Potential ameliorative effect of intracavernosal botulinum toxin type a treatment on erectile dysfunction in rats with bilateral cavernous nerve injury

Objective: After radical prostatectomy (RP), erectile dysfunction (ED) is frequently observed, primarily due to iatrogenic injury to the cavernous nerves. Disruption of innervation may lead to structural changes in the cavernous tissue such as hypoxia, increased fibrosis and endothelial cell atrophy. The relaxant effect of botulinum neurotoxin type A (BoNT-A) on smooth muscles and its suppressive effect on neuradrenaline release from sympathetic neurons suggest its potential utility in the management of ED following RP and in penile rehabilitation. In our study, we aimed to investigate the effectiveness and mechanisms of action of intracavernosal BoNT-A injection therapy on erectile function and penile rehabilitation in a rat model of bilateral cavernous nerve injury (BCNI). Materials and Methods: Twenty-eight male Sprague-Dawley rats weighing 250-350g were randomly divided into 4 groups. Group A received sham operation + intracavernosal saline, group B received sham operation + intracavernosal BoNT-A, group C received BCNI + intracavernosal saline, and group D received BCNI + intracavernosal BoNT-A. In the BCNI groups, bilateral crushing of the cavernous nerves was performed using a clamp, while in the sham operation groups, the cavernous nerves were not manipulated. In the BoNT-A groups, 1 ml volume of onabotulinum toxin A, equivalent to 10 units, was injected intracavernosally, while in the other groups, intracavernosal saline injection of the same volume was administered. Erectile function was evaluated on day 14 by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) following in-vivo electrical stimulation of the cavernous nerve. In vitro relaxation responses of isolated corpus cavernosum tissues obtained from rats were measured in an organ bath using acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS). Western blot and immunohistochemistry analyses were performed for eNOS, nNOS, Bax, TGF-β, and HIF-1α proteins in penile tissues stored at -80°C after euthanasia. Masson's trichrome staining was performed on tissue sections. Results: BoNT-A injection statistically significantly improved the impaired ICP/MAP and total ICP values after BCNI at all voltage levels. At 10-3 and 10-4 M ACh concentrations, cavernous tissues isolated from group D rats showed a greater in-vitro endothelium-dependent relaxation response than group C rats (p<0.05). There was no statistically significant difference between C and D groups in terms of in-vitro SNP and EFS relaxation responses (p<0.05). Masson trichrome staining showed that smooth muscle/collagen ratio decreased after BCNI and BoNT-A injection corrected this ratio (p<0.001). Western-blot analysis showed that BoNT-A reduced the increased Bax protein expression in the cavernous tissue after BCNI (p<0.05). Immunohistochemical analysis demonstrated a significant increase in staining intensity for TGF-β and HIF-1α, and a significant decrease in staining intensity for nNOS after BCNI, which were reversed by BoNT-A. There was no difference between groups in immunohistochemical evaluation of eNOS protein. Conclusion: Our study suggests that intracavernosal injection of BoNT-A in the early post-RP period may be beneficial for symptomatic treatment of ED and preservation of penile structural features. Further preclinical and clinical studies are needed.