Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury

Abstract

Background: Intracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear.

Objectives: To investigate the mechanism of action and fate of IC injected ADSC in a rat model of CN crush injury.

Design, setting, and participants: Sprague-Dawley rats (n=110) were randomly divided into five groups. Thirty-five rats underwent sham surgery and IC injection of ADSC (n=25) or vehicle (n=10). Another 75 rats underwent bilateral CN crush injury and were treated with vehicle or ADSC injected either IC or in the dorsal penile perineural space. At 1, 3, 7 (n=5), and 28 d (n=10) postsurgery, penile tissues and major pelvic ganglia (MPG) were harvested for histology. ADSC were labeled with 5-ethynyl-2-deoxyuridine (EdU) before treatment. Rats in the 28-d groups were examined for erectile function prior to tissue harvest.

Measurements: IC pressure recording on CN electrostimulation, immunohistochemistry of the penis and the MPG, and number of EdU-positive (EdU+) cells in the injection site and the MPG.

Results and limitations: IC, but not perineural, injection of ADSC resulted in significantly improved erectile function. Significantly more EdU+ ADSC appeared in the MPG of animals with CN injury and IC injection of ADSC compared with those injected perineurally and those in the sham group. One day after crush injury, stromal cell-derived factor-1 (SDF-1) was upregulated in the MPG, providing an incentive for ADSC recruitment toward the MPG. Neuroregeneration was observed in the group that underwent IC injection of ADSC, and IC ADSC treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum.

Conclusions: CN injury upregulates SDF-1 expression in the MPG and thereby attracts intracavernously injected ADSC. At the MPG, ADSC exert neuroregenerative effects on the cell bodies of injured nerves, resulting in enhanced erectile response.

Fig. 1

Diagrams depicting intracavernous (IC) and perineural injections. IC injection was applied to Sham plus adipose-derived stem cell (ADSC), Crush-control, and Crush+ADSC groups. Perineural injection was applied to the Crush plus perineurally injected ADSC (ADSC_p) group. The structures in the diagram represent the dorsal artery (red), the dorsal vein (blue), the dorsal nerve (yellow), the corpus cavernosum (green/red), and the urethra (purple).

Fig. 2. Electrostimulation of cavernous nerves after 4 wk. (a) Top: the effects of intracavernous and perineural injection of adipose-derived stem cells (ADSC) on the increase of intracavernous pressure (ICP) on electrostimulation of the cavernous nerve (CN) (each group: n = 10). Bottom: ratio of ICP to mean arterial pressure. The red bars are representative ICP recordings of a 50-s electrical stimulation of the CN

Sham+ADSC = sham operation and IC injection of ADSC; Crush control = bilateral CN crush and intracavernous (IC) injection of phosphate-buffered saline; Crush+ADSC = bilateral CN crush and IC injection of ADSC; Crush+ADSC_p = bilateral CN crush and perineural injection of ADSC.

Fig. 3. Masson’s trichrome staining in a penile midshaft specimen. Representative images of the corpus cavernosum in each cohort: (a) Sham plus adipose-derived stem cells (ADSC), (b) Crush control, (c) Crush+ADSC, (d) Crush plus perineurally injected ADSC (ADSC_p); original magnification: ×100. The graph depicts the ratio of smooth muscle-to-collagen in penile midshaft sections

Sham+ADSC = sham operation and IC injection of ADSC; Crush control = bilateral CN crush and intracavernous (IC) injection of phosphate-buffered saline; Crush+ADSC = bilateral CN crush and IC injection of ADSC; Crush+ADSC_p = bilateral CN crush and perineural injection of ADSC. ^#p = < 0.05.

Fig. 4. Neuronal nitric oxide synthase (nNOS) staining in a penile midshaft specimen. Rats (n = 5 in each group) were treated with sham operation (sham control) or bilateral cavernous-nerve (CN) crush, and then intracavernous injection (IC) of adipose-derived stem cells (ADSC) or phosphate-buffered saline (PBS). Tissues were harvested at the indicated time points after injections. Sections were stained for nNOS (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Original magnification: ×400. The graph demonstrates the effect of intracavernous ADSC injection on neuroregeneration in the dorsal nerve over time

Sham control = sham operation without injection; Crush control (C) = bilateral CN crush and IC injection of PBS; Crush+ADSC (C/A) = bilateral CN crush and IC injection of ADSC. ^#p < 0.05. ^*p < 0.001.

Fig. 5. Dynamics of 5-ethynyl-2-deoxyuridine (EdU)-positive adipose-derived stem cells (ADSC) present in penile tissues and major pelvic ganglia (MPG) over time. Rats (n = 5 in each group) were treated with sham operation or bilateral cavernous-nerve (CN) crush and then transplanted with EdU-labeled ADSC either through intracavernous (IC) or perineual injection. (a) Comparison of ADSC numbers in the penile injection site. At the indicated time points, penile tissues were processed for assessment of EdU-labeled cells per field (×100 magnification). (b) Comparison of ADSC numbers in the MPG. At the indicated time points, the MPG were processed for the determination of EdU-labeled cells per field (×100 magnification)

Sham+ADSC = sham operation and IC injection of ADSC; Crush+ADSC = bilateral CN crush and IC injection of ADSC; Crush+ADSC_p = bilateral CN crush and perineural injection of ADSC. ^#p < 0.05 compared with either of the other two groups at the given time point. ^*p < 0.001 compared with either of the other two groups at the given time point.

Fig. 6. Detection of stromal cell-derived factor-1 (SDF-1) expression in the major pelvic ganglion. Rats (n = 5 in each group) were treated with sham operation or bilateral cavernous-nerve (CN) crush, and then intracavernously injected with 5-ethynyl-2-deoxyuridine (EdU)-labeled adipose-derived stem cells (ADSC). One day later, both EdU and SDF-1 were clearly visible in the major pelvic ganglia (MPG) of the (a) Crush+ADSC group, but not of the (b) Sham+ADSC group. (c) Higher magnification of the boxed area in panel A further shows the close association between SDF-1 expression and ADSC. Sections were stained for EdU, SDF-1, and 4′,6-diamidino-2-phenylindole (DAPI). Original magnification: ×100. (d) The graph demonstrates the expression levels of SDF-1 in the MPG 1 d after CN sham or crush injury

^**p < 0.0001 versus Sham+ADSC.

Fig. 7

Presence of adipose-derived stem cells (ADSC) at the major pelvic ganglia 7 d after cavernous-nerve crush and intracavernous injection with 5-ethynyl-2-deoxyuridine (EdU)–labeled ADSC. S-100 protein was used to stain for nerve fibers, EdU staining for ADSC, and 4′,6′-diamidino-2-phenyleindole (DAPI) for nuclear staining. Original magnification: ×100.