Combined effects of brain-derived neurotrophic factor immobilized poly-lactic-co-glycolic acid membrane with human adipose-derived stem cells and basic fibroblast growth factor hydrogel on recovery of erectile dysfunction

Abstract

Erectile dysfunction (ED) is the most frequent long-term problem after radical prostatectomy. We aimed to evaluate whether the use of combination therapy with basic fibroblast growth factor (bFGF)-hydrogel on corpus cavernosum and with adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF)-immobilized poly-lactic-co-glycolic acid (PLGA) membrane on the cavernous nerve (CN) could improve erectile function in a rat model of bilateral cavernous nerve crush injury (BCNI). Rats were randomly divided into five groups (n=15 per group): a normal group (N group), a group receiving saline application after bilateral cavernous nerve crush injury (BCNI), a group undergoing bFGF-hydrogel injection in the corpus cavernosum after BCNI (bFGF), a group receiving ADSC application covered with BDNF-membrane after BCNI (ADSC/BDNF), and a group undergoing coadministration of bFGF-hydrogel injection and BDNF-membrane with ADSCs after BDNF (bFGF+ADSC/BDNF). Four weeks postoperatively, the erectile function was assessed by detecting the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP). Smooth muscle and collagen contents were measured using Masson's trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. The protein expression of the α-smooth muscle actin (α-SMA) and the cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by western blot and cGMP assay, respectively. In the bFGF+ADSC/BDNF group, the erectile function was significantly elevated compared with the BCNI and other treated groups and showed a significantly increased smooth muscle/collagen ratio, nNOS content, α-SMA expression, and cGMP level. In particular, there were no statistical differences in the ICP/MAP ratio, smooth muscle/collagen ratio, and α-SMA and cGMP levels between the bFGF+ADSC/BDNF group and normal group. Application of the BDNF-immobilized PLGA membrane with human ADSC into the CN and bFGF-incorporated hydrogel into the corpus carvernosum improved nearly normal erectile function in a rat model of postprostatectomy ED. This result suggests that a combined application of bFGF+ADSC/BDNF might be a promising treatment for postprostatectomy ED.

FIG. 1.

Fluorescence micrographs of the CN following bFGF+ADSC/BDNF application. Magnification is ×400. PKH26-labeled ADSCs (1×10⁶ cells) (red) are shown around the CN. Merged ADSCs (yellow, arrow) are shown in colocalization of PKH26-labeled ADSC and CN by double immunostaining with beta-III tubulin (green). DAPI was used to stain the nuclei (blue). ADSCs, adipose-derived stem cells; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CN, cavernous nerve. Color images available online at www.liebertpub.com/tea

FIG. 2.

ICP/MAP ratio change at 4 weeks following surgery. (A) Representative recordings of ICP for each group in response to CN stimulation at 10 V. The solid bars are representative ICP recordings with a stimulus interval of 50 s. (B) The ratios of ICP to MAP during electrical stimulation were calculated for each group. Each bar shows the mean ratio (±SEM) for n=15 rats per group. (^#p<0.05 compared with the BCNI group; *p<0.01 compared with the BCNI group; ^$p<0.05; N.S., not significant). BCNI, bilateral cavernous nerve crush injury; ICP, intracavernous pressure; MAP, mean arterial pressure. Color images available online at www.liebertpub.com/tea

FIG. 3.

Histological change in smooth muscle/collagen ratios of the corpus cavernosum at 4 weeks following surgery. (A) Representative micrographs of Masson's trichrome-stained rat penile sections. Smooth muscle was stained red, and collagen fibers were stained purple-blue. Magnification is ×200. (B) Relative quantification of smooth muscle/collagen ratios of penile tissue in each group. Smooth muscle atrophy was decreased in the AB/bFGF group relative to the other treatment groups. Quantitative analysis of smooth muscle and collagen content was performed with an image analyzer. Each bar shows the mean values (±SEM) from n=15 rats per group. (*p<0.05 compared with the BCNI group; ^#p<0.05; N.S., not significant). Color images available online at www.liebertpub.com/tea

FIG. 4.

Double immunohistochemistry using nNOS (red) and beta-III tubulin (green) for tissue sections obtained from around the dorsal penile nerve in each group. Magnification is ×400. nNOS, neuronal nitric oxide synthase. Color images available online at www.liebertpub.com/tea

FIG. 5.

Protein expression of α-smooth muscle actin (α-SMA) in the corpus cavernosum. Expression of endogenous α-SMA protein in penile tissue was quantified by western blot. β-actin was used as an internal control to assure equal loading. The bar graph represents the relative expression of α-SMA to β-actin expression. (mean±SEM; *p<0.05 and ^#p<0.01 compared with the BCNI group; ^$p<0.01; N.S., not significant). Color images available online at www.liebertpub.com/tea

FIG. 6.

cGMP levels in the penile tissue. The cavernous cGMP level was determined by using a cGMP direct immunoassay kit (n=15 per group). The bar graph represents the mean±SEM. (*p<0.05 compared with the BCNI group; ^#p<0.01). cGMP, cyclic guanosine monophosphate. Color images available online at www.liebertpub.com/tea