Sildenafil citrate has been shown to be highly effective in the treatment of erectile dysfunction and it is used to treat impotence of various etiologies with good tolerance.

The penis is composed of three columns of erectile tissue each enclosed by its own dense fibrous connective tissue. Corpora cavernosa, two of the three columns of erectile tissue, are positioned dorsally, and the third column, the corpus spongiosum, is positioned ventrally. The corpus spongiosum surrounds the urethra. The three corpora are surrounded by loose connective tissue and smooth muscle cells. The vascular spaces of the corpora cavernosa are larger centrally and smaller peripherally. All corpora include vascular spaces that receive blood from branches of the deep dorsal and cavernosal arteries of the penis.

Figure 1. Schematic drawing of a transverse section of the rat penis showing the corpus cavernosum and corpus spongiosum surrounding the urethra. Dorsal nn, dorsal nerves; and dorsal aa, dorsal arteries.

Sildenafil citrate, a pyrazolo-pyrimidinyl-methylpiperazine compound, is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5. It enhances the relaxant effect of nitric oxide (NO) on the corpus cavernosum by inhibiting PDE5, which is responsible for degradation of cGMP in the tissue. Inhibition of PDE5 by sildenafil citrate increases cGMP levels in the corpora, causing smooth muscle relaxation and blood flow into the penis during the local release of NO due to sexual stimulation. This is the main action of sildenafil citrate on the physiologic mechanisms of erection. However, we do not know the long-term effects of the use of sildenafil citrate on the corpora of the penis. In addition, histopathological and ultrastructural effects of the use of sildenafil citrate on corpus cavernosum components are not known. Therefore, we decided to investigate the histopathological and ultrastructural effects of sildenafil citrate in the rat corpus cavernosum using light and electron microscopical techniques.

Twenty male 10–12-week-old Wistar rats with an average weight of 250 g were used. Rats were fed standard diet and were divided into two groups of 10 rats each. The first group was used as control and the second group was treated with 2 mg/kg body weight/day sildenafil citrate orally via gavage on alternate days (3 days in a week) for 4 weeks. All male rats were coupled with female rats overnight for sexual activity only on the days when sildenafil was given. The impact of sildenafil citrate on sexual function was evaluated.

The Ethics Committee at Celal Bayar University, School of Medicine, approved our study protocol.

After 4 weeks, all rats were anesthetized with methoxyflurane and sacrificed by cervical dislocation. Penile tissue from the middle part of the penis was collected from each rat. Tissue was fixed using 10% formalin during 24 h for light microscopy, and 2.5% glutaraldehyde during 4 h for electron microscopy.

All specimens were fixed in a solution of 10% formalin during 24 h. Specimens were washed and soaked in a graded series of ethanol. Then they were embedded in paraffin. Sections (5 ?m thick) were cut and prepared for both histochemical and immunohistochemical staining. Periodic acid Schiff (PAS) staining was used for histological diagnosis.

Specimens were processed for light microscopy and sections were incubated at 60°C overnight and then dewaxed in xylene for 30 min. After soaking in a decreasing series of ethanol, sections were washed with distilled water and phosphate-buffered saline (PBS) for 10 min. Sections were then treated with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37°C for 15 min and washed with PBS. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H₂O₂ for 15 min to inhibit endogenous peroxidase activity. Then, sections were washed with PBS and incubated for 18 h at 4°C with primary antibody, a polyclonal anti-inducible NOS (iNOS) in a 1:100 dilution (Zymed, San Francisco CA, USA). Afterwards, sections were washed 3 times for 5 min each with PBS, followed by incubation with biotinylated IgG (Dako) and then with streptavidin-peroxidase conjugate (Dako). All incubation steps were separated by 3 washing steps. After washing 3 times for 5 min with PBS, sections were incubated with a solution containing 3-amino-9-ethylcarbazole (Dako) for 5 min to visualize immunolabelling and then with Mayer’s hematoxylin. Sections were covered with mounting medium and were analyzed light microscopically with a BX 40 microscope (Olympus, Tokyo, Japan). Control samples were processed in an identical manner, but the primary antibody was omitted. Two observers blinded to clinical information evaluated the staining scores independently.

1 mm³ of specimens were fixed with 2.5% glutaraldehyde for 4 h. Then, postfixation was performed using osmium tetroxide for 1.5 h. Specimens were processed by standard dehydration in a graded series of ethanol before infiltration and embedding in Epon 812 (Fluka, Buchs, Switzerland). Ultrathin sections were cut on an ultracut R ultramicrotom (Leica, Nussloch, Germany) and stained with uranyl acetate-lead citrate before viewing with a 9S electron microscope (Zeiss, Oberkochen, Germany).

Measurements of the surface area of blood vessels were performed to assess vascular density in all samples. Three micrographs from each sample were randomly selected and for the estimation of surface areas of blood vessels, the unbiased stereology method was used. A cycloid test system, with its minor axis parallel to the vertical direction, was randomly translated in x and y directions on the micrograph. The test system has a known length of cycloid per point (l/p). In order to estimate surface density, we performed two measurements on each image: first, the number of intersections between the cycloid lines and the boundary of interest (I); and second, the number of points that were found within the reference space (P). Surface areas of blood vessels were then estimated with the formula: surface area of blood vessel 2?I/((l/p)?P).

The thickness of the basal lamina was also measured using micrographs. All measurements were performed by three investigators who were blinded to the micrographs. Each investigator reported his/her results separately. The mean of the results of the three investigators was used in the statistical analysis. All data were expressed as the mean±standard error of the mean. Data comparisons were performed using an ANOVA test, with P<0.05 for the level of significance.

The histological analysis revealed that the corpus cavernosum, which is the main anatomic structure used during erection and consists of numerous vascular spaces with wide and irregular shaped lumina that are lined by endothelial cells, was elongated and the number of vascular spaces was increased in sildenafil-treated rats. The amount of connective tissue in the corpus cavernosum was clearly increased and dense collagen and smooth muscle fibers were observed in treated rats. The lamina basalis of blood vessels of treated rats in the corpus cavernosum appeared to be thicker than in control rats after staining with PAS.

Figure 2. Light micrographs of penile tissue of control rats (A,C) and rats treated with sildenafil (B,D) after PAS staining. The dorsal vein (V) and corpus cavernosum (CC) show increased density of collagen bundles (asterisk) in the stroma of the corpus cavernosum and a thicker basement membrane (arrows) around arteries and vessels is present in treated rats. Magnifications: (A,B) ×100; and (C,D) ×200.