Ó Springer 2006

International Urology and Nephrology (2006) 38:129–132 DOI 10.1007/s11255-005-3150-4

Effects of ethanol on intracorporeal structures of the rat C¸etin Yesilli1, Go¨rkem Mungan2, Ilker Sec¸kiner1, Bu¨lent Akduman1, Gamze Numanog˘lu3 & Aydin Mungan1 1

Department of Urology, Karaelmas University, School of Medicine, Kozlu/Zonguldak, Turkey; 2 Department of Biochemistry, Karaelmas University, School of Medicine, Kozlu/Zonguldak, Turkey; 3Department of Pathology, Karaelmas University, School of Medicine, Kozlu/Zonguldak, Turkey

Abstract. Objective: Previous studies demonstrated that acute in vitro exposure of corpus cavernosal tissue to ethanol decreased its response to field stimulation and pharmacological stimulation. In the present study we investigated the effects of chronic ethanol consumption on the ultrastructure of cavernosal smooth muscle cells, elastic fibres and collagen content. Material and methods: Fourteen adult wistar rats were divided into a control group (n=7, fed a standard diet and tap water) and an alcoholic group (n=7, fed a standard diet and 5% (v/v) ethanol in drinking water and by increasing the ethanol concentration for every week, at the end of 6th week 30% (v/v) ethanol concentration was attained. Same dose was given until 12th week. At the end of 12th week blood samples were obtained and the ethanol concentrations were determined. The cavernosal tissues were obtained and immunohistochemical examinations were performed. Results: Immunohistochemical analysis revealed that chronic ethanol exposure markedly decreased the content of smooth muscle cells, elastic fibres and collagen type 4. Conclusion: Our findings suggest that in this animal model chronic ethanol exposure decreases the percentage of staining for smooth muscle actin, elastin, and collagen type 4 which are the key structures fundamental for erection. Key words: Cavernosal structure, Collagen type 4, Elastin, Ethanol, Impotence, Smooth muscle actin

Introduction Penile erection results from increased arterial flow, sinusoidal smooth muscle relaxation and decreased venous return [1]. Failure of any of these vascular mechanisms may lead to erectile dysfunction. Previous studies have shown that intracavernosal smooth muscle cells (SMC) form the structural basis for sinusoidal relaxation, which is locally neurally controlled by the active corporeal lacuna endothelium [2–4]. In addition, elastic fibres (EF) and collagen are important elements for rigid erection [5–8]. Ethanol has various effects on male sexual activity. Ethanol-induced male sexual dysfunction is caused by ethanol’s effect on the central nervous system mainly through acute ethanol

ingestion and chronic alcoholism, in decreased testosterone and increased estrogen levels, and alcoholic polyneuropathy [9]. Chronic alcoholism is a well-known risk factor for impotence [10]. Generally, low levels of alcohol ingestion increase male sexual activity by increasing libido; however, higher alcohol levels impair the penile tumescence and reduce sexual performance [11]. It has been shown that ethanol had significant effects on both contraction and relaxation of rabbit corpus cavernosum [12–14]. However, in these in vitro studies only cavernosal contractility has been determined. In this present in vivo study we attempted to determine the effect of chronic ethanol exposure on the ultrastructure of cavernosal SMC, EF and collagen type 4 content.

130 Materials and methods Animals Healthy adult male Wistar rats weighing 200–250 g body weight were used. Rats were kept in clean cages in a temperature-controlled room with 12-h light/ dark schedule. All animals were killed by decapitation under ketamine hydrochloride (50 mg/kg).

membrane. Weigert for elastic fibers (long method) was used histochemically to visualize the elastic fibers. Smooth muscle actin (SMA), collagen type 4 and Weigert for elastic fibers were evaluated in a 40 magnification high power field. Myometrial tissue served as the positive control for antismooth muscle actin and skin sections were used as the positive control for collagen type 4. For each case, the positive staining was detected in all areas of the corpus cavernosa section.

Ethanol exposure Statistics Animals were divided into the following groups; Group 1 included control (n=7, rats) that received a standard diet and 5% (v/v) ethanol in drinking water for 1 week and ethanol concentration was increased 5% for every week and at the end of 6th week 30% (v/v) ethanol concentration was attained. Same dose was given until 12th week. At the end of 12th week blood samples were obtained and the ethanol concentrations were determined. Group 2 (n=7, rats) were fed with a standard diet and tap water. All animals were weighed before they were killed, and during chronic experiment animal weights and ethanol consumption were observed regularly throughout the ethanol-exposure period. Tissue Tissues from rats in groups 1 and 2 were obtained at the end of 12 weeks. The penis was excised en bloc with the aid of ketamine hydrocholoride 50 mg/kg subcutaneously. The tissues were placed into 10% formalin solution and sent to the immunohistochemical laboratory.

Values are presented as mean±standard deviation (SD). The significance of differences between groups was analyzed with Mann–Whitney U test at 95% confidence level. Statistical significance was considered when p<0.05.

Results At the end of 12 weeks, mean blood ethanol concentration was 91.4±8.6 mg/dl in alcoholic group and 5.95±3.81 mg/dl in control group. There were no significant differences in animal weights between two groups. The results of the immunohistochemical analysis of SMC, EF and collagen type 4 contents of the cavernosal tissues are presented in Table 1. We observed statistically significant differences between two groups for cavernosal content of SMC (p<0.002), EF (p<0.002) and collagen type 4 (p<0.002).

Discussion Immunohistochemical and histochemical examination After staying in 10% formalin solution for 24 h, three samples were obtained for every material and blocked with paraffin for haematoxylin-eosin staining. Sections with a thickness of 4 lm were cut from paraffin blocks and placed on glass slides coated with poly-L-lysine (Lab vision corporationneomarkers). Monoclonal anti-smooth muscle actin (Lab vision corporation-neomarkers) was performed immunohistochemically to visualize the smooth muscle cells, and collagen type 4 Ab (Lab vision corporation-neomarkers 1/50–1/100) was performed immunohistochemically to detect basal

It is generally accepted that the elevation of intracorporeal pressure leads to erection and this is achieved by the relaxing contractile action of the smooth muscle cells, so their degeneration may be important factor in erectile dysfunction. In addition, elastic fibres (EF) and collagen fibres are important elements for rigid erection [5–8]. The importance of corporeal smooth muscle and its functional significance has been appreciated in animal experiments and human clinical studies. It was suggested that these smooth muscle cells contribute to the firmness of the erect penis by increasing the intracavernous pressure in a way that could not be achieved by a vascular

131 Table 1. Values of cavernosal smooth muscle actin (SMA), elastic fibers (EF) and collagen type 4 among the groups Mean±SD

p value

Control group Alcoholic group SMA 53.2±3.40 EF 6.85±2.85 Collagen type 4 3.0±1.9

25.6±1.50 2.71±2.50 1.42±1.80

0.002 0.002 0.002

mechanism alone [6, 15]. In several studies it has been reported that cavernous tissue in impotent men showed a marked decrease in smooth muscle and elastic fiber content compared to that in normal potent men [2, 5, 16]. In chronic alcoholism, male sexual dysfunction may be caused from decreased testosterone and increased estrogen levels, and alcoholic polyneuropathy [9]. It is known that acute ethanol intoxication can cause urinary retention in benign prostatic hyperplasia [17]. In animal studies, the mechanism of urinary retention induced by ethanol partly showed that ethanol significantly impaired detrusor contractility in vivo and in vitro [18–20]. Several in vitro studies have revealed that ethanol had significant effect on both contractility and relaxation of rabbit corpus cavernosal smooth muscle (CCSM) [12–14]. It has been suggested that increasing the acetaldehyde, which is the principal metabolic by-product of ethanol metabolism may affect the function of CCSM through the mechanisms of inhibition of extracellular calcium ion influx and nitric oxide (NO) formation [14]. However, the mechanism of ethanol’s effect has not been understood clearly. The aim of our study was to determine the content of the fundamental structures of the corpus cavernosum by immunohistochemical analysis in ethanol-exposed rats. We observed a statistically significant difference in the content of SMC in control versus the alcoholic group (P=0.002) In addition we also observed alterations in the collagen content and EF. The collagen content and EF play an important role in achieving firmness, compliance and elasticity of the corpora cavernosa during erection. Elastic and easily expandable erectile tissue provides adequate compressive and stretching forces to drain the subtunical venules and cause high venous outflow resistance [18]. Changes in those structures may cause decreased relaxation of erectile tissue and affect the normal

filling of the vascular spaces [19, 20]. In our study we observed a significant decrease in the content of EF (p=0.002). Most microscopic studies to date agree on the abundance of collagen within the erectile tissue of potent men. This extracellular matrix no doubt plays an important role in the extreme structural changes of the penis during erection and detumescence [6–8]. The abundance of type 4 collagen may be explained by the prevalence of endothelial cells, which envelop the trabeculae of the cavernous spaces. These cells are responsible for the secretion of type 4 collagen that forms the basement membrane of blood vessels [21, 22]. The prevalence of endothelial cells and its secretory product, type 4 collagen, attest to the notion that the penis is a specialized organ of vascular origin [23, 24]. It has been found that in organic and psychogenic impotence there is a diminution in collagen 4 content [25]. In our study we also observed that in the alcoholic group the collagen 4 content decreased (P=0.002). Thus, according to our results we can speculate that ethanol might affect the number of those cells (SMC, EF and collagen 4) and lead to erectile dysfunction due to the above-mentioned reasons. In conclusion, our findings suggest that chronic ethanol exposure in this animal model affects the percentage of staining for smooth muscle actin, elastic fiber and collagen type 4 which are the key structures fundamental for penile erection. Chronic alcoholism may lead to erectile dysfunction. Acknowledgement We gratefully thank Assistant Prof. Dr. Ferruh Ayoglu from the Department of Public Health for statistical analysis.

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