Prolonged childbirth is a risk factor for urinary incontinence whose mechanisms are not well established. In rats, the vaginal distension (VD) model, which mimics prolonged childbirth, stretches the pelvic nerves, abolishes external urethral sphincter (EUS) [1] activity and induces urine leakage, a behavioral sign of stress urinary incontinence [2]. The present study aimed to determine in rats after VD if the electromyographic (EMG) activity of the EUS and behavioral urinary continence recover simultaneously.

The experimental protocol was approved by the university committee on Laboratory Animals and performed in accordance with the guidelines of the Mexican Council on Laboratory Animals Care (NOM-062-Z00-1999), and the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies, USA). We employed adult nulliparous Wistar female rats (250–300 g). The animals were randomized to undergo 4 h of VD or sham VD (SH). In VD animals, to simulate the difficulty of the fetus passing through the pelvic cavity and the injury induced by a prolonged second stage of human childbirth, a modified 10 Fr. Foley balloon catheter was inserted into the vagina and filled with 4 ml water for a period of 4 h. In SH animals the catheter was placed into the vagina for 4 h but was not inflated. Rats were divided into two experiments. In experiment 1, micturition behavior was recorded for 6 h, before and 3 to 13 days after VD (SH, n=5; VD, n=8) employing a closed-circuit video system that includes infrared cameras and digital video recording. Animals were placed in a wire floor cage and voided volume was collected in a plastic container. The ventral abdominal area of the animals was videotaped to determine urine loss during spontaneous rat behaviors. Stereotyped female micturition behavior occurred when rats walked to the edges of the cage, placed the rump toward the wall, and expelled urine, flowing in a stream [2]. In this case urine was found in the four edges but mainly in the corners. Leakage was considered as any small volume of urine expelled (~0.02 ml) without the stereotyped female behavior of micturition, and the number of drops were analyzed.
     In experiment 2, EUS EMGs were recorded immediately and 3-12 days after VD (VD, n=5 per day; SH, n=8) employing an electrophysiological recording system. EUS EMG responses were induced by mechanical stimulation of the clitoral sheath (pudendo-pudendal reflex) or the bladder (bladder-EUS reflex). During both stimulations, amplitude (µV) and frequency (Hz) of a 1-second sample at the middle of the EUS EMGs was analyzed.

SH rats showed the stereotyped female behavior of micturition and did not drip urine in any body posture throughout the study. In contrast, 100% of VD rats dripped urine in the absence of the stereotyped voiding behavior at day 3 (38 ± 7 drops) and 5 (39 ± 8 drops) after VD. The leakage was associated with behaviors implicating stress, such as standing to reach food, sneezing, scratching and vertical exploration. At day 7 (28 ± 5 drops) and 9 (13 ± 2 drops) after VD, the dripping disappeared in 70% of animals and at day 13 in 100%. In SH animals, EUS EMG activity during the pudendo-pudendal reflex was 87.4 ± 11 µV and 344 ± 50 Hz, and during bladder-EUS reflex was 52.2 ± 6 µV and 255 ± 25 Hz. In the VD group, EUS EMG activity was abolished immediately until day 5 after VD. At day 6, EMG activity during the pudendo-pudendal reflex was recovered in 26% (14.5 ± 5 µV) and 16% during the bladder-EUS reflex (14.3 ± 7 µV) with respect to EUS EMG amplitude of SH animals. At day 7 and 9 after VD the amplitude of EUS EMG activity was recovered to 40-60% and 30-35% during the pudendo-pudendal and bladder-EUS reflexes, respectively. At day 10-12 both reflexes were recovered to 100%.

The stereotyped behavior of micturition of female rats suggests that they feel bladder fullness and volition to expel urine, then move to the edges of the cage. Therefore, any urine expelled without the stereotyped behavior of micturition was considered as a sign of urinary incontinence. The fact that VD completely abolished the EUS EMG activity at the first 5 days after VD, which correlates with the maximum expression of stress urinary incontinence (leakage associated with stress behaviors), corroborate that EUS tonic contraction contributes to maintain urinary continence [3]. EUS inactivity can be induced during VD by injury of the EUS muscle, its nerve or neuromuscular junctions. Given that urine leakage did not occur all the time, we assume that rats can maintain certain degree of urinary continence after VD, suggesting that the other components of urinary continence are not severely affected by VD, particularly urethral smooth muscle and epithelial cells and their innervation from the major pelvic ganglia [2]. EUS activity gradually recovered after VD and required about 10-12 days to restore to the values of SH animals suggesting that this time period is the window for regenerative processes of the injured structures after VD. Given that EUS activity recovery precedes recovery of behavioral expression of urinary continence, suggests that EUS activity is necessary to correct urinary continence in rats after VD.

Urinary continence recovery after VD in conscious rats is preceded by EUS neuromuscular circuitry recovery.

Jiang, H.H., et al., Electrophysiological function during voiding after simulated childbirth injuries. Experimental Neurology, 2009. 215(2): p. 342-348.
Palacios, J.L., et al., Neuroanatomic and behavioral correlates of urinary dysfunction induced by vaginal distension in rats. American Journal of Physiology-Renal Physiology, 2016. 310(10): p. F1065-F1073.
Jiang, H.H., L.B. Salcedo, and M.S. Damaser, Quantification of neurological and other contributors to continence in female rats. Brain Research, 2011. 1382: p. 198-205.