Overflow urinary incontinence, which occurs with nerve injury during pelvic surgery for conditions such as uterine and colon cancers, significantly decreases the patient’s quality of life (QOL). We had previously reported a novel neurogenic overflow urinary incontinence model due to accessory nerve (ACN) injury, which showed hypertrophy and fibrosis of the bladder [1]. However, the detailed mechanisms underlying the fibrosis of the bladder due to ACN injury remain unclear. In this study, we aimed to evaluate the time-series expression of fibrosis-related genes in the bladder after bilateral ACN injury (BAI).

Ten-week-old male Wistar/ST rats were randomly categorized into sham and BAI groups. In the BAI group, we identified and crushed the ACN on both sides for 1 min using reverse-action tweezers. In the sham group, we only identified the ACN. After 72 h postoperatively, we evaluated bladder function using cystometrogram and performed Masson’s trichrome staining in the sham and BAI groups. Next, we set other rats for the observation period of 4, 8, 12, 24, and 72 h in the sham and BAI groups (Sham-4h, Sham-8h, Sham-12h, Sham-24h, Sham-72h, BAI-4h, BAI-8h, BAI-12h, BAI-24h, and BAI-72h groups). In all groups, after the observation period, the bladder was removed, and the mRNA expression levels of Tgf-β1 and Mcp-1 were evaluated using real-time PCR analysis.

Symptoms of overflow urinary incontinence were observed 72 h after BAI. The total area, collagen area, as well as the ratio of the collagen area to the total area of the bladder were significantly increased in the BAI group compared to those in the sham group 72 h postoperatively (p < 0.05; Fig. 1). Next, we investigated bladder weight and fibrosis-related gene expression levels in the bladder at an earlier than 72 h. The bladder weight to body weight ratio was significantly increased in the BAI-4h, BAI-8h, BAI-24h, and BAI-72h groups compared with that in the corresponding sham groups (p < 0.05). During the observation period, the mRNA expression levels of Tgf-β1 were tended to increase in the BAI groups compared with those in the corresponding sham groups, and this increase was significant in the BAI-12h group than in the Sham-12h group (p < 0.01; Fig. 2A). The mRNA expression levels of Mcp-1 were significantly increased in the BAI-4h, BAI-8h, BAI-12h, and BAI-24h groups compared with those in the corresponding sham groups (p < 0.05); however, there was no difference between the BAI-72h and Sham-72h groups (Fig. 2B).

The results of histological evaluation suggested that BAI causes hypertrophy and fibrosis of the bladder only 72 h after the operation. Therefore, to explore the mechanisms underlying fibrosis of the bladder, we investigated bladder weight and fibrosis-related gene expression in the bladder at an earlier than 72 h. The bladder weight to body weight ratio increased after 4 h of BAI, suggesting that organic changes in the bladder had already occurred. The mRNA expression levels of Mcp-1 were significantly increased 4 h after BAI and then decreased gradually, suggesting that MCP-1 may be involved in the initial inflammation of the bladder due to pelvic nerve injury. On the other hand, the mRNA expression levels of Tgf-β1 were significantly increased later than the time required for Mcp-1 and then maintained at high levels, suggesting that TGF-β1 may be involved in continuous fibrosis of the bladder due to pelvic nerve injury.

Pelvic nerve injury causes overflow urinary incontinence, hypertrophy, and fibrosis of the bladder. This might be involved in the upregulation of fibrosis-related genes at an earlier phase.

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.