y. and JW. Downie, Sexually dimorphic micturition in rats: relationship of perineal muscle activity to voiding pattern. American Journal of Physiology Regulatory, Integrative and Comparative Physiology, 2005. 289(5): R1307-18.