Chronic Cigarette Smoking Impairs Erectile Function through Increased Oxidative Stress and Apoptosis, Decreased nNOS, Endothelial and Smooth Muscle Contents in a Rat Model

Abstract

Cigarette use is an independent risk factor for the development of erectile dysfunction (ED). While the association between chronic smoking and ED is well established, the fundamental mechanism(s) of cigarette-related ED are incompletely understood, partly due to no reliable animal model of smoking-induced ED. The present study was designed to validate an in vivo rat model of chronic cigarette-induced ED. Forty 12-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats served as control group and were exposed only to room air. The remaining 30 rats were passively exposed to cigarette smoke (CS) for 4 weeks (n = 10), 12 weeks (n = 10), and 24 weeks (n = 10). At the 24-week time point all rats were assessed with intracavernous pressure (ICP) during cavernous nerve electrostimulation. Blood and urine were collected to measure serum testosterone and oxidative stress, respectively. Corporal tissue was assessed by Western blot for neuronal nitric oxide synthase (nNOS). Penile tissues were subjected to immunohistochemistry for endothelial, smooth muscle, and apoptotic content. Mean arterial pressure (MAP) was significantly higher in 24-week cigarette exposed animals compared to the control animals. Mean ICP/MAP ratio and cavernosal smooth muscle/endothelial contents were significantly lower in the 12- and 24-week rats compared to control animals. Oxidative stress was significantly higher in the 24-week cigarette exposed group compared to control animals. Mean nNOS expression was significantly lower, and apoptotic index significantly higher, in CS-exposed animals compared to control animals. These findings indicate that the rat model exposure to CS increases apoptosis and oxidative stress and decreases nNOS, endothelial and smooth muscle contents, and ICP in a dose dependent fashion. The rat model is a useful tool for further study of the molecular and cellular mechanisms of CS-related ED.

Fig 1. Smoke exposure equipment.

The rat was housed in an enclosed plastic cage (28x28x19 cm) and subjected to a constant influx of cigarette smoke using a small air pump. One lighted cigarette was placed into an inverted tube connected to a peristaltic pump communicating with the cage.

Fig 2. Western blot for neuronal nitric oxide synthase (nNOS).

(A) Western blot normalized to β-actin; (B) mean nNOS positivity. Mean nNOS was significantly higher in the control group compared with all others (* versus all other groups p < 0.05).

Fig 3. Endothelium content in the corpus cavernosum.

Representative images from rats in the control, 4-week, 12-week and 24-week groups. Endothelial cells are stained brown with the rat endothelial cell antigen-1 (RECA-1) antibody. The intense positivity of RECA-1 was higher in control than in 12-week and 24-week groups. Magnification is x200.

Fig 4. Smooth muscle content in the corpus cavernosum.

Representative images from rats in the control, 4-week, 12-week and 24-week groups were shown. Smooth muscle and connective tissue are stained with red and blue, respectively, using the trichrome method. The smooth muscle content was higher in control group than in 12-week and 24-week groups. Magnification is x100.

Fig 5. Analysis of apoptosis-positive cells by TUNEL stain.

The corpus cavernosum was stained with TUNEL stain (green) and DAPI (blue) for the visualization of apoptotic cells and cell nuclei, respectively. The result showed the increase of the number of apoptosis-positive cells from control to 4-week, to 12-week and to 24-week. Magnification is x800.