Basic and Translational Science Rho-kinase Levels in Testicular Ischemia- reperfusion Injury and Effects of Its Inhibitor, Y-27632, on Oxidative Stress, Spermatogenesis, and Apoptosis Selahittin C ¸ ayan, Barıs ¸ Saylam, Nalan Tiftik, Nil Do  gruer € Unal, Duygu D€ us ¸mez Apa, Ozan Efesoy, Burak C ¸ imen, Murat Bozlu, Erdem Akbay, and Kansu B€ uy€ ukafs ¸ar OBJECTIVE To investigate testicular Rho-kinase levels and the effects of its inhibitor, Y-27632, on oxidative stress, spermatogenesis, and apoptosis in testicular ischemia-reperfusion rat model. METHODS The study included 29 adult Wistar-Albino male rats weighing 150-200 g. The rats were divided into 3 groups. Group 1 underwent sham operation (n ¼ 10). In group 2, left testicular torsion- detorsion was performed (n ¼ 9). In group 3, Rho-kinase inhibitor Y-27632 (5 mg/kg) was injected intraperitoneally 30 minutes before detorsion (n ¼ 10). Two months later, bilateral orchiectomy was performed in all the groups. Rho-kinase levels by Western blotting, apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling method, testicular damage and spermatogenesis with modified Johnsen score, testicular total antioxidative status, and total oxidative status were measured. RESULTS In the torsion-detorsion (T/D) group, Rho-kinase level increased significantly, compared with the sham group (P ¼ .025). In the Y-27632 treatment group, Johnsen scores were significantly higher, and apoptosis indexes were significantly lower, compared with the T/D group (P ¼ .001). Signifi- cantly higher total antioxidative status levels and lower total oxidative status levels were observed in the Y-27632 treatment group, compared with the T/D group (P ¼ .001 and P ¼ .002, respectively). CONCLUSION Testicular ischemia-reperfusion significantly increased Rho-kinase levels in rats, and adminis- tration of Rho-kinase inhibitor, Y-27632, before detorsion might prevent ischemia-reperfusion injury. UROLOGY 83: 675.e13e675.e18, 2014. Ó 2014 Elsevier Inc. T esticular torsion is a common urologic emergency among newborns, children, and adolescents. The salvage rate is directly proportional to the duration of torsion, and early diagnosis followed by detorsion is the current management for the preservation of spermatogenesis and fertility. 1 Although reperfusion is essential for the survival of ischemic tissue, there is good evidence that reperfusion itself causes the pathophysio- logical cascades, including an activation of neutrophils, inflammatory cytokines, and adhesion molecules with a massive intracellular Ca 2þ release, the generation of oxygen-derived free radicals, and increased thromboge- nicity. 2 Reactive oxygen species (ROS) could cause deoxyribonucleic acid (DNA) damage, endothelial injury, and germinal cell necrosis. These free radicals react with lipids in the cell and mitochondrial membranes, changing their permeability and disrupting cell integrity. 3 Under normal conditions, free radicals are produced and their effects are counterbalanced by the endogenous antioxi- dant system. When ROS generation exceeds the defense mechanisms capacity to control, oxidative stress is gener- ated and contributes to reversible or irreversible cell injury. In particular, sperm are highly sensitive to oxidative stress and lipid peroxidation because of their high content of polyunsaturated fatty acids in the plasma membrane. The fatty acids are an essential requirement for the male germ cell to maintain sperm functions. 4 Ischemia-reperfusion injury in tissues induced extra- vascular recruitment of leukocytes is a multistep process comprising leukocyte rolling, adhesion, and transmigration. 5 Although numerous cellular pathways contribute to the inflammation in reperfusion injury, underlying mechanisms have yet to be elucidated, and the number of pathways involved has been rising continuously. Accordingly, it has been reported that Rho-kinase (ROCK) is expressed Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, University of Mersin School of Medicine, Mersin, Turkey; the Department of Pharmacology, University of Mersin School of Medicine, Mersin, Turkey; the Department of Biochemistry, University of Mersin School of Medicine, Mersin, Turkey; and the Department of Pathology, University of Mersin School of Medicine, Mersin, Turkey Reprint requests: Selahittin C ¸ ayan, M.D., Department of Urology, University of Mersin School of Medicine, 33079 Mersin, Turkey. E-mail: selcayan@mersin.edu.tr Submitted: July 5, 2013, accepted (with revisions): November 20, 2013 ª 2014 Elsevier Inc. 0090-4295/14/$36.00 675.e13 All Rights Reserved http://dx.doi.org/10.1016/j.urology.2013.11.032