Experimental Testicular Torsion and Antioxidant Therapy

Under normal condition, free radicals are produced and their effects are counterbalanced by the endogenous antioxidant system. When ROS generation exceeds the defense mechanisms' capacity to control, oxidative stress is occurred and contributes to reversible or irreversible cell damage [7, 140] . All living aerobic cells are normally exposed to some ROS, but if ROS levels rise, oxidative stress may occur, which results in oxygen and oxygen-derived oxidants, and in turn increases the rates of cellular damage. Oxidative stress has been shown to be a major cause of male infertility; a large proportion of infertile men have elevated levels of seminal ROS [141]. ROS can directly damage spermatozoa by inducing peroxidation of the lipid-containing sperm plasma membrane, which decreases its integrity, and may also affect sperm motility by damaging the axonemal structure [142-144].

Like all cells living under aerobic conditions, spermatozoa produce ROS, mostly originating from normal metabolic activity. High concentrations of ROS cause sperm pathology such as adenosine triphosphate (ATP) depletion leading to insufficient axonemal phosphorylation, lipid peroxidation, and loss of motility and viability. However, spermatozoa and seminal plasma contain a battery of ROS scavengers, including enzymes, such as SOD, catalase, and GSH peroxidase/ reductase system, and also a variety of substances with SOD- or catalase-like activities, such as ascorbic acid, GSH, pyruvate, taurine, hypotaurine, albumin, and carnitine. The fine balance between ROS production and scavenging as well as the right timing for ROS production is of paramount importance for the acquisition of fertilizing ability by spermatozoa. Excessive ROS generation that overcomes the ROS scavenging ability of human spermatozoa appears to be related to male infertility, such as testicular torsion [141, 144-146].

There are two available antioxidant strategies. In the first strategy, the superoxide radical and hydrogen peroxide are removed using specific enzymes such as SOD, catalase, and GSH peroxidase (Px), either by administration of these enzymes or by increasing their in vivo activities. In the second strategy, radical generation is prevented. However, these systems are not always fully operative [118]. Normally, a balance exists between concentrations of ROS and antioxidant scavenging systems. One of the rational strategies to counteract the oxidative stress is to increase the scavenging capacity of seminal plasma with antioxidants.

Experimental spermatic cord torsion has been widely studied under several different aspects, including the effect of ischemia on testicular structure, I/R, contralateral testis injury, therapy with pharmacologic agents, and measures to avoid or reduce these injuries. There are conflicting results because of several factors such as animal type and species, age, model of ischemic injury, ischemia and/or reperfusion time, and technique for the evaluation of testicular injury [26]. Several studies have claimed that the contralateral testis is not affected by unilateral torsion [147-150], while others favored the opposite view [12, 151-153]. However, animal studies have demonstrated that significant testicular damage occurs in both testes, and contralateral testicular damage may be avoided by early removal of the torsed testis and pretreatment with antioxidant therapy. Experimental models of testicular torsion are valuable tools to evaluate the relationship between the degree and duration of torsion with the blood flow and resultant damage to the torsed and/or untorsed testis. The results, however, have been conflicting. With 720° of torsion, several studies have reported reduction in testicular blood flow varying from 61.7 to 100%, with ischemic injury after periods of time as diverse as 1 and 8 h [26] . Numerous studies have evaluated the efficacy of antioxidants in male infertility [146, 154, 155]. Furthermore, pretreatment with antioxidants and ROS scavengers has been shown to prevent reperfusion injury in testes.

Korean red ginseng is a potent antioxidant and free radical scavenger. Kim et al. [156] investigated the effects of it on testicular damage in a rat testicular I/R injury model. Researchers have evaluated superoxide generation (by using a lucigenin-enhanced chemiluminescence assay) and the blood levels of ROS (by using the free oxygen radical test-FORT). The FORT provides an indirect measure of hydroperoxides, which are a useful measure of oxidative stress because they indicate the presence of intermediate oxidative products of lipids, amino acids, and peptides. In this study, Korean red ginseng attenuated the increase in the testis FORT level and recovered the testis dysfunction caused by ischemia and subsequent reperfusion in the rat testis by suppressing superoxide production.

Coenzyme Q10 is an essential component for electron transport in oxidative phosphorylation of mitochondria. It is a potent antioxidant, a membrane stabilizer, and cofactor in the production of ATP by oxidative phosphorylation. It can reverse endothelial dysfunction by preventing oxidative and nitrative stress, scavenging free radicals, and inhibiting lipid peroxidation and inflammation. Erol et al. [157] demonstrated that coenzyme Q10 administration before reperfusion period of testicular torsion provided a significant reduction in the level of testicular MDA and expression of inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), and germ cell-specific apoptosis.

Aktoz et al. [ 158] showed a significant reduction in the activity of TUNEL, eNOS, and a rise in the expression of testosterone in testes tissue of I/R treated with quercetin therapy. The TUNEL method detects fragmentation of DNA in the nucleus during apoptotic cell death in situ. Quercetin prevented oxidant injury and cell death by scavenging oxygen radicals, protecting against lipid peroxidation, and chelating metal ions.

Montelukast is a new anti-inflammatory drug with antioxidant properties that interferes directly with leukotriene production (5-lipoxygenase inhibitors) and/or reception (leukotriene receptor antagonists). Ozturk et al. [159] investigated whether montelukast (MK-0476) could protect the testis from injury associated with testicular torsion and detorsion. To accomplish this, they measured the MDA, GSH, and MPO levels and performed for histological examination. Montelukast significantly reduced the I/R-induced elevation in testes tissue. MDA and MPO levels and testicular GSH levels were significantly higher when compared with the I/R/untreated rats.

In an experimental study by Shirazi et al. [160], the comparison of the protective effects of papaverine, lidocain, and verapamil on sperm quality of the testis after I/R-induced damage was reported. In conclusion, verapamil and lidocaine, as anti-oxidants, but not papaverine, as vasodilator, had beneficial effects on semen analysis parameters after testicular torsion. Gao et al. [103] investigated whether verapamil and hypothermia protect spermatogenesis of torsioned testes; they showed that either verapamil or local hypothermia can enhance testicular resistance against I/R injury and the combination of two can more efficiently prevent the germ cells from apoptosis.

Yang et al. [161] investigated antioxidant and lipid-lowering agent simvastatin on testicular I/R damage in rats. Simvastatin protected testes from torsion/detorsion injury in a dose-dependent manner. They speculated that this effect may involve attenuating nuclear factor kappa B (NF-kB) activation and decreasing oxidative stress induced by torsion/detorsion.

Apoptosis (programmed cell death) is characterized by a variety of changes resulting in the recognition and phagocytosis of apoptotic cells. Caspases (cysteinyl aspar-tate-specific proteinases) play a central role in the regulation of apoptosis in the human seminiferous epithelium, sperm differentiation, and testicular maturity. However, caspases have been implicated in the pathogenesis of multiple andrological pathologies such as impaired spermatogenesis, decreased sperm motility, increased levels of sperm DNA fragmentation, and apoptosis in testicular torsion [162]. In recent years, poly (adenosine triphosphate-ribose) polymerase (PARP) has been implicated in the process of apoptotic cell death and acts as a "death substrate" for caspases. Several PARP inhibitors such as nicotinamide, 3-aminobenzamide, 1,5-dihydroxyisoquinoline, or 4-amino-1,8-naphthalimide have been examined in testicular torsion/detorsion model [163-165]. Kar et al. [166] wanted to show biochemical, histopathological, and apoptotic changes caused by unilateral spermatic cord torsion in ipsilateral and contralateral testis and the effect of the PARP inhibitor, nicotinamide, on these changes in early and late periods. I/R injury-related changes were assessed by levels of MDA and total and free GSH in the serum. Rats were divided into two major groups as early and late periods. Bilateral orchidectomy was performed by the end of the fourth hour in early and 2 months after I/R in late groups. Nicotinamide decreased tubular damage and apoptosis in early and late periods in both testicles.

Lysiak et al. [19] reported that I/R of the testis results in germ cell-specific apop-tosis, followed by a reduction in testis weight and daily sperm production. They said that this has been associated with an increase in the adhesion of neutrophils to tes-ticular subtunical venules and an increase in ROS. In the study, they demonstrated a partial rescue with the catalase, Cu-Zn SOD, catalase plus SOD, and M40403 (a nonpeptide mimic of SOD) by infusing right femoral vein on 30 days after I/R of the testis, and the administration of ROS scavengers significantly reduced the I/R injury. Moon et al. [167] investigated the expression of tyrosine kinase receptors A and B, and p75 growth factor receptor in I/R rat testis. They reported that tyrosine kinase receptors A and B, but not p75 growth factor receptor, are involved differently in the survival of testicular cells during acute I/R injury. Tyrosine kinase receptor A increases especially related to germ cell survival.

Recently published studies demonstrated that MAPK family is of vital importance for signal transduction pathways and belong to the extracellular signal-regulated kinase family and are serine/threonine protein kinases activated by a variety of cell surface receptors. Indeed, the MAPK, MAPK3/MAPK1 (also named extracellular signal-regulated kinase [ERK] 1/2), MAPK8 (also named c-Jun-NH2-terminal kinase [JNK]), and MAPK14 (also named p-38), has a role in the pathogenesis of testicular I/R injury [126, 128, 168-170]. Active MAPKs are responsible for the phosphoryla-tion of a variety of effector proteins, including several transcription factors, such as NF-kB, activating protein (AP) 1, and the production of proinflammatory cytokines, including TNF-a. These cytokines have multifunctional effect such as proinflamma-tory response, immunoregulatory response, apoptosis, and certain testicular pathologies, especially testicular torsion [169]. NF-kB, anuclear transcription factor, controls a number of cellular processes including the immune response, inflammation, proliferation, apoptosis, and calcium homeostasis and also plays a role in the testicular I/R damage. Minutoli et al. [168] studied the involvement of MAPKs (ERK 1/2, JNK and p38) activation in NF-kB knockout mice in testicular I/R model. In addition, it has been demonstrated that PD98059 is a flavonoid and inhibits MAPK3/MAPK1, blunted MAPK3/MAPK1 and MAPK8, and decreased TNF expression and improved the testicular damage caused by I/R injury in both testes [169]. Recently, in the other study, same researchers reported that testicular I/R may cause two different pathways of organ damage: (1) an early pathway involving NF-kB, MAPK3/MAPK1, MAPK8, MAPK14 and TNF-a which produces an inflammatory derangement of the testis and (2) a more delayed response involving MAPK3/MAPK1 only, TNF-a[ BAX, and caspase-3 and -9 that likely activates apoptosis [128]. Moon et al. [170] demonstrated that both the Akt/protein kinase B and ERK1/2 activation increased in damaged seminiferous tubules, suggesting that these signaling pathways contribute to the survival of testicular cells after testicular I/R injury.

Hypoxia-inducible factor (HIF)-1 is a transcription factor that regulates response to hypoxia and oxygen homeostasis in many tissues involving testis. Powell et al. [171] showed that HIF-1 played a role in the cellular response to hypoxia after torsion.

Al-Maghrebi et al. [172] recently reported that increased survivin expression in I/R injury was correlated with oxidative stress, apoptosis, and spermatogenic damage. Down-regulation of testicular survivin expression is a potential new target for the prevention of germ cell death during testicular torsion.

Sildenafil, vardenafil, and tadalafil are the phosphodiesterase type 5 inhibitors. They are new class of vasoactive drugs that have been developed for the treatment of erectile dysfunction. The beneficial effect of intraperitoneal vardenafil [173] and sildenafil [174,175] on I/R injury in a rat model of testicular torsion has been investigated. Verdanafil treatment exerted antiischemic effect, increased total antioxidant enzymes levels, and decreased MDA levels, germ cell apoptosis, and eNOS and iNOS levels. Similarly, catalase and SOD activities are increased, and MDA concentrations are decreased with sildenafil treatment. However, the effect of oral vardenafil [176] and oral sildenafil and vardenafil [177] has been investigated and they are found not to have protective effect on testicular I/R injury.

Regulation of testicular function was involved in several growth factors, such as epidermal growth factor (EGF), insulin-like growth factor (IGF) I, transforming growth factor-P, fibroblast growth factor, insulin, and erythropoietin. These factors are able to influence Leydig cell steroidogenesis by binding to specific plasma membrane receptors [178] . In addition, pretreatment with some growth factors, such as EGF, vascular endothelial growth factor (VEGF), and IGF, has been shown to prevent I/R damage in testis. Uguralp et al. [179] investigated the effects of sustained and local administration of EGF on improving bilateral testicular tissue after torsion. MDA levels were significantly lower in the EGF groups than in the control groups. GSH-Px levels in the control group were significantly higher than in the EGF group. Their study showed that local and sustained EGF release after testicular torsion improves bilateral testicular injury. VEGF, an angiogenic peptide, mediates angiogenesis and vasculogenesis and promotes endothelial cell survival. VEGF expresses in testis, prostate, and seminal vesicles and presents in high concentration in semen. Thus, it could play an important role in male reproductive system. Recently, it has been suggested that VEGF has the protective effect on different I/R injury models. Hashimoto et al. [ 180] reported that the spontaneous increase in VEGF in I/R testis may lead to a reduction of ischemic testicular damage. Tungkiran et al. [181] evaluated the effect of VEGF injection into the testis, especially on spermatogenesis and apoptosis. They measured mean seminiferous tubular diameter, germinal epithelial cell thickness, mean testicular biopsy score, and apoptosis (caspase-3-positive cells). Researchers found that VEGF administration caused a significant decrease in testicular caspase-3-positive cells compared with I/R group. However, administering VEGF before reperfusion might have the potential to reduce the long-term histopathologic damage after testicular torsion. Other growth factors are IGF-1 and IGF-2. They are locally produced in the testis and may regulate the balance between pro- and antiapoptotic proteins at a cellular level. It was interesting that IGF-1 levels decreased in the contralateral testis following testicular torsion [182] and IGF-1 administration seems to lower the levels of germ cell apoptosis, which may be important for protecting the testes from torsion/detorsion injury [183],

Previous studies have demonstrated that NF-kB played a critical role in I/R injury. NF-kB transcriptionally activates many genes involved in the inflammatory process, including intercellular adhesion molecule 1 (ICAM-1), vascular cellular adhesion molecule 1 (VCAM-1), and E-selectin (ELAM-1), which are important mediators of the inflammatory process in reperfusion injury. Apoptosis always occurs in the ischemic-reperfused tissue, and the functions of the tissue never remain unchanged. Many factors, including ROS, IL-1, and TNF-a, cause IkB phosphorylation. Sulfasalazine acts as a potent inhibitor of NF-kB by inhibiting I kappa B phosphorylation, thereby preventing its translocation into the nucleus and decreasing adhesion molecule expression. Zhao et al. [184, 185] concluded that sulfosala-zine prevented apoptosis in spermatogenic cells after the testicular torsion inhibiting NF-kB activation.

Tumor necrosis-a is produced by round spermatids, pachytene spermatocytes, and testicular macrophages in the testis. The type 1 TNF receptor has been found on Sertoli and Leydig cells and numerous studies suggest a paracrine mode of action for TNF-a in the normal testis. IL-1 a has been reported to be produced by Sertoli cells, testicular macrophages, and possibly postmeiotic germ cells. IL-1 receptors have been reported on Sertoli cells, Leydig cells, testicular macrophages, and germ cells suggesting both autocrine and paracrine functions. While these proinflammatory cytokines have important roles in normal testicular homeostasis, an elevation of their expression can lead to testicular dysfunctions. A pivotal role for IL-1P in the pathology of testicular torsion has been recently described whereby an increase in IL-1P production after reperfusion of the testis is correlated with the activation of the stress-related kinase, JNK, and ultimately resulting in neutrophil recruitment to the testis and germ cell apoptosis [186].

Several studies have suggested that the bcl-2 family of proteins, including pro-(bax, bak, bid, bad, and bim) and antiapoptotic (bcl-2, bcl-xs) molecules, plays a crucial role in the regulation of germ cell apoptosis. In addition, bax/bcl-2 system is important for the evolution of normal spermatogenesis. Sukhotnik et al. I 187] reported that in ipsilateral testis, bax/bcl-2 ratio did not change significantly, and the elevation of germ cell apoptosis was not marked; in the contralateral testis, germ cell apoptosis increased after 6 h of detorsion, achieved statistical significance after 24 h, and decreased after 72 h of detorsion and was initiated by decreased bcl-2 messenger ribonucleic acid (RNA) levels and elevated bax/bcl-2 ratio within the first 6 h of detorsion.

I/R of the testis results in germ cell-specific apoptosis and can lead to decrease spermatogenesis. Germ cell-specific apoptosis after I/R of the testis has been shown to be correlated with and dependent on neutrophil recruitment to the testis after I/R. In experimental animals models, cell adhesion molecules such as E- and P-selectins, ICAMs, CD44 play an important role in neutrophil recruitment to the testis after torsion-/detorsion and resulting germ cell-specific apoptosis. After torsion/detorsion of the testis, cytokines (both TNF-a and IL-1P) increase and they regulate selectins expression. Therefore, selectins expression in the testis may be a diagnostic indicator of I/R of the testes and blockade of selectins function may have therapeutic values [188-190]. Studies that used E-selectin-deficient mice have demonstrated that E-selectin expression is critical for neutrophil recruitment to subtunical venules in the testis after I/R and for the resultant germ cell-specific apop-tosis. Celebi and Paul [188, 189] administered a function-blocking monoclonal anti-mouse E-selectin or P-selectin antibody (FBMABs), and these FBMABs inhibited significantly inflammation and neutrophil migration to the I/R-induced testis. They suggested that anti E-selectin antibody alone or combination of anti E- and P-selectin antibody have been a new point of view by antiadhesion therapy to the clinical management. In addition, Moon et al. [191] showed increased expression of CD44 in rat testis I/R model.

Lysiak et al. [192] demonstrated that the proinflammatory cytokines, TNF-a and IL-1P , were stimulated after I/R as was the phosphorylation of JNK. The downstream transcription factors of JNK, ATF-2, and c-Jun were also phosphorylated at specific times after I/R of the testis. Activation of the JNK stress-related kinase pathway was correlated with an increase in E-selectin expression and neutrophil recruitment to the testis after I/R. Intratesticular injection of IL-10 also caused JNK phosphorylation and neutrophil recruitment to the testis. These results speculated that testicular I/R injury stimulated IL-10 expression, which lead to activation of the JNK signaling pathway and ultimately E-selectin expression and neutrophil recruitment to the testis. This provided the first evidence of a cytokine/stress-related kinase signaling pathway to E-selectin expression in vivo.

Recently, Tsounapi et al. [21] investigated the protective effects of sivelestat sodium hydrate, a neutrophil elastase inhibitor, on ipsilateral and contralateral testes after unilateral testicular I/R injury in rats. They demonstrated that unilateral testicular I/R resulted in marked increases in bilateral testicular MDA levels, MPO activity, and enhanced expression levels of heat shock protein (HSP) 70. Sivelestat ameliorated the I/R-induced ipsilateral testicular damage bilaterally through its ability to inhibit neutrophil elastase. By inhibiting neutrophil elastase, it has a direct action to the accumulated and activated leucocytes, offering efficient protection against the production of oxygen radicals and cytokines.

Trapidil is an antianginal drug which is a phosphodiesterase and platelet-derived growth factor inhibitor. It has effects of nitroglycerine-like vasodilatation, inhibition of platelet aggregation via blockage of thromboxane A2, reduction of lipid peroxidation, IL-6 and IL-12, and procoagulant activity by inhibiting the CD40 pathway in monocytes. Bozlu et al. [20] investigated the protective effects of trapidil on long-term (60 days after I/R) histologic damage; and Somuncu et al. [193] also investigated on MDA, NO, and total sulfhydryl levels. These studies demonstrated that trapidil decreased free oxygen radical formation and attenuated histopathological damage testes.

There is a consistent basal expression of erythropoietin mRNA, and this expression is increased by hypoxia; this expression has been also detected in Sertoli and peritubular myoid cells in testis. It has also been observed that testosterone production has been stimulated by erythropoietin in rat Leydig cell and that it influences steroidogenesis. Testosterone production has been increased by intravenous eryth-ropoietin administration in patients with renal failure. It has been suggested that this effect of erythropoietin might act directly on human Leydig cell function, which requires activation of a protein kinase-C-dependent pathway [178]. Moreover, erythropoietin has free radical scavenger, antiapoptotic and anti-inflammatory activity. In addition, recently published studies have shown a protective role of erythropoietin on experimental I/R injury in testis [178, 194, 195]. Moreover, one of the recently published studies has reported that administration of darbepoetin-a, a novel erythropoietin protein, caused reduction of MDA and NO levels and an increase of GSH levels in testis torsion [196].

Ozturk et al. [197] demonstrated that platelet-activating factor (PAF) antagonist BN-52021 decreased MDA values and the testicular injury score and increased SOD, catalase, and GSH-Px values in the BN-52021-treated group compared to in the I/R group in rats with reperfusion damage due to unilateral testicular torsion.

Another experimental study was designed to evaluate the effects of VIP on lipid peroxidation and histopathology in both testes after unilateral testicular torsion and detorsion. It has antioxidant, anti-inflammatory, antiapoptotic, neurotrophic, and immunomodulatory actions. Immunohistochemical studies have demonstrated the presence of VIP and its receptors in the testis, as well as other parts of the male urogenital tract of various animal species and humans. Can et al. [198] demonstrated that VIP could protect testicular tissue from detorsion injury.

Recent studies showed that ethyl pyruvate (anti-inflammatory, antioxidant, and antiapoptotic) [127]. angiotensin-converting enzyme inhibitor (lisinopril), angiotensin II type 1 receptor blocker (losartan) [199] . and PAF antagonist BN-52021 (reduced ICAM-1 expression) [197] reduced germ cell-specific apoptosis and oxidative stress on testicular torsion/detorsion model in rats. Sozubir et al. [200] reported that Insl3 mutant mice predisposes to testicular pathologies such as torsion, atrophy. Thus, Insl3 is a candidate signaling molecule in human testicular torsion.

NO, a water- and lipid-soluble, freely diffusible gaseous molecule with a short half-life, is formed from l-arginine and molecular oxygen by a family of NOS. It has been demonstrated that the three isoforms of NOS (iNOS, eNOS, and neuronal nitric oxide synthase [nNOS]) involved in the pathogenesis of testicular I/R injury in association with germ cell death, through either necrosis or apoptosis. Expression of eNOS in testes has been demonstrated in Leydig cell, Sertoli cell, spermatocyte, spermatids, and endothelial cells [122, 201, 202]. Although several investigators have reported that NO which has dual effects on cell survival and death is an important signal transduction molecule in I/R injury. Nevertheless, there are conflicting results in testicular torsion. Dokucu et al. [203] reported the protective effect of NO on contralateral testis in unilateral testicular torsion. In Barlas and Hatibo lu's study [204], l-arginine, precursor ofNO, was used to increase NO synthesis; this caused a decrease in reperfusion injury. l-nitro arginine methyl ester (l-NAME), a competitive inhibitor of NO synthase, was used to reduce NO formation; this concluded a negative effects on testicular I/R injury. However, Ozokutan et al. [205] also used l-arginine and (l-nitro monomethyl arginine) l-NMMA, a competitive inhibitor of NO synthase. Inhibition of NO synthesis with l-NMMA significantly improved I/R injury, increasing NO production with l-arginine-increased testicular damage. In Dokucu's study [206] . they suggested that molsidomine has a protective effect against I/R injury in rat testes. Molsidomine, a precursor of NO, reduced MDA and histopathologic score and increased expression of Sonic hedgehog and HIF a-1 in testes I/R injury. l-NAME, the NOS inhibitor, reversed the protective effect of molsidomine against I/R injury. The effect of molsidomine may be related to reducing the effects of oxidative stress in testes. Also, Ozturk et al. [207] demonstrated that NO regulated adhesion molecules expression such as tenascin, lectin, and ICAM-1, which was the proof of inflammation in torted testicle; furthermore, molsidomine played an important role in the prevention of I/R injury.

Gabexate mesilate, a synthetic serine protease inhibitor, has anticoagulant activity. It inhibits trypsin, plasmin, kallikrein, and thrombin; it has been used for the treatment of acute pancreatitis and disseminated intravascular coagulation and for anticoagulation during hemodialysis. Gezici et al. [208] found that the treatment with gabexate decreased MDA levels significantly when compared to the I/R/ untreated group. In addition, gabexate protected the levels of antioxidant enzymes such as SOD, catalase, and GSH-Px. In addition, the Tc-99m pertechnetate uptake ratio and the perfusion index were significantly increased after gabexate treatment.

So far, it has been found that capsinoids possess the biological properties of antitumor, antioxidant, and antiobesity. Chili peppers are the major source of nature capsaicinoids, which consist of capsaicin, dihydrocapsaicin, etc. Sarioglu et al. [209] showed that capsaicin effectively prevented apoptosis in the contralateral tes-tis after ipsilateral torsion.

Rosuvastatin is one of the synthetic statins, which is used for hyperlipidemia treatment. It has been experimentally shown that it has antioxidant and anti-inflammatory effect, decreasing leukocyte adhesion and thrombocyte aggregation. Recently, Karakaya et al. [210] measured testis basal blood flow with laser Doppler flowmeter and they reported that rosuvastatin could protect tissue perfusion in the experimental testicular torsion model.

Romeo et al. [211] aimed to evaluate the effects of raxofelast, a vitamin E-like antioxidant, on lipid peroxidation and histopathology in both testes after unilateral testicular torsion and detorsion. Conjugated dienes (CD) levels, an index of lipid peroxidation, and testis histopathology were evaluated. Testicular I/R in untreated rats produced high testicular levels of CD. Furthermore, histological examination revealed marked damage to the testis interstitium with severe hemorrhage and edema. The administration of raxofelast lowered CD levels and significantly reduced histo-logical damage. Antonuccio et al. [126] supported this study evaluating testicular JNK, ERK, and TNF-a activation by Western blot analysis, and mRNA expression and CD using a spectrophotometer technique. These data suggested that the hydro-philic vitamin E-like antioxidants are good candidates for testicular torsion.

However, Turan et al. [212] investigated the role of lipid peroxidation in ipsilat-eral and contralateral testicular reperfusion injury following unilateral testicular torsion and the effect of vitamin E in the management of this injury. There were no significant differences between right and left testes within groups or between right or left testicular MDA values in different groups. The results suggested that vitamin E given before or after detorsion of testes is not useful in preventing testicular reperfusion injury.

Recently, surprising results were observed in the coadministration of vitamin C and dopamine on I/R injury after experimental testicular torsion in rats. Investigators could not find more beneficial effects from the combined administration of vitamin C and dopamine than vitamin C alone, although it was better than the dopamine administration alone [213].

Lycopene is a potent antioxidant. It was reported that lycopene treatment in men with idiopathic infertility provided an improvement in male infertility, especially in sperm motility, concentration, and morphology. In addition, regular lycopene intake for 9 months in oligospermia patients improved the sperm characteristics. Moreover, testicular I/R model in rats, lycopene increased the motility in bilateral testes and decreased the rate of abnormal sperm in ipsilateral testis, but did not rise sperm concentration in bilateral testes. Lycopene also restored antioxidant enzyme activities, but not reduced glutathione level [6] .

Ginkgo biloba (EGB 761), a free radical scavenger, is investigated by Akgul et al. [214]. Ginkgo biloba decreased MDA, nitrate-nitrite concentrations on unilateral testicular torsion model. Moreover in another study, treatment with Ginkgo biloba showed significant decrease in apoptotic cells, eNOS, and iNOS activities in testes [215].

l-carnitine, a naturally occurring enzymatic antioxidant, is a necessary factor in the utilization of long chain fatty acids to produce energy. l-carnitine exhibits a wide range of biological activities including anti-inflammatory, antiapoptotic, neu-roprotective, cardioprotective, and gastroprotective properties. Furthermore, these effects are attributed to its antioxidative and free radical scavenging activity [18, 216-219]. In addition, it plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of l-car-nitine found in the human body, and initiation of sperm motility occurs in parallel to l-carnitine increase in the epididymal lumen. It is known that l-carnitine prevents the formation of ROS and scavenges free radicals and it protects cells from peroxidative stress. Moreover, it plays a key role in sperm metabolism by providing readily available energy for use by spermatozoa, which positively affects sperm motility, maturation, and the spermatogenic process [146] [ The use of l-carnitine and its derivatives in therapy has been proposed in recent years for treatment of male infertility, and a number of controlled and uncontrolled human and animal studies published to indicate their possible application. In several clinical studies with infertile patients, seminal plasma total carnitine level was found to be low and carnitine supplementation improved reproductive function [220-222] . An increase in spermatozoa motility has been observed in treated patients affected with idio-pathic forms of oligospermia, asthenozoospermia, and oligoasthenoteratozoo-spermia [223-225] or affected with bacterial inflammation of the accessory sexual gland [226,227]. However, the mechanisms by which carnitine controls male fertility has not yet been clearly identified. In our study, this is the first time we showed that, l-carnitine has a protective effect in experimental testicular torsion/detorsion model in rats; l-carnitine decreased MDA levels and improved histological damage in testicular tissue [228]. In addition, it has been reported that l-carnitine has the protective effect on apoptosis I/R injury in testis [229-231]. Recently, the combined use of l-carnitine (an antioxidant and free radical scavenger) and meloxicam (COX-2 inhibitor) was investigated in the treatment of cellular damage caused by testicular torsion. Meloxicam caused the most dominant inhibitory effect on the expression of specific genes of inflammation as well as combination therapy. Because the effect of these inflammatory genes was still evident 4 days after detorsion, combination therapy using these agents could be administered until the late postoperative period to achieve the most reproductive treatment of testicular torsion at the cellular level. This process would prevent the autoimmune activity against sperm cells and protect the innocent contralateral testis from the insult of the anti-sperm antibodies [232] .

N-acetylcysteine is a small molecule containing a thiol group. It was first introduced as a mucolytic drug in the 1960s, after that N-acetylcysteine has been found to have an antioxidant effect, acting as a free radical scavenging agent and a precursor of GSH. Recently, Akta et al. [233] suggested that early administration of N-acetylcysteine may have a protective effect in the rat experimental testicular torsion/detorsion models. N-acetylcysteine significantly reduced thiobarbituric acid reactive substances (TBARS) levels and histological parameters of spermatogenesis and improved in bilateral testes when compared with torsion and torsion/detorsion groups. In the other two studies [234, 235] N-acetylcysteine prevented the oxidative effects of reperfusion injury after torsion, regulating antioxidant enzyme activities and germ cell apoptosis. Another study showed protective effect of erdosteine in testis I/R injury [236]. Also, erdosteine is a mucolytic agent, and owing to the presence of two sulfhydryl group in its metabolites, can act as a free radical scavenger and this component of the mechanism of action is likely to be involved in the testicular injury induced by I/R.

Curcumin is a major active component of turmeric, which is extracted from the powdered dry rhizomes of Curcuma longa. Curcumin exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, and antitumoral effects. It reduces production of proinflammatory cytokines and activation of NOS, cyclooxygenase-2, lipooxygenase, AP-1, and NF-kB. There are a few studies investigating curcumin on testis I/R injury. Basaran et al. [237] demonstrated the protective effect of curcumin on testicular I/R injury for the first time. They showed that the curcumin treatment diminished iNOS and eNOS immunoreactivity in ipsilateral and contralateral testes. However, it had no effect on testicular MDA levels. Later, Wei et al. [238] reported that the rats treated with curcumin had significant decreases in MDA level and xanthine oxidase activity and had significant increases in heme oxygenase-1 protein expression level and testicular spermatogenesis in ipsilateral testes, compared with I/R group.

Nigella sativa L. and thymoquinone are effective free radical scavengers. They have antioxidant activity and protect against the damage caused by free radicals. Gokge et al. [239] showed that thymoquinone administration reduced histological damage, MDA, total oxidative stress, and oxidative stress index values, but did not affect the total antioxidant capacity and MPO activity in the I/R testes.

Edaravone, a free radical scavenger and anti-inflammatory agent, is used for the clinical treatment of cerebral ischemia, protecting neurons. Its effect is especially on hydroxyl radicals. To induce testicular I/R, Tamamura et al. [240] clamped testicular artery in the rat testis for 30 min. Blood flow and NO release were monitored with a laser Doppler flowmeter and an NO-selective electrode, respectively. Tissue MDA, nitrate-nitrite, 8-hydroxydeoxyguanosine (8-OHdG), MPO, and HSP 70 and its mRNA were measured. In this research, investigators showed that testicular I/R resulted in marked increases in nitrate-nitrite, MDA, 8-OHdG and MPO levels, and in the expression levels of HSP 70 and its mRNA in rat testes. Edaravone ameliorated the I/R-induced testicular damage through its ability to scavenge free radicals and anti-inflammation.

Uguralp et al. [241] evaluated the effects of resveratrol on testicular I/R injury. Resveratrol, a nonflavinoid polyphenol found in great amounts in grapes, mulberries, peanuts, and in red wine, exhibits a wide range of biological activities such as antioxidant, anti-inflammatory, and antitumor properties. In the study, the testicular tissue levels of MDA and GSH and histological changes were determined. In rats treated with resveratrol, MDA levels were significantly decreased, GSH levels were significantly increased, and the mean testicular tissue injury score in the resveratrol group was significantly lower than in torsion and detorsion groups. This study demonstrated that intraperitoneal administration of resveratrol in rats may protect testis against injury associated with reperfusion. Moreover, in the other study resveratrol decreased germ cell apoptosis in the ipsilateral testes [242] . In addition, tea polyphenols had a protective effect against testicular torsion [243] .

Another experimental study was designed to evaluate the effects of garlic extract on testicular tissue after testicular torsion/detorsion. Garlic extract, an antioxidant, attenuated the generation of toxic-free radicals and inhibited the xanthine oxidase-mediated I/R injury cascade [244] .

a-Lipoic acid acts as antioxidant not only through free radical quenching, but also indirectly through recycling of other cellular antioxidants. Free lipoic acid has not been detected in human beings because it is bound to proteins. However, after therapeutic applications, free lipoic acid can be found in the circulation. Guimaraes et al. [140] studied the protective effects of a-lipoic acid in experimental testicular torsion. a-Lipoic acid pretreatment promoted decreased TBARS concentrations and increased GSH levels in testes tissue and total antioxidant power in plasma.

Etensel et al. [245, 246] investigated the protective effects of dexpanthenol after torsion at the first, fifth minute, and first hour and testicular atrophy at 60th day on testicular I/R injury model. Dexpanthenol (provitamin B5) is the biologically active alcohol of pantothenic acid (vitamin B5). Dexpanthenol has approved FDA and it is safe, cost-effective, and readily available agent. They reported that dexpanthenol attenuated lipid peroxidation and tissue damage and significantly prevented testicu-lar atrophy at 60th day of testicular torsion.

Avlan et al. [247] investigated the effects of selenium on ipsilateral and contralateral testicular damage after unilateral testicular torsion/detorsion. The highest MDA and the lowest SOD values were determined in both testes in torsion/detorsion group. There were statistically significant differences in MDA levels and SOD enzyme activities in torsion/detorsion group compared with control group. These results suggested that I/R injury occurred in both testes after unilateral testicular torsion/detorsion and that selenium administration before detorsion prevented reperfusion injury in testicular torsion. These findings were also confirmed histopathologically.

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts and has been used as a folk medicine for many years. CAPE was known to have antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, and antitumoral effects. It has been investigated that the effects of CAPE in rats subjected to testicular torsion/detorsion [248-250] . Pretreatment with CAPE attenuated the testicular injury, as well as the increase in the tissue levels of MPO, TBARS, NO levels, and iNOS activity caused by torsion/detorsion in the testis. Testis tissues showed a significant increase in GSH-Px activity compared to the torsion group when CAPE was applied. In conclusion, investigators concluded that CAPE treatment exerts a protective effect on testicular I/R.

Adivarekar et al. [251] showed that administration of lomodex (low molecular weight dextran) and MgSO4 prior to detorsion resulted in prolonged testicular salvage with a potential of subsequent improvement in semen quality, fertility, and reduction in long-term morbidity and showed preservation of tubular morphology. In another study, Abes et al. [252] reported that ATP-MgCl2 administration before or after detorsion may prevent reperfusion injury in testicular torsion.

Metal aspartates including zinc aspartate are inhibitors of ROS. Their inhibitory activities are a consequence of both the scavenging of the free radicals and the inhibition of xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities. Ozkan et al. [253] demonstrated that zinc aspartate reduces I/R injury by its antioxidant effects after unilateral testicular torsion/detorsion and affects the antioxidant enzyme systems such as SOD, catalase.

Another experimental I/R study was designed by Wei et al. [254]. They presented the beneficial effects of taurine on I/R testis model. Taurine is a potent anti-oxidant and antiapoptotic and also exerts cytoprotective effect on I/R injury of other organ. Taurine has been found in Leydig cells, vascular endothelial cells, and other interstitial cells in the testis. In addition, sperm and seminal fluid are rich in taurine. It has also been shown to be involved in sperm motility, capacitation, acrosome reaction, and osmoregulation. Taurine acts as an antioxidant and prevents sperm lipid peroxidation. Wei and collaborates demonstrated that taurine decreased ROS generation by diminishing the neutrophil recruitment to the testis.

Ibuprofen is one of the most useful nonsteroidal anti-inflammatory agents available to humans. It is known to inhibit cyclooxygenase, modulate channel activities, and activate peroxisome proliferator-activated receptor (PPAR), an orphan nuclear receptor that acts as a ligand-activated transcription factor, thereby inhibiting proin-flammatory cytokine production. In addition, ibuprofen has antiradical and antioxi-dant effects and scavenges ROS. It protects the lipids of biological membranes from oxidation and consequently inhibits the accumulation of lipid peroxidation products such as MDA. Several studies have showed that ibuprofen could scavenge hydroxyl and superoxide radicals and that it possessed radioprotective [255] and neuroprotective effects such as Alzheimer disease [256, 257]. Recently, ibuprofen was successfully used to decrease I/R injury in multiple organ systems, including the retina, liver, heart, and brain. Nonetheless, to our knowledge, the effect of ibuprofen on testicular I/R injury had not been previously reported. Our results showed that because of its anti-inflammatory and antioxidant effects, ibuprofen pretreatment may have protective effects in the experimental testicular torsion/detorsion model in rats [258] .

The peroxisome proliferator-activated receptor-gamma (PPAR-y) is a member of the nuclear hormone receptor superfamily that is involved in several physiological processes, such as glucose homeostasis, cellular differentiation, regulation of lipid, and lipoprotein metabolism, as well as in pathological states, including atherosclerosis, inflammation, cancer, infertility, and demyelination. Thiazolidinediones are synthetic PPAR-y ligands used in the control of type 2 diabetes, whereby rosiglita-zone is the most potent and selective agent in this class, which binds the receptor with a higher affinity than other thiazolidinediones, such as pioglitazone or ciglita-zone. In recent studies, it was demonstrated that rosiglitazone ameliorated the lesions associated with I/R of kidney, heart, and gut, possibly through its anti-inflammatory and antioxidant properties. Inan et al. [259] aimed to evaluate the effects of rosiglitazone on lipid peroxidation, eNOS immunoreactivity, and the his-topathology in both testes after unilateral I/R in rats. Investigators reported rosigli-tazone prevented I/R injury in both testes. In addition, Mogilner et al. [260] found that diclofenac did not change histologic parameters of spermatogenesis, but decreased germ cell apoptosis in both testes following testicular I/R. It has been shown that PPAR beta/delta (p/5) is expressed in testis. Minutoli et al. [261] tested PPAR-p/5 agonist (l-165,041) and antagonist (GW9662) and evaluated testicular ERK, TNF-a, and IL-6, and also investigated PPAR-p/5 activation, mRNA expression, and organ damage. They found that l-165,041 administration increased the PPAR-p/5 message and protein, inhibited ERK, TNF-a, and IL-6 expression, and decreased histological damage. Concomitant administration of GW9662 reversed the protection exerted by PPAR-p/5 agonist.

Salmasi et al. [262] reported that morphine increased the ipsilateral intratesticu-lar antioxidant markers (SOD, catalase, GSH-Px) during the reperfusion phase after unilateral testicular torsion. The ipsilateral MDA levels in the morphine group were significantly lower than in the I/R group. The observed effects of morphine may have been a result of its impact on neutrophils and its direct scavenging action against peroxynitrite.

Pentoxifylline, a methyl xanthine derivative, has antioxidant effect. It has been used for its regulatory effects on microvascular blood flow. It was reported that unilateral testicular torsion and detorsion caused an increase in the MDA levels of both testes. Pentoxifylline decreased MDA levels on both side and attenuated reperfusion damage on both side, possibly with its effects on blood flow and neutrophils [263-265] .

The effect of methylene blue on the experimental testis torsion/detorsion models has been investigated. Both Greenstein [266] and Inan [267] suggested that methylene blue didn't protect testes from histological damage in case of ipsilateral testis after I/R injury. Nevertheless, surprisingly Inan et al. reported that methylene blue had harmful effect and increased MDA levels on the contralateral testis.

Propofol, which is widely used as an intravenous anesthetic, has been shown to have an antioxidant activity on several tissues. Unsal et al. [268] investigated the prevention of reperfusion injury with propofol after testicular torsion. They assessed tissue MDA level, xanthine oxidase, catalase, and GSH-Px activities. Pretreatment with propofol prevented a further increase in MDA levels and significantly decreased catalase activity following detorsion. GSH-Px activities were not affected either by torsion/detorsion or propofol pretreatment. Histologically, torsion caused some separation between germinal cells in the seminiferous tubules, which became much more prominent in the detorsion group and attenuated with propofol pretreatment. In brief, propofol decreased free radical formation and attenuated histopathological damage in the testis after reperfusion. Yagmurdur et al. [269] suggested that propo-fol prevented testicular damage by scavenging reactive oxygen and nitrogen species and inhibiting lipid peroxidation and attenuated germ cell-specific apoptosis [270] . Recently, Harvey et al. [271] used propofol sedation performing manual testicular detorsion to a 14-year-old adolescent boy. Patient's testicular pain is completely reduced.

Nezami et al. [272] stated that the use of immunophilin ligands (cyclosporine and FK 506-tacrolimus) had significantly decreased testicular torsion damage. The results of biochemical studies suggest that reduction of oxidative stress along with attenuated neutrophil accumulation by immunophilin ligands may have a major role in their cytoprotective effects and their potent biologic properties in inhibiting programmed cell death and necrosis.

Mexiletine is a congener of lidocaine and safe antiarrhythmic agent, preventing I/R injury in myocardium. Also, mexiletine is a potent antioxidant agent, most of the reports about its protective effects on I/R injury on heart. Ozen et al. [273] investigated its effects on the electrical field stimulation (EFS)-induced contractions in rabbit vasa deferentia after torsion/detorsion. However, mexiletine had no preventive effect on this inhibition.

Allopurinol has been shown to have preventive effects on testicular I/R injury [274-276] . Kehinde et al. [276] tried five antioxidant (acetyl salicylic acid, ascorbic acid, allopurino, quercetin, and SOD), but they found that only allopurinol had a beneficial effect at 3 months after experimental torsion in a rabbit model. Moreover, in Abasiyanik and Dagdonderen's study [274], allopurinol treatment prevented only MDA increase, but no histopathologic results. However, Silva et al. [277] reported that allopurinol didn't protect the testes on 60 days after I/R injury.

Melatonin is one of the most powerful endogenous antioxidants known. Antioxidant effects of melatonin can occur by either a direct or an indirect mechanism. Melatonin itself exerts direct antioxidant effects via scavenging the hydroxyl radical, peroxyl radical, singlet oxygen, peroxynitrite anion, and superoxide anion. Additionally, it acts as an indirect antioxidant by stimulating several antioxidative enzymes, including GSH-Px, GSH reductase, glucose-6-phosphate dehydrogenase, and SOD. Conversely, it inhibits a prooxidative enzyme, NOS. Barun et al. [278] investigated whether EFS-evoked biphasic contractions are altered in ipsilateral and contralateral rat vasa deferentia obtained from animals exposed to the unilateral tes-ticular torsion/detorsion procedure. They also evaluated the effects of melatonin on these contractile responses. The investigators suggested that melatonin produces an inhibition on EFS-evoked biphasic twitch responses in the ipsilateral and contralateral rat vasa deferentia following unilateral testicular torsion/detorsion in the rat. In another study, Abasiyanik and Da donderen [274] compared beneficial effects of melatonin and allopurinol in experimental testicular torsion. Their results showed that melatonin treatment prevented testes from injury both biochemically and histo-pathologically. In addition, Ozturk [279], Duru [280], and Yurtgu et al. [281, 282] showed preventive effects of melatonin administration on reperfusion damage in experimental testis I/R. In their studies, it was demonstrated that MDA levels were lower in both torsioned and contralateral testes. Furthermore, Yurtgu et al. [281] reported that melatonin increased serum inhibin B levels which reflect Sertoli cell function and the state of spermatogenesis. Jeong et al. [283] compared the preventive effects of cyclosporine A combined with prednisolone and melatonin on damage to contralateral testis after ipsilateral torsion/detorsion between pubertal and adult rats. Their study stated that the preventive effects of cyclosporine A combined with pred-nisolone on contralateral testicular damage were characteristic only in pubertal rats, while the preventive effects of melatonin were characteristic in pubertal and adult rats. These results suggested that damage to the contralateral testis induced by an immunological mechanism may be more significant during puberty than during adulthood. Kanter [284] confirmed these experimental studies with immunohis-tochemistry. They reported that melatonin treatment increased the immunoexpression of proliferating cell nuclear antigen and testosterone and decreased germ cell apoptosis in I/R testis. Recently, Kucer et al. [285] found that melatonin treatment improved sperm morphology and epididymal sperm quality in I/R injury.

Yazawa et al. [286] demonstrated the suppressing effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells and vascular neutrophil adhesion after I/R in testis. This inhibition was suppressed by intravenous administration of mifepriston, a glucocorticoid receptor antagonist. In recent years, Cho et al. [287] suggested that medical treatment, such as drug treatment and surgery treatment should be performed as early as 2 h after testicular torsion. Because apoptosis occurs at least partially via the phosphoinositide-dependent protein kinase-1/serum- and glucocorticoid-inducible kinase 1/forkhead transcriptional factor FOXO3a signaling cascade, these cascades impair the testosterone secretion capacity of the testes after only 2 h of torsion.

Yapanoglu [288] and Aksoy [289] studied about the effects of dehydroepiandros-terone on testicular torsion damage in rats. They found that dehydroepiandrosterone may be a protective agent for preventing biochemical, apoptotic, and histopatho-logic changes related to oxidative stress in testicular injury. Unal et al. [290] investigated the preventive effects of trimetazidine on I/R injury in rats. They also reported that trimetazidine may be a protective agent for preventing biochemical and histo-pathologic changes related to oxidative stress in testicular injury. Sava§ et al. [291] reported that human chorionic gonadotropin treatment improved contralateral histo-pathologic injury and increased the serum testosterone levels. In addition, the positive effect of hyperbaric oxygen therapy [292] and surfactant tetronic 1107 [293] on testicular torsion model in rats has been shown.

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