Analyzing BoNT-induced urinary retention in vivo.

A. Schematic illustration of the bladder injection model. Mice were anesthetized with isoflurane and a low midline laparotomy was made. The bladder was exteriorized and decompressed of urine to thicken the bladder wall. BoNT/B was injected into the lateral walls of the bladder bilaterally. B. Mice injected with BoNT/B were subjected to 4-hour nighttime voiding spot assay every 24 hours for 4 days. Representative images for urine spots (black areas) are shown. C. Voiding spots quantified into total void areas (inch²) demonstrate similar pre-injection urine volumes across WT, Syt1^M3, and Syt2^M3 mice (n = 5 for each group). All groups showed a decline in voided volumes in sham controls that went through the surgery and injection procedures without toxins, likely from surgical manipulation of the bladder and the use of post-operative pain control narcotics. n = 3 for each group. D. At 24–48 hours post-injection, WT and Syt2 KI mice had significantly diminished voided volumes compared to Syt1 KI mice, with progressive recovery over 96 hours. *P<0.05, **P<0.005, ns = not significant. E. Number of voiding spots per 4-hour nighttime evaluation showed no difference across genotypes, and across pre- and post-injection time points, demonstrating that voiding frequency is unchanged across groups. ns = not significant.