The influence of smoking exposure and cessation on penile hemodynamics and corporal tissue in a rat model

Abstract

Background: While epidemiological studies have clearly documented that smoking cessation significantly enhances sexual health, the underlying mechanism remains largely unknown. Thus, we wished to explore possible mechanisms by using a rat model of smoking-associated erectile dysfunction (ED).

Methods: Forty 8-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats were exposed only to room air (N group). The remaining 30 rats were passively exposed to cigarette smoke over a 12-week period. At the end of 12 weeks, the smoking (S, n=10) group underwent immediate erectile function testing and were sacrificed. The remaining 20 rats were exposed to room air only for 4 (Q4W, n=10) or 8 (Q8W, n=10) weeks and then underwent erectile function testing and sacrifice. Erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP). After blood collection for serum testosterone determination, rats were sacrificed to obtain corporal tissue for immunohistochemistry.

Results: Mean ICP/MAP ratio was significantly lower in the S group compared to the N and Q8W groups (0.52±0.11, 0.94±0.05, and 0.94±0.12, respectively, P=0.0189). Smooth muscle/collagen ratio was also significantly lower in the S group compared to the N and Q8W groups (11.8±0.94, 17.5±1.82, and 16.4±0.60, respectively, P=0.0008). Oxidative stress and apoptotic indices were significantly higher in the S group compared to the N and Q8W groups. Neuronal and endothelial nitric oxide synthases were significantly less expressed in the S group compared to the N and Q8W groups.

Conclusions: Smoking cessation is associated with partial recovery of penile hemodynamics in a rat model of smoking associated ED.

Keywords: Cigarette; cessation; erectile dysfunction (ED); rat; smoking.

Figure 1

Measurement of erectile function. Representative graphs are shown for rats with exposure only to room air for 12 weeks (N), smoke exposure for a full 12 weeks followed by immediate erectile function testing (S), smoke exposure for 12 weeks followed by 4 weeks of room air only exposure before erectile function testing (Q4W) and smoke exposure for 12 weeks followed by 8 weeks of room air only exposure before erectile function testing (Q8W). The red curve and blue bar represent intracavernous pressure (ICP) values and the duration of cavernous nerve stimulation (50 seconds), respectively.

Figure 2

Oxidative stress in corpora cavernosa. Representative images from rats with exposure only to room air for 12 weeks (N), smoke exposure for a full 12 weeks followed by immediate erectile function testing (S), smoke exposure for 12 weeks followed by 4 weeks of room air only exposure before erectile function testing (Q4W) and smoke exposure for 12 weeks followed by 8 weeks of room air only exposure before erectile function testing (Q8W). Free radical-induced oxidative DNA damage products are stained brown with 8-OHdG antibody. Positive staining was more intense in the S group than in the N and Q8W groups. (Scale bars: left, 500 µm; right, 100 µm).

Figure 3

Neuronal nitric oxide synthase (nNOS) in corpora cavernosa. Representative images from rats with exposure only to room air for 12 weeks (N), smoke exposure for a full 12 weeks followed by immediate erectile function testing (S), smoke exposure for 12 weeks followed by 4 weeks of room air only exposure before erectile function testing (Q4W) and smoke exposure for 12 weeks followed by 8 weeks of room air only exposure before erectile function testing (Q8W). nNOS-positive fibers are strained brown with nNOS antibody. Positive staining for nNOS was less intense in the S group than in the N and Q8W groups. (Scale bars: left, 500 µm; right, 100 µm).

Figure 4

Western blot for endothelial nitric oxide synthase (eNOS). Representative images from rats with exposure only to room air for 12 weeks (N), smoke exposure for a full 12 weeks followed by immediate erectile function testing (S), smoke exposure for 12 weeks followed by 4 weeks of room air only exposure before erectile function testing (Q4W) and smoke exposure for 12 weeks followed by 8 weeks of room air only exposure before erectile function testing (Q8W). Mean eNOS was significantly lower in the S group compared with N and Q8W groups; significant differences between the N and Q4W groups were also noted. (*, P<0.05; **, P<0.01).

Figure 5

Smooth muscle content in corpora cavernosa. Representative images from rats with exposure only to room air for 12 weeks (N), smoke exposure for a full 12 weeks followed by immediate erectile function testing (S), smoke exposure for 12 weeks followed by 4 weeks of room air only exposure before erectile function testing (Q4W) and smoke exposure for 12 weeks followed by 8 weeks of room air only exposure before erectile function testing (Q8W). Smooth muscle and connective tissue are stained with red and blue, respectively, using the trichrome method. The smooth muscle content was less in the S and Q4W groups than in the N and Q8W groups. The scale bar represents 250 µm.

Figure 6

Analysis of apoptosis-positive cells by TUNEL stain. Representative images from rats with exposure only to room air for 12 weeks (N), smoke exposure for a full 12 weeks followed by immediate erectile function testing (S), smoke exposure for 12 weeks followed by 4 weeks of room air only exposure before erectile function testing (Q4W) and smoke exposure for 12 weeks followed by 8 weeks of room air only exposure before erectile function testing (Q8W). Apoptotic cells and cell nuclei in the corpora cavernosa were visualized with TUNEL staining (green, arrows) and 4′,6-diamidino-2-phenylindole staining (blue), respectively. The S group had a greater number of apoptotic cells than the N and Q8W groups. The scale bar represents 100 µm.