Testicular torsion is a urological emergency with the overall consent in treatment protocol including early exploration, detorsion, and fixation of the both testes; and the exicision of testis to avoid damage to the contralateral testis if it is necrotic . Testicular torsion and detorsion induces biochemical and morphological changes caused greatly by ischemia/reperfusion (I/R) injury in the testicular tissue . I/R injury is associated with the activation of neutrophils, inflammatory cytokines and adhesion molecules resultant with increased thrombogenicity, release of massive intracellular Ca2+, and generation of reactive oxygen species (ROS) . Injury starts in the ischemia period and exacerbates during reperfusion. Previously, several chemicals such as allopurinol , gabexate mesilate , and dexpanthenol  were used to prevent this kind of injury in animal experimental testicular torsion/detorsion (T/D) model.
Erythropoietin (EPO) is produced by kidney in response to hypoxia and stimulates erythroid progenitor cells via erythropoietin receptors (EPO-R) to increase the number of mature erythrocytes within the circulation, thereby augmenting oxygen carrying capacity [7, 8, 9]. In recent years, expression of functional EPO-R has also been demonstrated in non-hemopoietic cells and organs . Many studies have discovered the potential role of EPO as a multiple functionel endogenous mediator offering protective effect against I/R injury in various tissues and organs in animals [9, 11, 12].
A novel recombinant erythropoietic protein darbepoetin alfa is a long-acting EPO analogue. It is succesfully used in the current medical practice for the treatment of various anemic conditions in spite of erythropoietin. The aim of the present study was to experimentally investigate the preventive effect of darbepoetin alfa on testicular T/D injury in rats.Materials and Methods
The experimental protocol was approved by the local animal ethics review committee of our institution. Thirty male Wistar albino type rats weighing 280 to 320 gr were used in this study. The animals were maintained on pellet food and water ad libitum. Rats were divided into 3 groups containing 10 rats in each. Group 1, sham operation; Group 2, T/D; Group 3, T/D + darbepoetin alfa.
All surgical procedures were performed under ketamine (50 mg/kg) and xylazine HCl (8 mg/kg) anesthesia. T/D and sham operations all were performed on the testis through a left scrotal incision. In group 1, the left testis was brought out through the incision and then replaced with a fixation to the scrotum by 5/0 silk suture. In group 2, the left testis was exposed and torsion created by rotating it 720 clockwise direction and then left in the same position for 2 hours by fixing it to the scrotum with 5/0 silk suture. After 2 hours of torsion period, 4 hours of detorsion was performed. In group 3, rats underwent the same surgical procedure as group 2 but darbepoetin alfa (Aranesp, Amgen) 25 gr/kg was injected intraperitonally 30 minutes before detorsion. All injections were diluted with sterile saline and adjusted to a final consantration of 50 gr of darbepoetin alfa in 1.0 cc with similar volumes of saline which had been given to group 1 and group 2 rats. At the end of the experiments (sixth hour) bilateral orchiectomy was performed to measure the tissue levels of malondialdehyde (MDA), nitric oxide (NO) and glutathione (GSH), and to perform histological examination in testes.
Biochemical AnalysesSample preperation
Testes tissues for the estimation of tissue antioxidant levels were prepared at 4C. Tissues were weighted and cut into small pieces. A 10% homogenate was made in ice-cold potassium phosphate buffer solution (PBS, pH 7.4) containing 5 mM EDTA using a glass homogenizer. The homogenate was centrifugated at 15000 rpm for 10 minute at 4C and thus supernatant was obtained for measurement the estimation of different antioxidants and lipid peroxides.
The total protein levels for homogenates was estimated by the Biuret method .Tissue MDA determination
MDA levels in the samples were determined by a previously described method . 0.5 ml of sample was pipetted into a 10 ml centrifuge tube and 2.5 ml of trichloroacetic acid (20%) and 1.0 ml of thiobarbituric acid (0.6%) solution was added. The tubes were heated for 30 minutes in a boiling water bath and the reaction mixture was then cooled in an ice-bath followed by the addition of 4.0ml of n-butanol. The tubes were mixed with a vortex and centrifuged at 3000 rpm for 10 minute. The absorbance of the organic layer was measured at 535nm.Tissue NO determination
The production of NO was determined indirectly by measuring the nitrite levels based on Griess reaction . Samples were initially deproteinized with 75 mmol/L ZnSO4. After clean up, an aliquot of the sample was treated with copperized cadmium in glycine buffer at pH 9.7 to reduce nitrate to nitrite. The concentration of nitrite in this aliquot thus represented the total nitrate plus nitrite. In Griess reagent, a chromophore with a strong absorbance at 545 nm is formed by reaction of nitrite with a mixture of naphthlethylendiamine and sulphanilamide.Tissue GSH determination
Reduced GSH was determined by method of Beutler et al . The supernatants were mixed with 4 ml Phosphate buffer and DTNB (Dinitro2,2-Dithiobenzoic Acid) (0.01 M). After shaking, its absorbance was measured at 412 nm within 10 min of the addition of DTNB against blank. Quantity of GSH in tissue sample was calculated using standart GSH and results.Histological Examination
All testes were fixed in formalin and embedded in parafin blocks. Tissue sections were stained with Hemotoxylene&Eosin (H&E). The light microscope histological examination was done by a pathologist in a blinded fashion. Testicular tissue injury was graded on a system described by Cosentino et al . Grade 1 showed normal testicular architecture with an orderly arrangement of germinal cells. Grade 2 injury showed less orderly, noncohesive germinal cells and closely packed seminiferous tubules. Grade 3 injury exhibited disordered, sloughed germinal cells with shrunken, pycnotic nuclei and less distinct seminiferous tubule borders. Grade 4 injury defined seminiferous tubules that were closely packed with coagulative necrosis of the germinal cells.Statistical Analysis
Statistical analyses were accomplished by using of SPSS computering programmes (version 13.0). All results were reported as means S.D. The comparison of the results from the various experimental groups and their corresponding controls was carried out using a one-way analysis of variance (ANOVA) followed by pairwise multiple comparison procedures (Tukey test). The differences were considered significant when P<0.05.Results
Table 1 shows the ipsilateral and contralateral MDA, NO and GSH values for all groups. The MDA and NO levels of ipsilateral testis tissues were significantly increased in the T/D group compared with the sham and treatment groups (P<0.05). Significant decrease was found in the GSH level in the T/D group compared to the sham group and treatment group (P<0.05). The values of these parameters in the contralateral testes were not significantly different from the sham group levels.
Individual and mean ipsilateral testicular injury scores are shown in Table 2. The rats in sham group had essentially normal testicular architecture (Fig. 1a). The highest histologic grade (mean; 2.9 0.56) was determined in T/D group (Fig. 1b). Most of testes in the treatment group by darbepoetin alfa showed grade 2 injury (Fig. 1c) and its histologic grade (mean; 2.2 0.42) was significantly lower than T/D group (P<0.05). All contralateral testes showed normal seminiferous tubule morphology.Discussion
ROS including superoxide (O2?), hydroxyl (OH), hydrogen peroxide (H2O2), singlet oxygen (1O2) and nitric oxide (NO), which are generated during ischemia and reperfusion, play a major role in microvascular dysfunction and exert direct tissue damage, leading to a lipid peroxidation, protein denaturation, and DNA oxidation . Superoxide dismutase (SOD), Catalase (CAT), GSH, and glutathione peroxidase (GPx) are involved in defense against these processes . This cascade of events is known as reperfusion injury. Many researchers have focused their efforts on preventing I/R injury by means of pharmacologic intervention prior to reperfusion [4-6].
Recombinant human EPO (rHuEPO) has been shown in general to be an exceedingly safe drug, as millions of patients have recieved it over the last 15 years for treatment of anemia. Until about 8 years ago, it was a generally accepted belief that erythropoietin acts only on erythroid precursor cells, but because erythropoietin receptors (EPO-R) have been found in many other tissues including brain, spinal cord, heart and testis, there is an emerging consensus that erythropoietin may help nonerythroid cells to survive and proliferate [20, 21]. EPO has multiple protective effects, such as anti-apoptotic, anti-oxidant, anti-inflammatory and angiogenic . Parsa et al demonstrated that EPO preserved myocardium in the ischemic zone and enhanced cardiac contractile function following ischemic events . Patel et al showed that EPO reduced renal dysfunction and injury caused by renal I/R in mice . Neuroprotective effect of EPO demonstrated in numerous experimental studies in animals [12, 23]. In addition, in the first clinical trial EPO administration to patients with ischemic stroke showed significant improvement in clinical outcome parameters and a trend towards smaller infarct size . There are several reports documanting that exogenous EPO administration reduced the injury caused by I/R of the spinal cord , eye , gut , lung  and liver  in animals. In summary, EPO is increasingly regarded as a general tissue-protective agent. 
The exact mechanisms of the EPO's protective effect in I/R injury are not fully known. The receptor-associated tyrosine kinase (janus-kinase 2) is the main intracelluler signaling pathway for the effect of EPO on haematopoiesis and neuroprotection. [30, 31] Ates et al demonstrated that inhibition of tyrosine kinase by genistein blocks the preventive effect of EPO on renal I/R injury . Abdelrahman et al provided evidence that the EPO related protection of tissues exposed to oxidative stress is secondary to the inhibition of caspases 3, 8, and 9 and, hence, an antiapoptotic effect of EPO .
The antioxidant function of EPO had not been investigated until a few workers observed that when rHuEPO was administered to hemodialysis patients [33-35] and to patients suffering from anemia of chronic renal failure [36, 37] for a considerable period of time, there was a significant reduction in lipid peroxidation and an enhancement of SOD, CAT, and other antioxidant activities . EPO may act as a direct as well as indirect antioxidant . By scavenging oxyradicals, it may serve the role of a direct antioxidant, and by stimulating the other antioxidant defensive mechanism(s), it may act as an indirect antioxidant . It is possible that EPO exerts EPO-R-independent cytoprotective actions via antioxidation .
Darbepoetin alfa is a novel erythropoiesis-stimulating agent by EPO-R, and it have an extended circulating half-life and thus showing increased biological activity in vivo . We preferred using darbepoetin alfa in our experiment for its longer activity period. In this study, we investigated if darbepoetin alfa could attenuate MDA, NO and GSH levels and histological injury score in reperfused rat testis.
MDA is the end product of lipid peroxidation and is a well-known parameter for determining the raised free radical formation in reperfused tissue . Level of MDA significantly increased in T/D group when compared to the sham group. Treatment with darbepoetin alfa significantly decreased MDA levels.
NO is an important mediator of cell death either through apoptosis or necrosis, depending on the intensity and the duration of injury . Our study showed that I/R injury due to testicular torsion and detorsion elevated NO production in ipsilateral testicular tissue. Treatment with darbepoetin alfa before reperfusion significantly decreased the concentration of testicular NO level when compared with the I/R group.
The tissue is able to counteract the oxidative stress stuation derived from a rapid ROS generation through the antioxidant defenses; the GPx system appears to constitute the first line of defense, since there is a depletion of reduced glutathione [44, 45]. We observed that GSH level was decreased in T/D group compared with sham group. Although the GSH level in the treatment group was lower than in the sham group, it was significantly higher when compared to T/D group.
Histopathologic examinations were in accordance with elevated ipsilateral testicular tissue MDA and NO levels. Mean testicular injury score increased in T/D goup. Treatment group showed lesser histopathologic damage than T/D group.
According to our biochemical and histological results, treatment with darbepoetin alfa decreased damage in ipsilateral testis caused by I/R.
There are concerns such as hypertension and thromboembolic complications about erythropoietic therapy for patients who are not anemic. But, a few doses administration of rHuEPO for prevent tissue injury is not likely to be harmfull .
The effect of unilateral torsion on contralateral testes is controversial. In our study, testes were harvested after 2-hour torsion and 4-hour detorsion. Biochemical analyses and histologic examination showed no damage in contralateral testes. We suggest in early periods of detorsion, there is no damage to contralateral testes after unilateral testicular T/D.
In conclusion, the results of the present study suggest that treatment with the long acting EPO analogue darbepoetin alfa may have protective effect on testicular I/R injury. We evaluated the early antioxdant effect of darbepoetin alfa on I/R injury. Anti-inflammatory, anti-apopitotic and long-term effects of darbepoetin alfa on testicular injury should be evaluated with later studies.