MISCELLANEOUS The Protective Effects of Pulsed Magnetic Field and Melatonin on Testis Torsion and Detorsion Induced Rats Indicated by Scintigraphy, Positron Emission Tomography/Computed Tomography and Histopathological Methods Serdar Savas Gül1*, Serkan Gürgül2, Murat Uysal3, Fikret Erdemir4 Purpose: The aim of the present study was to show the protective effect of pulsed magnetic field (PMF) appli- cation and melatonin administration on damage in testis in a one-sided torsion detorsion induced rat model using testicular scintigraphy with 99mTc pertechnetate, PET/CT with 18F-FDG and histopathological methods. Materials and Methods: Sixty male rats were used in the study; 30 rats were randomly divided into five groups for one day applications of sham control, torsion, melatonin, pulsed magnetic field (PMF) and melatonin plus PMF. Similarly, the other 30 rats were divided into the same five groups (n = 6) for one week treatment, but the animals were sacrificed after one week. Rats were exposed to 50 Hz, 1 mT PMF for two hours. PET/CT with 37 MBq 18F-FDG and testicular scintigraphy with and 37 MBq 99mTc pertechnetate examinations were carried out, and testicular tissue was examined using histopathological methods. Results: In one day treatment, melatonin administration significantly increased perfusion and glucose metabolism compared to torsion group (P < 0.01). Perfusion and glucose metabolism was also higher in the PMF and mela- tonin plus PMF groups than torsion group (P < 0.01). In one week treatment, melatonin administration resulted in a significantly higher perfusion and glucose metabolism rates compared to torsion group (P < 0.01 and P < 0.001, respectively). In addition, perfusion and glucose metabolism significantly increased in PMF and melatonin plus PMF groups compared to torsion group (P < 0.01 and P < 0.001, respectively). Furthermore, caspase-3 immuno- reactivity and pathological changes increased in the torsion group (P < 0.05). Melatonin and melatonin plus PMF treatment reduced the rate of immunoreactivity and pathological findings compared to the torsion group (P < 0.05). Conclusion: According to these results it can be concluded that PMF application has a therapeutic benefit as effective as melatonin administering. In addition, it was indicated that PET/CT with 18F-FDG and testicular scin- tigraphy with 99mTc pertechnetate could be efficiently used in determining the treatment efficiency in testicular torsion. Keywords: positron emission tomography/computed tomography; pulsed magnetic field; scintigraphy; testis torsion INTRODUCTION Testis torsion is a surgical condition with acute on-set especially in childhood and young adolescent men, and it needs to be diagnosed and treated. It refers to inhibition of blood flow into testis and associated structures depending upon twisting of spermatic cord around its axis. It has an incidence rate of 1/4,000 in men younger than 25 years of age.(1) The main treatment in testis torsion is manual or surgical detorsion of testis. Even with successful intervention, testis atrophy and in- fertility develops in 40-60% of these patients.(2) The major pathology after testis torsion is testis is- chemia. In testis where developed torsion was detor- sioned, injury continues after reperfusion. At start, is- chemic damage occurs in testis, and during restoration of blood flow reperfusion damage may also occur.(3) Free oxygen radicals that formed during reperfusion 1Gaziosmanpasa University, Faculty of Medicine, Department of Nuclear Medicine, Tokat 60100, Turkey. 2Gaziantep University, Faculty of Medicine, Department of Biophysics, Gaziantep 27000, Turkey. 3Gaziosmanpasa University, Faculty of Medicine, Department Anatomy, Tokat 60100, Turkey. 4Gaziosmanpasa University, Faculty of Medicine, Department of Urology, Tokat 60100, Turkey. *Correspondence: Gaziosmanpaşa University, Faculty of Medicine, Department of Nuclear Medicine, Tokat 60100, Turkey. Tel: +90 356 2120045, Fax: +90 356 2120045, E-mail: gopnukleertip@gmail.com. Received February 2018 & Accepted May 2018 ID: 4404 causes testis injury.(4) The main damage of free oxy- gen radicals to testis is in the form of cell viability loss through lipid peroxidation and break of membrane in- tegrity.(5) Since sperms have high levels of unsaturated fatty acids in their plasma membranes, they are highly sensitive to oxidative stress and especially to lipid per- oxidation. Accordingly, loss of sperm motility due to negative effect of free oxygen radicals on sperms via this mechanism, especially adenosine triphosphate loss within cell and axoneme damage, loss of sperm viabil- ity and morphological changes impairs sperm capacity and acrosome reaction.(6,7) Many studies in literature established torsion models and investigated histolog- ical changes in serum and tissue levels. Antioxidants were also extensively studied for treatment purposes. It is known that antioxidants act in a way to counterbal- ance the effect of free oxygen radicals and block their negative effects. Many antioxidant agents, mainly me- latonin, have been used to treat the oxidative damage from free oxygen radicals.(8-10) Despite these classical studies, magnetic field applica- tion was not investigated in testis torsion and, except for diagnostic purposes, nuclear medicine imaging methods were not commonly used to determine the changes and efficiency of treatments using the same parameters. The aim of the present study was to evaluate the protective effect of pulsed magnetic field (PMF) and melatonin applications on damage during and after one sided testis torsion using positron emission tomography/computed tomography (PET/CT) with (18) Flour fluoro-2-deoxy- D-glucose (18F-FDG) examination, testicular scintigra- phy with (99m) Technesium (99mTc) pertechnetate and histopathological methods. MATERIALS AND METODS This study was conducted with permission of the Local Ethics Committee (2014 HADYEK-50). All experi- ments and protocols described in the present study were performed in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted by Nation- al Institutes of Health (U.S.). All procedures were ap- proved by the Medical Faculty Experimentation Ethics Committee of Gaziosmanpaşa University. Study Population Sixty male Wistar-Albino rats, 90 days of age and weighing 200–250 g each, were used in this study. The rats were raised under a 12h light/12h dark cycle (light from 07:00 to 19:00) in quiet rooms with 22–24°C am- bient temperature and they had free access to standard rat nutrients and purified drinking water ad libitum. For one day treatment groups, 30 rats were divided into five groups; sham control, torsion, melatonin, PMF and me- latonin plus PMF. Similarly, for one week group, other 30 rats were divided into the same five groups (n = 6). In sham control group, testes were taken out via scro- tal cutting, put back without any other procedure, and the skin was appropriately closed. In torsion group, no treatment was carried out after torsion procedure. After the experiment, rats were sacrificed using intraperito- neal administration of high dose pentobarbital (100 mg/ kg). Procedures Testis Torsion and Detorsion In the day of experiment, rats were taken to operation room. After anesthesia using intraperitoneal adminis- tering of 50 mg/kg ketamine (Ketalar®, Parke- Davis) and 10 mg/kg xylazine (Alfazyne®, Alfasan), rats were stabilized in operation table. For rats which would be subjected to torsion, detorsion and sham operations, standard scrotal incision was realized after local anes- thesia application to left side ilioinguinal area carried out using (2% Citanest®, Astra Zeneca). In all groups, left testis was taken out after scrotal incision. In testis torsion groups, torsion was carried out via twisting left Table 1. Statistical data to left/right testis ratio from one day treatment groups (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. ‡p < 0.01, *p < 0.001 and €p < 0.01 compared with torsion. One Day Groups N Mean Std. Deviation Std. Error 18F FDG glucose metabolism Sham 6 0.998‡ 0.024 0.010 Torsion 6 0.754 0.075 0.030 Melatonin 6 1.088‡ 0.085 0.034 PMF 6 1.065‡ 0.107 0.044 Melatonin plus PMF 6 1.142‡ 0.153 0.062 Total 30 1.009 0.165 0.030 99mTc Pertechnetate testicular perfusion Sham 6 1.001* 0.051 0.020 Torsion 6 0.750 0.054 0.022 Melatonin 6 1.171€ 0.116 0.047 PMF 6 1.311€ 0.317 0.129 Melatonin plus PMF 6 1.175€ 0.159 0.065 Total 30 1.082 0.252 0.046 Figure 1. Schematic representation of work plan (Abbreviations: PMF, pulsed magnetic field; 99mTc, (99m)Technesium; 18F FDG, (18)Flour fluoro-2-deoxy-D-glucose). Figure 2. Testis torsion and detorsion model images. Effects of magnetic field and melatonin on torsion and detorsion-Gül et al. testis 720° clockwise. In order to maintain the torsion, testis was fixed to scrotum at tunica albuginea via silk suture after twisting. After the operation, incision was closed. After two hours of torsion, left testis was detor- sioned. Then, tissues were closed appropriately. All details about the study were shown in Figure 1. Testis torsion and detorsion model was shown in Figure 2. Melatonin Treatment In one day melatonin group, a single dose of 40 mg/kg melatonin was administered intravenously, while in one week melatonin group a daily 40 mg/kg of melatonin was administered intravenously. PMF Application Protocol Before PMF application, rats were acclimated to their environment for a week. Habituation to the treatment conditions was accomplished by placing the rats in a restrainer at least three times for 30 min. PMF was con- ditioned using Helmholtz coil apparatus in a earthed shielded Faraday cage (90x90x55 cm3) (Figure 3A). Coils of 60 cm diameter and 30 cm clearance were constructed by electrically and thermally insulated cop- per wire of 2.5 mm diameter with 50 turns. Resistance was 0.78 Ω and inductance was 8.8 mH. Coils were connected to a signal generator (ILFA Electronic Ltd., Adana, Turkey) to produce magnetic field with a peak amplitude of 1 mT. The peak value of the magnetic field was measured using a gaussmeter equipped with a Hall-effect probe (FW Bell 5180, Pacific Scientific OECO, Milwaukie, OR) (Figure 3B). The time-varying magnetic field consisted of quasi-triangular waveform, a rise time of 0.3 ms and a fall time of 9.7 ms. The maximum induced electrical field between the coils was 0.25 V/m calculated based on Faraday’s law. After two- hour testis torsion and detorsion, rats were placed in a 26x17x13 cm restrainer and housed in the center of the Helmholtz coils. The horizontal, uniform, and homoge- nous PMF (1 mT; 15 Hz) was applied. Rats (F-MF;M- MF) were then exposed to a horizontal and sinusoidal MF (50Hz,1mT) for two hours, while the control rats (FC; MC) were kept at the same laboratory conditions as intact groups. The ambient geomagnetic field was recorded as 50 μT. No significant temperature change was detected during the experiments between two ac- tivated Helmholtz coils. The temperature (20–22 °C) and humidity (40–60%) were monitored continuously throughout the experiment. All PMF applications were carried out at the same hours of the day (9:00–11:00 a.m.). Sham treatment was performed under the same environmental conditions using another apparatus in- cluding only Helmholtz coils in a Faraday cage. The polycarbonate cages were cleaned after every test ses- sion to avoid any biochemical effects. After two hours of PMF exposure, scintigraphic evaluation was made on rats. PMF application was carried out by two inves- tigators (SG and MU) who were unaware of the study groups. Nuclear Medicine Imaging All groups had testicular perfusion imaging using 99mTc pertechnetate testicular scintigraphy and glucose metab- Figure 3. A) Schematic drawing of PMF exposure system. B) Magnetic field amplitude with Hall-effect probe. Figure 4. Testicular scintigraphy using 99mTc pertechnetate; one day sham control (1a), torsion (1b), melatonin (1c), PMF (1d), me- latonin plus PMF (1e), and one week sham control (2a), torsion (2b), melatonin (2c), PMF (2d), melatonin plus PMF(2e). Figure 5. Examination of glucose metabolism using PET/CT with 18F-FDG; one day sham control (1a), torsion (1b), melatonin (1c), PMF (1d), melatonin plus PMF (1e), and one week sham control (2a), torsion (2b), melatonin (2c), PMF (2d), melatonin plus PMF (2e). Effects of magnetic field and melatonin on torsion and detorsion-Gül et al. olism imaging using PET/CT with 18F-FDG. 18F-FDG PET/CT was performed to assess glucose metabolism in bilateral testes. Rats received intravenous injections of 1 mCi (37 MBq) of FDG 1 hour before the acquisi- tion of the PET/CT image. PET imaging was performed using a combined PET/CT scanner (Siemens Biograph 2 PET/CT, New Jersey, USA). Attenuation correction of PET images with the CT data was performed. Right after CT data acquisition, a standard PET imaging protocol was taken from the cranium to the mid-thigh with an acquisition time of 3 min/bed in 3-dimensional mode. CT and PET images were matched and fused into transaxial, coronal and sagittal images. The testicular scintigraphy was performed with intrave- nous injection of 1 mCi (37 MBq) of 99mTc pertech- netate. The perfusion phase (5 second/frame, for 2 minutes) and static imaging (5 minute anterior view, immediately after dynamic imaging) using a dual head variable angle nuclear gamma camera (Symbia Sie- mens, Hoffman Estates, USA) equipped with a low energy high resolution collimator and 140 keV 99mTc photopeak) demonstrated arterial flow in the bilater- al testes. Images obtained were evaluated. Region of interest (ROI) were drawn in both testis regions after imaging and left/right testis activity rates were deter- mined using semi-quantitative method. Image analysis was carried out by a researcher (SSG) who was not in- formed about the source of the images. Histopathological Examination During the torsion period, color changes due to edema and venous stasis which were macroscopic results of ischemia were observed in all testes to which surgi- cal procedure was applied. Histopathological evalua- tions carried on tissue samples were recorded in forms prepared to this aim. There were normal distributions among all groups. Left and right testis tissues in all groups were studied for glucose metabolism and per- fusion. In addition, histopathological evaluations were carried out. Testis tissue samples were taken and submersed in 10% formaldehyde solution. These tissues underwent routine histological procedures and were embedded in paraffin. Five and twenty-five micron dissections were taken from these paraffin embedded tissues using rotary microtome. Histopathological evaluations were carried out after hematoxylin eosin staining of testis tissue sam- ples. Numbers of spermatogonium and spermatocyte, and volume and diameter of seminiferous tubules were determined. In addition, the Johnsen score was used to evaluate the morphological damage of testis tissue as a result of testis torsion. Johnsen score was determined and damage in seminiferous tubules was assessed for the purpose of evaluation of spermatogenesis. The Johnsen scoring system is principally based on the pro- gressive degeneration of germinal epithelium and a suc- cessive loss of the most mature cell types during testic- ular damage evaluated using the following categories: 1 (No cells at all within the tubules), 2 (No germinal cells, only Sertoli cells), 3 (Only spermatogonia), 4 (No spermatozoa and spermatids, less than 5 spermatocytes, but numerous spermatogonia per cross-section), 5 (No spermatozoa and spermatids, numerous spermatocytes and spermatogonia), 6 (No spermatozoa, 5 ± 20 sper- matids, numerous spermatocytes and spermatogonia per cross-section), 7 (No spermatozoa, numerous sper- matids, spermatocytes and spermatogonia), 8 (Less than 5 ± 10 spermatozoa per tubular cross-section), 9 (Numerous spermatozoa, germinal epithelium disor- ganized with sequestration of germinal cells, tubular lu- men was obturated) and 10 (Complete spermatogenesis, numerous spermatozoa, germinal epithelium of regular height, tubular lumen of normal diameter).(11) Finally, H-score of caspase-3, an enzyme active in apoptosis, was carried out immunohistochemically to determine the level of this enzyme.(12) Evaluation of the immuno- histochemical labeling was performed using H-Score analyses as previously described. Caspase3 immuno- reactivities were semi-quantitatively evaluated using the following categories: 0 (no staining), 1+ (weak but detectable staining), 2+ (moderate or distinct staining), Figure 6. Microscope images of hematoxylin eosin stained tissues in one day sham control (a), torsion (b), melatonin (c) and pulsed magnetic field (d) groups Figure 7. Microscope images of caspase-3 stained tissues in one day sham control (a) and torsion (b) groups. Effects of magnetic field and melatonin on torsion and detorsion-Gül et al. and 3+ (intense staining). For each tissue, an H-score value was derived as follows: First, the sum of the per- centages of cells that stained at each intensity catego- ry was calculated, which was then multiplied by the weighted intensity of the staining using the formula as follows: H-score = ∑Pi (i+ l). In this formula, ‘i’ repre- sents the intensity scores, and ‘Pi’ is the corresponding percentage of the cells. Five randomly selected areas were evaluated under a light microscope on each slide (40x objective). Two investigators (MU and SSG), who were not informed about the type and source of the tis- sues, determined the percentage of cells at each inten- sity within these areas at different times. The combined average score of both observers was used. Statistical Analysis All data were analyzed by IBM SPSS Statistical Soft- ware version 18.0. The normal distribution of data and the homogeneity of variance were evaluated using the Kolmogorov Smirnov test and the Levene test, respec- tively. Because of the normal distribution in the study groups, One-way ANOVA test was used for comparing groups in terms of glucose metabolism, testicular perfu- sion, Johnsen score and H-score values, and Tukey test was used as post-hoc test. Results of the experiments were given as mean ± standard deviation and P < 0.05 was considered statistically significant. RESULTS A. One-day Groups Evaluation of “left testis/right testis” perfusion with tes- ticular scintigraphy using 99mTc pertechnetate Findings of testicular scintigraphy in different groups were shown in Table 1 and Graphic 1. There was a significant decrease in perfusion based on scintigraphy between torsion and sham groups (P < 0.001). Perfu- sion of left testis was significantly impaired compared with the right one. Melatonin, PMF and melatonin plus PMF application significantly increased the perfusion compared with that in the torsion group (P < 0.01), but this increase was more prominent in the PMF group. When compared with the sham group, it was apparent that melatonin, PMF and melatonin plus PMF groups had an increase in perfusion, but these differences were not statistically significant (P = .08, P = .09 and P = .09, respectively). Images from one day treatment were given in Figure 4. Evaluation of “left testis/right testis” glucose metabo- lism with PET/CT using 18F-FDG Results of PET/CT in different groups were shown in Table 1 and Graphic 1. A significant decrease was Graphic 1. Comparison of the results from one day treatment groups (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. ‡p < 0.01, *p < 0.001 and €p < 0.01 compared with torsion. Graphic 2. Comparison of the results from one week treatment groups (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. §p < 0.05, ‡p < 0.01, ɸp < 0.01 and *p < 0.001 compared with torsion; #p < 0.01, ¥p < 0.05 and £p < 0.01 compared with sham; ⸸p < 0.01compared with melatonin plus PMF. Graphic 3. Comparison of one day treatment groups using Johnsen score after histopathological examination (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. Graphic 4. Comparison of one day treatment groups using H-score after histopathological examination (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. Effects of magnetic field and melatonin on torsion and detorsion-Gül et al. detected between torsion and sham groups in glucose metabolism assay using 18F-FDG PET/CT (P < 0.01). It was revealed there was a decrease in glucose metabo- lism of left testis compared with the right one after one day treatment period and the difference was significant (P < 0.01). Glucose metabolism was significantly high- er in melatonin, PMF and melatonin plus PMF groups than in torsion group (P < 0.01), but this increase was more prominent in the melatonin plus PMF group. There was no significant difference among melatonin, PMF, melatonin plus PMF and sham groups for glucose metabolism. PET/CT images from one day treatment were given in Figure 5. B. One Week Treatment Groups Evaluation of “left testis/right testis” perfusion with tes- ticular scintigraphy using 99mTc pertechnetate Findings of testicular scintigraphy in different groups were shown in Table 2 and Graphic 2. A significant decrease was observed in perfusion based on scinti- graphic method between torsion and sham groups (P < 0.01). Perfusion rates in melatonin, PMF and mela- tonin plus PMF groups, on the other hand, were signif- icantly higher than that in torsion group (P < 0.001). Melatonin, PMF and melatonin plus PMF application eliminated the perfusion loss and led to a significant in- crease in perfusion (P <0.01). Additionally, melatonin plus PMF group had perfusion increase compared with the melatonin and PMF groups (P < 0.01). Evaluation of “left testis/right testis” glucose metabo- lism with PET/CT using 18F-FDG Results of PET/CT in different groups were shown in Table 2 and Graphic 2. A significant decrease was determined in glucose metabolism via PET/CT using 18F-FDG between torsion and sham groups (P < 0.05). After one week of treatment period, left testis metab- olism was shown to be significantly lower than right testis metabolism. Glucose metabolism in melatonin, PMF and melatonin plus PMF groups was significantly higher than that in torsion group (P < 0.01), but this in- crease was more prominent in the melatonin and mela- tonin plus PMF groups. When compared with the sham group, it was apparent that melatonin and melatonin plus PMF groups had a significant increase in glucose metabolism (P < 0.01 and P < 0.05, respectively). Histological and immunohistochemical results were presented in Table 3. Histopathological analyses using hematoxylin eosin staining of one day treatment group revealed that cell damage was more prominent in tor- sion group compared to sham group and melatonin plus PMF group (P < 0.05) (Figure 6). Caspase-3 staining showed a higher level of apoptosis in torsion group than in sham group (P < 0.05) (Figure 7). Johnsen score in- dicated the cell damage in testis tissue. Johnsen score was 9.6 for sham group 2.2 for torsion group, 4.0 for melatonin group, 2.1 for PMF group and 3.9 for mel- atonin plus PMF group (Graphic 3). H-score indicat- ed the cell damage in testis tissue. H-score was 18 for sham group, 56 for torsion group, 24 for melatonin group, 49 for PMF group and 30 for melatonin plus PMF group (Graphic 4). DISCUSSION Table 2. Statistical data to left/right testis ratio from one-week treatment groups (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. §p < 0.05, ‡p < 0.01, ɸp < 0.01 and *p < 0.001 compared with torsion; #p < 0.01, ¥p < 0.05 and £p < 0.01 compared with sham; ⸸p < 0.01compared with melatonin plus PMF. One Week Groups N Mean Std. Deviation Std. Error 18F FDG glucose metabolism Sham 6 0.997§ 0.252 0.103 Torsion 6 0.630 0.193 0.079 Melatonin 6 1.442‡, # 0.144 0.059 PMF 6 1.199‡ 0.270 0.110 Melatonin plus PMF 6 1.427‡, ¥ 0.228 0.093 Total 30 1.139 0.355 0.640 99mTc Pertechnetate testicular perfusion Sham 6 0.994ɸ 0.059 0.024 Torsion 6 0.724 0.143 0.058 Melatonin 6 1.245*, £, ⸸ 0.135 0.055 PMF 6 1.254*, £, ⸸ 0.114 0.046 Melatonin plus PMF 6 1.413*, £ 0.026 0.010 Total 30 1.126 0.264 0.048 Groups N Mean Std. Deviation Johnsen score Sham 6 9.8 0.3 Torsion 6 2.3*, # 0.6 Melatonin 6 4.1* 0.9 PMF 6 2.2*, # 0.5 Melatonin plus PMF 6 3.9* 0.7 Total 30 4.5 0.6 H-score Sham 6 18 3 Torsion 6 56*, # 7 Melatonin 6 26 6 PMF 6 48*, # 8 Melatonin plus PMF 6 30* 5 Total 30 36 6 Table 3. Statistical data to Johnsen score and H-score from one day treatment groups (Abbreviations: PMF, pulsed magnetic field). One-way ANOVA test was used for comparing groups. *p < 0.05 compared with sham group; #p < 0.05 compared with melatonin and melatonin plus PMF group. Effects of magnetic field and melatonin on torsion and detorsion-Gül et al. Testis torsion is known as ischemia-reperfusion damage in clinical practice. The diagnosis and treatment of tes- tis torsion is critical for the maintaining of fertility since inappropriate treatments could lead to male infertility. Experimental testis torsion lowers blood flow without causing a significant change in central blood pressure, and it results in impairment of apoptosis, testicular atro- phy and spermatogenesis in germ cells.(13) Following a torsion of more than four hours, blood flow in ipsilater- al testis could not be completely restored even 24 hours after detorsion.(14) During ischemia, germ cell death takes place due to low level of oxygen to meet met- abolic needs, cellular energy stores are depleted, and toxic metabolites accumulate.(15) Experimental studies revealed that testis necrosis develop within two hours in arterial blocking and within six hours in venous block- ing.(16) The testes which are not treated within 12 hours after the development of symptoms could be lost.(17) The aim in the present study was to create an acceptable ischemia in torsioned testes before a treatment. In addi- tion, torsion and the experiment had to be ended before irreversible damage occurred in testes. Therefore, du- ration of ischemia was two hours. Experimental stud- ies showed that ischemic damage occurred as a result of testis torsion and was associated with number and duration of torsion. In addition, it was also shown that blood flow ceased irreversibly in 720° torsion of tes- tes and a complete ischemia developed. Nevertheless, experimental studies generally use testis torsion angles between 360° and 720°. In addition, left testis torsion is more commonly encountered since left testis has a longer spermatic cord than that of right testis.(18) There- fore, a 720° torsion-detorsion model of left testis was used in the present study. Oxidant and antioxidant mechanisms are in a balance state in body, and break down of this balance in fa- vor of oxidant mechanisms result in tissue damage. (19) In the case of a torsion, detorsion is classical way of treatment, but detorsion is known to cause a more comprehensive damage in testis through oxidative damage due to reperfusion. As a result of reperfusion of ischemic tissue, toxic free oxygen radicals such as nitric oxide, superoxide anions, hydrogen peroxide and hydroxyl radical form.(20) Free oxygen radicals are short half-life chemical compounds and they consist of one or more uncoupled electrons, which render them to be unstable and quite reactive.(21) In order to be stable, free oxygen radicals attack lipids, amino acids and nucleic acids. Although in normal conditions free oxygen rad- icals play roles in cell differentiation, sperm capacita- tion, acrosome reaction and maintaining the fertility, their high levels due to various stresses negatively af- fect cells. Protein, carbohydrate, nucleic acid and lipid components of cells are potential targets of free oxygen radicals. As a result of their impact on abovementioned cell components, free oxygen radicals that increase as secondary to oxidative damage leads to negative con- sequences such as inflammation, apoptosis, breakdown of cell membrane integrity, fibrosis and proliferation. It has been reported in literature that oxidative damage play role in pathogenesis of arthritis, cancer, diabetes mellitus, carious infections, central nervous system dis- eases, cardiac diseases and testis torsion.(22-26) Testis torsion model has shown that oxidative damage occurs within hours or even within minutes following the perfusion. It has also been revealed in these mod- els that levels of antioxidant enzymes decreased while free oxygen radicals increased.(27) Free oxygen radicals formed during testicular reperfusion activate leucocytes and cause them to adhere to vein endothelium, conse- quently impairing blood circulation. Lack of restoration of perfusion in some of capillaries after ischemia/rep- erfusion is called no-reflow phenomenon.(28) Similar- ly, we established ischemia/reperfusion in the present study and examined testis perfusion and glucose me- tabolism. Testicular torsion–detorsion generates ROS that cause apoptosis, resulting in lipid peroxidation and metabolic alterations. It is known that the formation of intracellu- lar ROS or the depletion of cellular antioxidants may re- sult in apoptosis.(29) Apoptosis is a form of programmed cell death characterized by DNA fragmentation, cyto- plasmic shrinkage, membrane changes, and cell death without damage to neighboring cells. Caspase-3, also known as the primary executioner caspase, is respon- sible for morphological changes of apoptosis.(30) The ischemia/reperfusion (I/R) phenomenon occurs in tes- ticular torsion–detorsion in which torsion comprises the ischemic period, whereas detorsion comprises reperfu- sion injury.(10) In particular, reperfusion injury results in anoxia, leading to the generation of large quantities of ROS, pro-inflammatory cytokines and lipid perox- idation, followed by activation of the apoptosis path- way which causes even severe ischemic tissue damage. (31) In our study, it was observed that caspas-3 activities increased significantly in testis of rats subjected to tes- ticular torsion–detorsion. It was found that this increase aggravated testicular damage. It was also noticed that melatonin, PMF and melatonin plus PMF application significantly increased the perfusion compared with that in the torsion group. Therefore, reperfusion in- jury resulting in an increase in perfusion may be the main reason why melatonin and PMF treatments did not show a significant effect in terms of apoptosis. In addition, it supports our assumption that melatonin is more successful than PMF against apoptosis that occurs in the oxidative damage caused by reperfusion injury. As we have shown in this study, the antioxidant effect of melatonin may have demonstrated a better improve- ment in apoptosis, possibly by showing more protective efficacy than PMF against increased oxidative damage after reperfusion. It is well known that the general approach to decrease or eliminate the effects of free oxygen radicals occurring as secondary to oxidative damage is the use of antiox- idants. In this context, effects of antioxidants on some pathologies in various tissues such as atherosclerosis, hypertension, diabetes mellitus, renal disease, ulcera- tive colitis, chronic obstructive pulmonary disease and testis torsion were investigated. For this purpose, vita- min E, melatonin, retinol, β-carotene, omega-3, resver- atrol, allopurinol, N-acetylcystein, zinc, caffeic acid, vitamin C, coenzyme Q10 and melatonin were used as antioxidant agents in various studies.(32-35) Melatonin is known as the strongest antioxidant agent to reduce testicular damage after testis torsion. Melatonin is produced in pituitary gland with a circadian rhythm. It has been shown to have an antioxidant effect both directly by eliminating free oxygen radicals and indi- rectly by elevating the level of antioxidant enzymes. In addition, melatonin has an inhibitory effect on nitric oxide synthesis. Melatonin has been reported to prevent lipid peroxidation in many organs and tissues, not only in testes, and to protect against the effect of oxidative Effects of magnetic field and melatonin on torsion and detorsion-Gül et al. damage.(27) In this context, Erdemir et al. studied the effect of melatonin on antioxidants in systemic circu- lation after one sided testis torsion in rats. They admin- istered 50 mg/kg melatonin to a group rats after 720° of torsion for two hours and measured blood levels of malondialdehyde, superoxide dismutase, protein car- bonyl and nitric oxide in rats. Their results showed that melatonin significantly decreased the levels of antioxi- dant enzyme and lipid peroxidation products of torsion and brought them to levels of control group.(36) Sim- ilarly, melatonin administration lowered the levels of reactive oxygen radicals and minimized oxidative stress in the present study. Further, efficiency of early and late administration of melatonin was investigated. The PMF are electromagnetic stimulation which are in 30-300 Hz interval and they are considered not to have any adverse or harmful effects because of their low energy levels. They are assumed to assist ther- apies through their ionic activities and accelerating blood flow. Effects of electromagnetic fields on living organisms have been investigated since 1950s. Very low level of electricity is conducted to tissues and there are studies reporting its therapeutic and regulatory ef- fects. These effects basically involve cell division rate, mRNA and protein synthesis levels, permeability of cell membranes, changes in transfers of Ca+2, Na+, K+ ions, and as a result, increases in micro blood circula- tion. These changes affect both electrical and metabolic behaviors of cell. In addition, they directly or indirectly influence melatonin production, and change daily me- tabolism and hormone production of organism. Electro- magnetic fields of certain frequencies and amplitudes change the behaviors of T-lymphocyte cells and affect cytotoxicity.(37,38) Kumar et al. showed the therapeutic effect of pulsed magnetic field application on testicular function.(39) In the present study, whether PMF appli- cation would be beneficial in testis torsion was investi- gated. In addition, early and late period effects of PMF application were studied. Testicular scintigraphy using 99mTc pertechnetate is a nuclear medicine imaging technique commonly used for especially acute testis torsion. It gives informa- tion on perfusion in torsioned testis.(40-42) PET/CT with 18F-FDG are used to evaluate glucose metabolism lev- els of cells. This technique is frequently used especially in oncology patients for diagnosis, staging, re-staging and evaluation of response to therapy.(43,44) Testicular scintigraphy using 99mTc pertechnetate and PET/CT with 18F-FDG techniques were used together in the present study, and thus perfusion and glucose metabo- lism in testis tissue were evaluated simultaneously. Numerous studies that have been conducted so far have shown that damage due to testis torsion cannot be completely resolved and there is not a routinely used therapeutic agent in clinical practice. In addition, al- though testis is the most easily torsioned organ in body, number of studies in the literature is not satisfactory on this issue. Therefore, experimental and clinical studies about treatment of testis torsion have been going on. Testis torsion-detorsion model was used in the present study. Protective effects of melatonin whose anti-oxi- dizing therapeutic efficiency is well known and PMF application which, to our best knowledge, has not been so far used for this aim were investigated using testic- ular scintigraphy with 99mTc pertechnetate, PET/CT with 18F-FDG and histopathological methods. It was revealed that PMF application alone improved perfu- sion and glucose use as efficiently as melatonin in one day and in one week treatment groups. Combined use of PMF and melatonin, on the other hand, increased perfu- sion only in one day treatment group, and increased glu- cose use only in one week treatment group compared to each of PMF and melatonin. In this study, we investi- gated the blood supply by testicular scintigraphy using 99mTc pertechnetate and the level of glucose use by 18F-FDG PET/CT in testicular tissue. Glucose use of testicular tissue is an active process. Simply, increasing blood supply in testis may not cause an increase in glu- cose use. Therefore, the two methods must be evaluated together. Limitations of the study: Histopathological examina- tions were not made in one-week treatment groups due to the problems experienced during the fixation of tis- sue. There is a need for a one-month treatment group work in order to better understand the effectiveness of PMF. CONCLUSIONS In conclusion, it was found that PMF application was as effective as melatonin administration both in early and late period after testis torsion. In addition, it was also shown that testicular scintigraphy with 99mTc pertech- netate and PET/CT with 18F-FDG methods could be effectively used both in diagnosis and in determining the efficiency of the therapy in testis torsion. However, randomized, prospective clinical studies are necessary to confirm our results. Limitations of the study: Histopathological examina- tions were not made in one-week treatment groups due to the problems experienced during the fixation of tis- sue. 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