171 Increased Level of Tumor Necrosis Factor-alpha (TNFA) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and TNFA Inhibition Can Enhance Penile Smooth Muscle Relaxation by Upregulating Expression of Nitrergic Neurons

Erectile dysfunction (ED) is induced due to inflammation in pelvic ganglia after cavernous nerve (CN) injury. Previously, we demonstrated that increase in inflammatory cells, particularly neurotoxic M1 macrophages, can lead to impaired smooth muscle relaxation following bilateral CN injury (BCNI). The aim of this study is to examine temporal changes of tumor necrosis factor-alpha (TNFA), after BCNI in vivo and its effect on ex vivo neurite outgrowth from major pelvic ganglion (MPG) with exogenous administration. In addition, we examined effect of TNFA signal inhibition on penile smooth muscle function ex vivo with TNFA receptor-1,2 knockout mice (TNFR-KO). Male Sprague-Dawley rats were randomized to undergo BCNI or sham surgery. Sham rats’ MPGs were harvested after 48 hours. MPGs of BCNI groups were harvested at 6 hours, 12 hours, 24 hours, 48 hours, 7 days, or 14 days after surgery (5 rats/group). qPCR was used to evaluate gene expression of neuronal nitric oxide synthase (nNOS) and tyrosine hydroxylase (TH). Western blot (WB) was used to evaluate protein amount of TNFA in MPGs, and immunofluorescence (IF) was used to localize TNFA. MPGs were harvested from healthy rats and cultured in Matrigel with different doses of exogenous TNFA (eTNFA) (0, 10, 20, 30 ng/mL). We measured neurite outgrowth from MPGs 3 days after culture. MPGs were processed for IF for TH and nNOS to compare treatment with TNFA to control. We used wild type mice (WT) and TNFR-KO to examine effect of TNFA inhibition on smooth muscle function after BCNI. WT and TNFR-KO were divided into BCNI and sham groups. MPGs were collected 48 hours after surgery and processed for qPCR to evaluate gene expression of nNOS. Penes were harvested to evaluate smooth muscle function to electrical field stimulation (EFS) with myography.