Overflow urinary incontinence (OUI) frequently occurs after pelvic surgery such as resection of uterine and colon cancer. Previously, we found that rats with bilateral accessory nerve injury (BAI) showed OUI symptoms [1] and abnormalities in detrusor contraction [2]. However, the effects of internal urethral function by BAI are not well known. Nitric oxide (NO) is known as one of the key molecules which regulate internal urethral function. We also reported that a novel NO donor “NORD-1”, which releases NO under irradiation of red-light, induced relaxation of intact rats’ internal urethra in vitro [3]. Thus, this study first investigated whether BAI affects internal urethral relaxation induced by NO. Next, we examined whether NORD-1 and red-light irradiation induced enough relaxation in BAI rats’ internal urethra.

We performed two experiments.
Experiment 1. Effects of BAI on the relaxation response of the internal urethra to NO 
We randomly divided ten-week-old male Wistar ST rats into BAI group and Sham group (each group, n=8). On day 1, each group received BAI or sham surgery. One week after surgery, rats were euthanized and ring tissue of the internal urethra was carefully harvested. The specimens were placed in an organ bath filled with Kreb’s solution. We evaluated the contraction response to 80 mM KCl Krebs’ solution and the relaxation response to cumulative administration of sodium nitroprusside dihydrate (SNP, 10^-9 M-10^-4 M) under phenylephrine (3×10^-5 M)-induced precontraction. We used repeated measures ANOVA with the Huynh-Feldt correction for two-group statistical comparison. In addition, we calculated Emax and EC50 and used Welch’s t-test.

Experiment 2. Relaxation of internal urethra caused by NORD-1 and red-light irradiation in BAI rats
We divided rats into two groups (each group, n=8) and conducted surgery in the same way as in experiment 1. After isolation of the internal urethra, the tissue was placed in NORD-1 (10^-5 M) with Kreb’s solution for 15 minutes. Then, we placed the tissue in an organ bath and caused precontraction with phenylephrine (3×10^-5 M). After reaching a plateau, the relaxation response was evaluated under red-light irradiation (16, 39, 78, and 149 mW/cm2). We used Welch’s t-test for each intensity.

In experiment 1, the contractile response to 80mM KCl did not change between the Sham and BAI groups. While Emax of the relaxation response to SNP did not change between two groups, EC50 was significantly higher in the BAI group than in the Sham group (log EC50, BAI; -5.6±0.2 vs. sham; -5.9±0.1, P<0.01). 
In experiment 2, red-light irradiation caused relaxation of the internal urethra specimen in all conditions and both groups. The tension returned to normal as soon as the light was turned off. Analytical results are shown in Figure 1. The ratios of the relaxation response in both groups were increased in a light-intensity-dependent manner. The relaxation response to light irradiation was lower in the BAI group than in the Sham group, but light irradiation induced relaxation up to about 70% in BAI rats.

Considering the reduction of EC50 in the relaxation response to SNP, it is possible that NO receptor-mediated signaling was attenuated in BAI rats. Emax in the relaxation response to SNP did not differ between the two groups. NO supplementation may be useful to achieve sufficient relaxation. In experiment 2, NO supplementation by NORD-1 and light irradiation also caused strong relaxation in BAI group. Moreover, the tension returned after stopping the light irradiation, suggesting that NORD-1 released NO only during irradiation.

BAI caused a decrease in the relaxation response of the internal urethra to NO. NORD-1 and light irradiation induced sufficient relaxation of the internal urethra. Using NORD-1, the release of NO can easily be controlled spatially and temporally. Therefore, administration of NORD-1 and irradiation with red light might be useful in OUI for controlling urethral relaxation after pelvic surgery.

Maeda K, Hotta Y, Shibayama M, et al. Impairment of accessory nerves around major pelvic ganglion leading to overflow urinary incontinence in rats. Neurourol Urodyn. 2021;40(2):624-631.
Maeda K, Hotta Y, Kawata R, et al. Detrusor underactivity causes neurogenic voiding dysfunction in a rat bilateral accessory nerve-injury model. J Pharmacol Sci. 2021;146(2):116-119.
Maeda K, Hotta Y, Ieda N, et al. Control of rat bladder neck relaxation with NORD-1, a red light reactive nitric oxide releaser: In vitro study. J Pharmacol sci. 2021;146(4):226-232.

Continence 12S (2024) 101470
DOI: 10.1016/j.cont.2024.101470