Testicular torsion is an urologic emergency occurring primarily in adolescent boys and young men, and leads to testicular injury. Prompt diagnosis and surgical intervention is necessary to restoration of testicular blood flow [1,2]. Re-institute of blood flow and oxygenation is essential to salvage ischemic tissues; on the other side, reperfusion may cause further damage paradoxically in the ischemic tissue, known as reperfusion injury [3,4]. Decrease in blood flow causes hypoxia, resulting in elevated levels of lactic acid, hypoxanthine, and lipid peroxides in ischemic tissues. Increase in blood flow after lipid peroxidation produces oxygen derived free radicals, which can cause tissue damage in reperfusion period . Enzymatic antioxidant defence systems such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) protect tissues from reactive oxygen species .
Previously, several drugs and chemicals were used to prevent reperfusion injury [7-9]. However, none of them have found routine clinical application.
Another method for preventing reperfusion injury is the gradual reperfusion (controlled reperfusion) of ischemic tissues [10-13]. Aim of this concept is reduce the reperfusion injury by use of gradually increased blood flow method instead of sudden reperfusion. Controlled reperfusion has been successfully used in clinical practice for treating patients with severe, prolonged lower-limb ischemia . This study was planned to evaluate the effects of gradual detorsion on testicular torsion model in rats.
Materials and Methods
The experimental protocol was approved by the local animal ethics review committee of our institution. Twenty one male Sprague-Dawley type rats weighing 285 to 330 g were used in this study. The animals were maintained on pellet food and water ad libitum. Rats were divided into 3 groups containing 7 rats in each. Group 1, sham operation; Group 2, sudden (conventional) detorsion; Group 3, gradual detorsion.
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 the testis was sutured to scrotum to prevent spontaneous detorsion and the scrotum closed. After 2 hours of torsion period, full detorsion was performed for 2 hours. In group 3, 360° detorsion was done for 20 minutes by fixing it to the scrotum with 5/0 silk suture after 720° torsion for 2 hours. Then the testis was done full detorsion for 100 minutes.
At the end of the experiments (fourth hour) left orchiectomy was performed to measure the tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and to perform histological examination in testes.Examination of Testicular Blood Flow
Imaging was performed with an Acuson Antares ultrasound machine (Siemens Medical Solutions, Mountain View, CA) with a 13-5 MHz linear array transducer. Both color and power Doppler techniques were used for testicular blood flow examination. The Doppler parameters were designed to maximize detection of slow flow. The vascularity in each testicular parenchyma was graded on a scale of 0 to 4 depending on the location and persistence of flow described by Coley et al. . (0 = no flow, 1 = intermittent flow in the peripheral 1/3 of the testis, 2 = constant flow in the peripheral 1/3 of the testis, 3 = intermittent flow in the central 2/3 of the testis, 4 = constant flow in the central 2/3 of the testis). Our observations show that partial torsion produces a damping of testicular arterial pulsatility in the rat model. Thus, we used time-averaged mean velocity (TAMV) as a parameter instead of peak-systolic velocity to measure arterial blood flow in testes. Multiple Doppler waveforms were obtained from at least 2 intratesticular arteries, and a mean velocity for each testis was determined.
All of the ultrasonographic examinations and measurements were performed by a single radiologist (A.B.) who was unaware of the presence and degree of testicular torsion. Measurements was performed in pretorsional period, 1 to 5 minutes before detorsion and 15 to 20 minutes after detorsion.Biochemical analysis
After weighing the tissue, homogenate, supernatant and extracted samples were prepared as described elsewhere  and the following determinations were made on the samples using commercial chemicals supplied by Sigma (St. Louis, USA). Protein measurements were analysed in homogenate, supernatant and extracted samples according to the method explained elsewhere . The tissue homogenate was used for MDA levels. The tissue supernatant was used for analysis of GSH-Px. SOD activity was assessed in the extracted sample, ethanol phase of the lyzate after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged.
The tissue MDA level was determined by a method  based on the reaction with thiobarbituric acid (TBA) at 90-100 °C. In the TBA test reaction, MDA or MDA-like substances and TBA react with the production of a pink pigment having an absorption maximum at 532 nm.
GSH-Px activity was measured by the method of Paglia and Valentine . The enzymatic reaction in the tube, which is containing following items: NADPH, reduced glutathione, sodium azide, and glutathione reductase, was initiated by addition of H2O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer.
Total (Cu/Zn and Mn) SOD activity was determined according to the method of Sun et al . The principle of the method is based on the inhibition of nitroblue-tetrazolium (NBT) reduction by the xanthine-xanthine oxidase system as a superoxide generator. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate.Histological Examination
All testes were fixed in Boins solution and embedded in paraffin blocks. Tissue sections were stained with hematoxylin&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, non-cohesive 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 testicular vascularity and TAMV values. The testicular blood flow was completely occluded by 720° rotation. Pre-torsional and post-detorsional values of testicular blood flow were not different statistically in group 2. The testicular blood flow during the 360° torsion period in group 3 was significantly different from levels of 0° and 720° torsion periods (P<0.05).
Table 2 shows the ipsilateral MDA, SOD and GSH-Px values for all groups. The MDA levels of testis tissues were significantly increased in the sudden detorsion group compared with the sham group (P<0.05). We found decrease of the MDA level in the gradual detorsion group, but it was not statistically significant amount compared with the sudden detorsion group. Significant decrease was found in the SOD and GSH-Px activities in the sudden detorsion group compared with the sham and gradual detorsion groups (P<0.05).
Individual and mean ipsilateral testicular injury scores are shown in Table 3. The rats in sham group had essentially normal testicular architecture (Fig 1a). The highest histological grade (mean; 2.42 ± 0.53) was determined in sudden detorsion group (Fig 1b). We found decrease of the mean injury scores (2.14 ± 0.37) in gradual detorsion group (Fig 1c), but it was not statistically significant amount.Discussion
It is believed that, reperfusion injury is related to the sudden availability of large amounts of molecular oxygen in ischemic tissue leading to the production of oxygen-derived free radicals which is cause extensive tissue injury . Truly, it is demonstrated that blood flow of some tissues such as testis [22-23], cremaster , kidney , bowel , limb  and myocard  are higher in the initial reperfusion phase than the level of pre-ischemic period, that is overflow. If oxygen-derived free radicals indeed are a major cause of reperfusion injury in the ischemic tissue, it may be possible to reduce injury by decreasing the accessibility of molecular oxygen at the time of reperfusion . Thus, gradual reperfusion may help to protection of ischemic tissues by reducing the sudden burst of available oxygen at the time of reperfusion . Reduction of oxygenized blood flow on initial reperfusion is aimed at counteracting the known biochemical changes that occur with I/R, such as the break down of aerobic metabolism, metabolic acidosis, an increase in intracellular calcium, and development of oxygen-derived free radicals with the onset of reperfusion . The concept of controlled (gradual) reperfusion was first described in cardiac surgery and thereafter clinically applied to peripheral vascular surgery and lung transplantation [26-28]. Vinten-Johansen et al. reported that gradual restoration of coronary blood flow during the initial 30 minutes of reperfusion to achieve a "gentle reperfusion" reduced infarct size and post-ischemic myocardial blood flow defects in a model of ischemia reperfusion . Duranni et al. demonstrated that gradually increasing blood flow in reperfusion phase decreases ischemic injury in ipsilateral and contralateral rat kidneys .
We have used the partial detorsion method for provide gradual reperfusion in testicular torsion model. Sonda et al demonstrated that the 720° rotation completely occluded blood flow to the rat testis during time of torsion . Torsion of 360° produces a moderate ischemic effect  and for a period of 12 hours causes no observable gross or microscopic changes in the testis . For this reason we chose incomplete (360°) detorsion after 720° torsion for avoid sudden reperfusion and oxygenation of the testis in this study.
As there is evidence that most additional tissue injury caused by uncontrolled (sudden) reperfusion occurs during the first 20 to 30 minutes, we chose a 20 minutes interval for incomplete detorsion (360°) [14,32].
There is no agreement about status of the testicular blood flow after detorsion. Nyugen et al. demonstrated that testicular blood flow was higher than pre-torsional level in first hour of detorsion [22,23]. These blood flow changes are correlated with histopathologic abnormalities . Lysiak et al demonstrated that mean microvascular blood flow and mean interstitial PO2 values return basal levels within 30 minutes after detorsion . Turner et al. showed that testicular blood flow reach to pre-torsional level in 4 hours . In this study, there was no significant difference in the testicular blood flow between pre-torsional period and 15th-20th minutes post-detorsion period in group 2.
Ischemia with consecutive reperfusion (reperfusion after ischemia) causes oxidative stress, which is characterized by an imbalance between reactive oxygen species and antioxidative defense system[3-8,8]. MDA is the end product of lipid peroxidation and is a well known parameter for determining the increased free radical formation in post-ischemic tissue . The MDA levels of testis tissues were significantly increased in the sudden detorsion group compared with the sham group. We found decrease of the MDA level in gradual detorsion group, but it was not statistically significant amount. Histological examinations were in accordance with the elevated testicular tissue MDA levels. Significant decrease was found in the SOD and GSH-Px activities in the sudden detorsion group compared with the sham and gradual detorsion groups.
The exact mechanisms of protective effect of the gradual reperfusion in I/R injury are not fully known. It has been implied that the sudden burst of radicals following abrupt reperfusion may be avoided by this method. Thus the generation of radicals (reactive oxygen species) may be spread over a longer period of time, slowing the rate of production enough to allow protection by endogenous scavenging mechanisms [13, 36]. Indeed, our results demonstrated that gradual detorsion of testis causes increase in SOD and GSH-Px activities when compared with sudden detorsion group. Gabig et al. showed that transient acidosis inhibits neutrophilic activity and this may diminish superoxygen radical production during gradual reperfusion . In addition, gradual blood reperfusion may cause a decrease in testicular temperature and this may decrease reperfusion injury by an effect similar to cold ischemia. Power et al. demonstrated that hypothermia attenuates reperfusion injury in rat testis .
Another similar surgical approach to decrease reperfusion injury is "ischemic postconditioning" which is a variation of "controlled reperfusion". This phenomenon is brief (short) intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia. Suggested mechanisms of protection include a reduction in neutrophil accumulation and decreased endothelial dysfunction, attenuation of oxidative stress, a reduction in apoptotic cell death, and attenuation of mitochondrial calcium accumulation .
"The mentioned mechanisms of protection include a reduction in neutrophil accumulation, decrease endothelial dysfunction, attenuation of oxidative stress, decline in apoptosis, and decrease of intracellular calcium overload."
In this study, the effect of gradual reperfusion to the ischemic rat testis was studied to ascertain its effects on the reduction of I/R injury. Gradual blood flow was provided by partial detorsion of torsioned testis. We found that testicular blood flow decreases moderately during 360° torsion period. This method causes arterial and venous partial obstruction, and has difference from classical gradual reperfusion which accomplishes by arterial micro-clamp. Venous outflow obstruction may theoretically cause increase of interstitial pressure and edema in testicular tissue. However, we did not determine any difference histologically between sudden and gradual detorsion groups in terms of edematous condition.
In the light of our biochemical and histopathological findings, we can say that gradual detorsion has a tendency to decrease testicular I/R injury in the rat model. The gradual detorsion is an easy surgical manipulation and combining this surgical technique with some other pharmacologic treatment modalities may yield better results. We have shown early effects of gradual reperfusion on ischemic testis in this study. To determine optimal duration of gradual reperfusion and optimal degree of detorsion, and long-term effects of this method warrant further investigations.References
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