Cryptorchidism is one of the most common surgical problems in pediatric urology. Management of high testis may vary but the most popular method in surgical treatment is the Fowler–Stephens maneuver (FSM). Division of the spermatic vessels aids in placing the high testis into the scrotum but ends by testicular atrophy in 20–50% of the cases. It is a well-known fact that ischemia causes necrotic damage in all parts of the testis. More recent studies show that low-grade ischemia may induce apoptosis. Ligation and division of testicular blood vessels produce an 80% decrease in testicular blood flow at 1 h after ligation of the vessels; however, after 30 days, testicular blood flow returns to its normal level. Thus, ligation and division of spermatic vessels induces testicular low-grade ischemic damage.

Nitric oxide (NO) is implicated in numerous pathophysiological processes including aging, apoptosis, diabetes, inflammation and oxidative stress. NO is an important intracellular and intercellular signalling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms. Multiple factors determine whether the final effect of nitric oxide signaling is beneficial or deleterious. Among these factors are environmental factors, which affect the formation of NO by NO synthase (NOS). NO is a freely diffusible, water and lipid-soluble gaseous molecule with a short half-life and is formed from l-arginine and molecular oxygen by a family of NOS enzymes. There are three major isoforms of the enzyme, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). The activity of these enzymes is rather different. For instance, nNOS and eNOS are Ca2+ dependent and are constitutively expressed in many tissues, whereas iNOS is Ca2+ independent and is induced in a variety of tissues after exposure to inflammatory cytokines or ischemic stimuli. In the testis, eNOS is expressed in Leydig cells and Sertoli cells, spermatocytes, and spermatids. Expression of iNOS requires transcriptional activation that is induced by various cytokines. Upregulation of NOS expression leads to excessive NO production for prolonged periods of time and accounts for oxyradical-mediated tissue damage. Although little is known about the mechanisms of its toxicity in germ-cell injury during testicular ischemia, NO is an important mediator of cell death either through apoptosis or necrosis, depending on the intensity and duration of injury.

NO at physiological concentrations inhibits proinflammatory platelet aggregation, integrin-mediated adhesion, and proinflammatory-induced gene expression, factors that control vascular inflammation and oxidative injury. However, NO at high concentrations can display pathogenic properties due to the production of peroxynitrite, NO radicals and other reactive oxidizing compounds causing a change in NO effects from protective to deleterious.

The aim of the present study was to investigate the effects of spermatic vessel ligation on testicular NO levels, expression of iNOS and eNOS and germ cell-specific apoptosis in both ipsilateral and contralateral testes.

The study was performed on 28 male 30-day-old Wistar albino rats. The animals were housed in a temperature- and light-controlled environment with ad libitum access to water and food pellets. All experimental procedures were approved by the Ethics Committee of the Faculty of Medicine, Celal Bayar University, Manisa, Turkey. All surgical procedures were performed under general anesthesia administered by intramuscular injection of ketamine and xylasine hydrochloride under sterile conditions. The artery and vein of vas deferens and cremasteric muscle were identified with the use of a preparation microscope and their integrity was assured prior to ligation of spermatic vessels. Spermatic vessels were ligated at approximately 1 cm distance of the testis, the site where spermatic vascular ligation is performed in the FSM. The testes were analyzed for the presence of ischemia after ligation of the vessels and the procedure was then ended.

The animals were randomly allocated into four groups containing seven rats each. Animals were sacrificed at 2 h (group 1), at 4 h (group 2), and at 24 h (group 3) after ligation. Sham-operated animals served as controls (group 4). All ipsilateral and contralateral testes were collected for examination. All testes were dissected into two halves and one halve was put in Bouin’s fixative solution for histological evaluation and the other halve was frozen in liquid nitrogen and stored at ?70 °C for biochemical evaluation.

For the estimation of tissue NO production, biochemical assessment of stable NO oxidative metabolites, nitrite (NO2?) and nitrate (NO?3), was performed. Assessment of tissue nitrite and nitrate levels was based on the Griess method. All procedures were performed at +4 °C. Testicular samples were homogenized in ten times the tissue volume of ice-cold Tris-HCl buffer (50 mMol/L, pH 7.4). After homogenization, samples were deproteinized with 75 mMol/L ZnSO4 and 55 mMol/L NaOH, and supernatants were used. One aliquot of supernatant was used for nitrite assessment by diazotization of sulfanilamide and coupling to naphthylethylene diamine. Another aliquot of supernatant was taken for the determination of total nitrite and nitrate levels which were reduced by copper-coated cadmium granules in glycine buffer at pH 9.7 and then by diazotization of sulfanilamide and coupling to naphthylethylene diamine. Absorbance of the colored reaction product was measured at 545 nm with a spectrophotometer (Shimadzu, Kyoto, Japan). Nitrate levels were taken as differences between absorbance values of two aliquots. A standard curve was obtained with solutions containing 2–10 ?mol/L sodium nitrate. Data in this study presents the sum of nitrite and nitrate levels, which are NO metabolites and expressed as ?mol/g wet tissue.

All specimens were fixed in Bouin solution for 24 h at room temperature. Afterwards, specimens were washed in 70% ethanol 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. Hematoxylin–eosin (HE) staining was used for histological diagnosis and for the determination of testicular diameters. The mean seminiferous tubular diameter was estimated by measuring two diagonal diameters of ten tubules in ten randomly selected fields (100 tubules in total) using an ocular micrometer.

For immunohistochemical staining, sections were first dried at 60 °C overnight and then incubated in xylene for 30 min. After washing in a decreasing series of ethanol, sections were washed in 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 by 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-iNOS antibody in a 1:100 dilution (Zymed, San Francisco, CA, USA) and a monoclonal anti-eNOS antibody in a 1:200 dilution (Biomol, Hamburg, Germany). Afterwards, sections were washed 3 times for 5 min each with PBS, followed by incubation with biotinylated IgG and then with streptavidin-peroxidase conjugate (Zymed). All incubation steps were separated by three washing steps. After washing for 3 times for 5 min with PBS, sections were incubated in a peroxidase substrate solution containing diaminobenzidine (Zymed) for 5 min and then stained with Mayer’s hematoxylin. Sections were covered with mounting medium and were analyzed with a BX 40 light microscope (Olympus, Tokyo, Japan). Control sections were processed in an identical manner, but the primary antibody incubation step was omitted. Two observers blinded to clinical information evaluated the staining scores independently. Staining intensity was graded as negative (?), mild (+), moderate (++) and marked (+++), respectively.