Sildenafil attenuates inflammation and oxidative stress in pelvic ganglia neurons after bilateral cavernosal nerve damage

Abstract

Erectile dysfunction is a common complication for patients undergoing surgeries for prostate, bladder, and colorectal cancers, due to damage of the nerves associated with the major pelvic ganglia (MPG). Functional re-innervation of target organs depends on the capacity of the neurons to survive and switch towards a regenerative phenotype. PDE5 inhibitors (PDE5i) have been successfully used in promoting the recovery of erectile function after cavernosal nerve damage (BCNR) by up-regulating the expression of neurotrophic factors in MPG. However, little is known about the effects of PDE5i on markers of neuronal damage and oxidative stress after BCNR. This study aimed to investigate the changes in gene and protein expression profiles of inflammatory, anti-inflammatory cytokines and oxidative stress related-pathways in MPG neurons after BCNR and subsequent treatment with sildenafil. Our results showed that BCNR in Fisher-344 rats promoted up-regulation of cytokines (interleukin- 1 (IL-1) β, IL-6, IL-10, transforming growth factor β 1 (TGFβ1), and oxidative stress factors (Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), TNF receptor superfamily member 5 (CD40) that were normalized by sildenafil treatment given in the drinking water. In summary, PDE5i can attenuate the production of damaging factors and can up-regulate the expression of beneficial factors in the MPG that may ameliorate neuropathic pain, promote neuroprotection, and favor nerve regeneration.

Figure 1

Cytokine expression is regulated by BCNR and SIL treatment. Protein extracts were incubated with a cocktail of biotinylated detection antibodies and then incubated in a membrane array containing 29 different anti-cytokine antibodies, in duplicates, followed by streptavidin peroxidase and chemoluminescence. The densitometric signal produced by each spot run in duplicate was proportional to the amount of cytokine bound as determined by densitometry analysis. (A) Representative membranes for Sham, BCNR and BCNR+SIL; (B) Densitometry analysis of cytokines * p < 0.05; *** p < 0.001 with respect to sham. # p < 0.05 with respect to BCNR.

Figure 2

Sildenafil down regulates the expression of inflammatory cytokine IL-1β in the pelvic ganglia after BCNR. (A) Protein extracts were subjected to Western blot analysis for IL-1β and normalized by GAPDH with the correspondent densitometric analysis. * p < 0.05; ** p < 0.01 respect to sham. ## p < 0.01 respect to BCNR; (B) Frozen pelvic ganglia sections were immunostained with a polyclonal antibody against IL-1β followed by a biotinylated secondary antibody and streptavidin conjugated with Texas red. Red: IL-1β, Yellow: FG. White: DAPI. Magnification 100×; Bar=100 µm.

Figure 3

Sildenafil up-regulates the expression of IL-6 in the pelvic ganglia after BCNR. Frozen pelvic ganglia sections were immunostained with a polyclonal antibody against IL-6 followed by a biotinylated secondary antibody and streptavidin conjugated with Texas red. Red: IL-6, Yellow: FG. White: DAPI. Magnification 100×; Bar = 100 µm.

Figure 4

Inducible nitric oxide synthase (iNOS) expression is down regulated by Sildenafiltreatment after BCNR. (A) Total protein extracts of Sham, BCNR and BCNR+SIL were subjected to Western blot studies for iNOS and normalized by GAPDH with the correspondent densitometric analysis, * p < 0.05, ** p < 0.01, respect to sham. # p < 0.05 respect to BCNR; (B) Frozen section of the pelvic ganglia were immunostained with a polyclonal antibody against iNOS followed by a biotinylated secondary antibody and Vectastain^® ABC kit. 3,3'-Diaminobenzidine (DAB) was used as a chromogen and sections were counterstained with Hematoxylin. Magnification 400×. Bar= 50 μm.

Figure 5

(A) Heme oxygyenase 1 is up-regulated by BCNR and down-regulated by Sildenafil treatment. Total RNA was extracted from 7 days Sham, BCNR and BCNR+ SIL and was subjected to real time PCR by TaqMan for HO-1 and normalized by the Ribosomal protein, large 1/3 (RPLP1/3) housekeeping gene; (B) Pelvic ganglia protein extracts were subjected to Western blot analysis for HO-1 by employing a monoclonal antibody normalized by GAPDH with the correspondent densitometric analysis; (C) Frozen pelvic ganglia sections from 7 days Sham, BCNR and BCNR+SIL were immunostained for HO-1 by employing a polyclonal antibody, with subsequent detection by DAB. No Counterstaining was performed. Representative pictures. Magnification 200×; Bar=50 µm. Image Analysis determined by integrated optical density (IOD) per cells in the pelvic ganglia *** p < 0.001; ** p < 0.01; * p < 0.05 with respect to Sham. # p < 0.05 , ### p < 0.001 with respect to BCNR.