Dual Strategy with Intracavernosal Implantation of Adipose-derived Stem Cells and Daily Oral L-arginine Optimize Recovery of and Cavernosal Functions after Cavernous Nerve Injury in Rats

Abstract

ABSTRACT Introduction Radical prostatectomy for prostate cancer has a significant potential role in inducing cavernous nerve injury (CNI). Increasing hypoxia and fibrosis induces destructive changes within cavernous vasculature. Adipose-derived stem cells (ADSCs) can maintain self-renewal and enhanced multi-differentiation potential through the release of a variety of paracrine factors and extracellular vesicles, allowing to repair damaged tissues. Considerable attention has increasingly been paid to their application in tissue engineering and regeneration. In addition, ADSCs can be obtained with a minimally invasive procedure and easily expanded in vitro with low immunogenicity. Nitric oxide (NO) is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases (NOS). Orally administered L-arginine enhances the erectile response presumably by stimulating NO production by the corporal tissues increasing cGMP production. Objective To evaluate the efficacy and tolerability of ADSCs and daily oral L-arginine alone or combination in treating rats with bilateral CNI (BCNI)-induced ED. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups: 1) control; 2) BCNI (4-wk); 3) BCNI+ADSCs (1 × 106 cells by intracavernosal injection); 4) BCNI+ L-arginine (4wk,10 mg/kg/day, oral); and 5) combination treatment with ADSCs and L-arginine in BCNI. Rats in the ADSCs treatment groups were subjected to intracavernosal injection after BCNI. In vivo erectile response was measured by CN stimulation. The relaxant and contractile responses were also detected on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) as well as hypoxia (hypoxia-inducible factor-1α, HIF-1α), fibrosis [transforming growth factor-beta 1 (TGF- β1)]and apoptosis (Bax and Bcl-2) markers were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results Rats with CNI had the lowest ICP/MAP ratio at 63% and total ICP at 55% for 7.5 V (P < .001 vs controls) as well as in vitro electrical field stimulation-induced relaxant response at 10% (P < .05 vs controls) and endothelium-dependent acetylcholine-caused relaxation at 56% (P < .001 vs controls). The combined treatment showed more significantly improved erectile and relaxant responses more effectively than ADSCs or L-arginine treatment alone. Increased in the expressions of nNOS, HIF-1α, Bax, and Bcl-2 in the BCNI group were restored by all treatment regiments. eNOS and TGF- β1 protein expressions were similar among groups. Mono-treatment partially improved decreased smooth muscle mass while combined therapy completely recovered. Conclusions The intracavernous injection of ADSCs and oral administration of L-arginine, and, importantly, their combination could effectively protect against hypoxia-induced neurotoxicity, via their increasing endothelial cell function and smooth muscle content, anti-inflammatory, anti-apoptotic, anti-fibrotic properties in addition to reversing erectile function in the BCNI rat model. HIF-1α overexpression by the inhibition of the apoptotic pathway with the Bcl-2 family may have protective effects on the survival of cavernosal neurons. Disclosure No