Journal of Pediatric Surgery 49 (2014) 484–489
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The short term effects of resveratrol on ischemia–reperfusion injury in rat testis Esin Yuluğ a,⁎, Sibel Türedi a, Ersagun Karagüzel b, Ömer Kutlu b, Ahmet Menteşe c, d, Ahmet Alver d a b c d
Department of Histology and Embryology, Karadeniz Technical University, Faculty of Medicine, 61080, Trabzon, Turkey Department of Urology, Karadeniz Technical University, Faculty of Medicine, 61080, Trabzon, Turkey Program of Medical Laboratory Techniques, Vocational School of Health Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey Department of Medical Biochemistry, Karadeniz Technical University, Faculty of Medicine, 61080, Trabzon, Turkey
a r t i c l e
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Article history: Received 21 May 2013 Received in revised form 15 July 2013 Accepted 19 August 2013 Key words: Resveratrol Testicular torsion–detorsion Ischemia-modified albumin Rat
a b s t r a c t Purpose: The purpose of this study was to identify changes taking place in the rat testis at the 24th hour of reperfusion following testicular torsion and to evaluate the effects of resveratrol (RSV), a powerful antioxidant, in preventing these changes using novel biochemical parameters and histopathology. Methods: Eighteen adult male rats were divided into three groups: Sham-operated (S), torsion/detorsion (T/D), and T/D + RSV groups. In the T/D group, testicular ischemia was achieved by rotating the left testis 720° clockwise for 4 h. In the T/D + RSV group, 20 mg/kg RSV was administered intraperitoneally 30 min before detorsion. All rats were sacrificed 24 h after detorsion. Serum and tissue malondialdehyde (MDA) concentrations, ischemia modified albumin (IMA), total oxidative status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), and histopathological damage score were analyzed. Results: Serum MDA, IMA, TOS, and OSI levels rose significantly in the T/D group. Serum MDA and IMA values were lower in the T/D + RES groups, but not significantly. OSI and TOS values were lower in the T/D + RES group, and the difference was significant. TAS values decreased significantly in the T/D group and rose in the T/ D + RSV group, but not significantly. Ipsilateral tissue MDA values were significantly elevated in the T/D group and decreased in the T/D + RSV group, but not significantly. Apoptosis and histopathological damage increased significantly in the T/D group and decreased significantly in the T/D + RSV group. In the contralateral testis, apoptosis increased significantly in the T/D group. It decreased significantly in the T/D + RSV group. Conclusions: Our findings show that RSV had a protective effect against oxidative damage induced with a testicular T/D model, especially at the antiapoptotic and histopathological level. OSI may be a good guide to the clinical status of testicular T/D. © 2014 Elsevier Inc. All rights reserved.
Testicular torsion is a urological emergency particularly seen in neonates, children and adolescents [1,2]. At the time of testicular torsion the germinal cells are exposed to hypoxia. If torsion is prolonged, necrosis occurs in the germinal cells. This may lead to subfertility and infertility [3,4]. Testicular tissue blood flow must therefore be re-established as soon as possible. Testicular tissue blood flow is established with reperfusion. Exposure of tissue to oxygen at the time of reperfusion leads to production of reactive oxygen radicals (ROS) [5,6]. Testicular torsion and detorsion may be regarded as an ischemia/reperfusion (I/R) injury. The mechanism involved in testicular injury is not completely clear, although oxidative stress is thought to be responsible for I/R injury. Oxidative stress is the result of imbalance between excessive ROS production and antioxidant mechanisms [7]. Ipsilateral testicular torsion has been shown to also affect the contralateral testis [8–10]. Protection of the testis in testicular torsion is therefore very important.
Antioxidants obtained from natural sources have been employed in recent studies for the protection of the testis against ischemia/ reperfusion injury [5,11,12]. Resveratrol (trans-3,5,4′-tri-hydroxystilbene) (RSV) is a natural phytoalexin with antioxidant properties. It is found in many plants, particularly grapes and peanuts. RSV protects the cell by preventing lipid peroxidation in the cell membrane and DNA damage caused by excessive ROS production [13]. Recent studies have emphasized that RSV is an effective ROS scavenger and exhibits an antioxidant property in cells producing ROS [14]. It has been reported to have a protective effect against oxidative stress in the liver, spleen [15] and heart [13]. The purpose of this study was to investigate the potential protective effects of RSV, an antioxidant, using familiar and novel oxidative stress markers and the histopathology in an experimental testicular torsion and detorsion model in rats. 1. Material and methods
⁎ Corresponding author. Tel.: +90 462 3777736; fax: +90 462 3252270. E-mail address: [email protected] (E. Yuluğ). 0022-3468/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2013.08.028
The experimental protocol was approved by the Ethical Committee on Animal research at our university. All rats were housed in a light-
E. Yuluğ et al. / Journal of Pediatric Surgery 49 (2014) 484–489
and temperature controlled room. Standard laboratory chow and water were given ad libitum. 1.1. Experimental groups Eighteen male Sprague Dawley rats weighing 250–300 g were used. Rats were divided into three groups. Surgical procedures were performed under 50 mg/kg of ketamine hydrochloride (Ketalar, Pfizer, Turkey) anesthesia. 1.1.1. Sham operated group (S) This group was established in order to determine the effect of surgical stress on spermatogenesis. A mid-line incision was made in the scrotum and the left testis gently extracted. This was then fixed to the scrotum. 1.1.2. Torsion/Detorsion group (T/D) Torsion was achieved by rotating the left testis clockwise 720°. The testis was fixed. Four hours after torsion, detorsion was performed and the testis replaced in its normal position in the scrotum [16]. 1.1.3. The torsion/detorsion and RSV treatment group (T/D + RSV) This group was subjected to exactly the same procedure as the T/D group. Thirty minutes before detorsion, a single intraperitoneal dose of 20 mg/kg RSV (R5010-500 mg, Sigma-Aldrich, St. Louis, MO, USA) was administered. All rats were sacrificed by exsanguination on the 24th hour after the surgical procedure under 50 mg/kg ketamine hydrochloride anesthesia with removal of blood and the bilateral testis. 1.2. Measurement of Ischemia-Modified Albumin (IMA) Reduced cobalt to albumin binding capacity (IMA level) was analyzed using the rapid and colorimetric method of Bar-Or et al [17]. Two hundred microliters of patient serum was placed into glass tubes and 50 μL of 0.1% cobalt chloride (CoCl2.6H2O) (Sigma, USA) in H2O was added. After gentle shaking, the solution was left for 10 min, in order to ensure sufficient cobalt albumin binding. Fifty microliters of 1.5 mg/mL dithiothreitol (DTT) (Sigma, USA) in H2O was added as a colorizing agent and the reaction was quenched 2 min later by adding 1.0 mL of 0.9% NaCl. A colorimetric control was prepared for preoperative and postoperative serum samples. For the colorimetric control samples, 50 μL of distilled water was substituted for 50 μL of 1.5 mg/mL DTT. Specimen absorbencies were analyzed at 470 nm by a spectrophotometer (Shimadzu UV1601, Ausburn, Australia). The color of the DTT containing specimens was compared with that of the colorimetric control tubes. The results were reported as absorbance units (ABSUs). 1.3. Serum MDA activity assay Lipid peroxidation in rat serum samples was determined as malondialdehyde (MDA) concentration using the method described by Yagi [18]. Tetramethoxypropane was used as a standard, and MDA levels were given as nmol/mL.
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1.5. Measurement of Total Oxidant Status (TOS) TOS levels were determined using method as previously described by Erel [20]. Serum TOS levels were calculated in μmol H2O2 equivalent/L. 1.6. Measurement of Total Antioxidant Status (TAS) The total antioxidant status level was determined using method developed by Erel [21]. Serum TAS levels were calculated in mmol Trolox equivalent/L. 1.7. Calculation of Oxidative stress index (OSI) The TOS:TAS ratio was used as the OSI. To perform the calculation, the unit of TAS, mmol Trolox equivalent/L, was converted to μmol Trolox equivalent/L, and OSI was calculated as follows: OSI = [(TOS, μmol H2O2 equivalent/L)/(TAS, μmol Trolox equivalent/L) × 100] [22]. 1.8. Histopathological evaluation Half the bilateral testis tissue was fixed in Bouin's solution. Following routine histological procedures, tissues were embedded in paraffin. Sections 5 μm in thickness were stained with hematoxylin– eosin (H&E). Slides from the groups were evaluated in terms of general testicular structural changes and spermatogenesis. A light microscope with camera attachment (Olympus DP 71; Olympus, Tokyo, Japan) was used for evaluation. Johnsen's testicular biopsy score (JTBS) was used for evaluation of spermatogenesis [23]. Mean JTBS in each section was calculated by dividing the total value by the number of seminiferous tubules. 1.9. Evaluation of germ cell apoptosis The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to analyze DNA fragmentations in the cell nuclei. TUNEL analysis was performed with an in situ cell death detection kit, POD, (ROCHE, Mannheim, Germany) in line with the manufacturer's instructions. Color was then developed with a 3,3′-diaminobenzidine including kit (DAB, Sigma, St Louis, MO, USA). Germinal cells with brown nuclei were evaluated as apoptotic. Apoptotic and normal cell numbers in 10 tubules in each slides were counted using the Analysis 5 Research program (Olympus Soft Imaging Solutions, Münster, Germany) at × 400 magnification. Apoptotic index (AI) was calculated as total TUNEL positive spermatogenetic cell number divided by total normal spermatogenetic cell number [24]. 1.10. Statistical analysis All statistical analyses were performed using the computer software Prism 5.0 (Graphpad Software Inc. San Diego, CA, USA). Data were presented as mean (±) standard deviation (SD). Kruskal Wallis analysis of variance and Mann–Whitney U-test with corrected Bonferroni test were used for statistical analysis. Statistical significance was set at P b 0.05. 2. Results
1.4. Tissue MDA activity assay Testis tissues were weighted and homogenized in ice-cold 1.15% KCl (2 and 10% w/v, respectively). The homogenate was centrifuged at 2000 g for 10 min. MDA levels in tissue samples were determined by the method of Uchiyama and Mihara [19]. Tetramethoxypropane was used as a standard and tissue MDA levels were calculated as nmol/g wet tissue.
There was no significant difference between the groups in terms of ipsilateral and contralateral testis weights. 2.1. Biochemical findings Biochemical results for the experimental groups are given in Table 1. Plasma MDA rose significantly in the T/D group. In the
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Table 1 Levels of biochemical oxidant and antioxidant parameters in all groups. Groups
Plasma MDA (nmol/mL)
Left testis tissue MDA (nmol/g)
Right testis tissue MDA (nmol/g)
TAS (mmol Trolox equivalent/L)
TOS (μmol H2O2 equivalent/L)
OSI H2O2/Trolox
IMA (ABSU)
Sham T/D T/D + RSV
0.12 ± 0.03 0.27 ± 0.05a 0.13 ± 0.03b
303 ± 54.87 521.6 ± 81.84a 502.8 ± 53.16a
297.3 ± 54 323 ± 76.58 320.7 ± 76.48
1.34 ± 0.05 1.08 ± 0.13a 1.15 ± 0.07a
11.08 ± 0.12 12.69 ± 0.89a 9.79 ± 0.62a,b
0.81 ± 0.02 1.17 ± 1.14a 0.84 ± 0.05b
0.78 ± 0.03 0.93 ± 0.06a 0.90 ± 0.02a
a b
P b .05 compared with sham group. P b .05 compared with T/D group.
T/D + RES group, it decreased compared to the T/D group, but this decrease was not significant. IMA values increased significantly in the T/D group and decreased in the T/D + RES group, this was not significant. Tissue MDA in the ipsilateral testis was significantly higher in the T/D group. It decreased in the T/D + RES group compared to the T/D group, although not significantly. There was no difference between the groups in the contralateral testis. TAS values decreased significantly in the T/D group. Although they increased in the T/D + RES group compared to the T/D group, the increase was not significant. OSİ and TOS values increased significantly in the T/D group, decreasing significantly in the T/D + RES group.
2.2. Histopathologic findings Histopathological and TUNEL analysis results for the groups are given in Table 2. Normal testicular morphology was observed in the sham group. Occasional immature germinal epithelial cells were present in the seminiferous tubule lumen. In the T/D group, degeneration in the germinal epithelium and vacuolization between epithelial cells were observed. Irregularities in the basal membranes of the seminiferous tubules and germinal epithelial cells accumulation toward the lumen were present. Multinuclear giant cells were observed in the germinal epithelium. Seminiferous tubule germinal epithelial cells in the T/D + RSV group had a close to normal morphology. No multinuclear giant cells were observed. Occasional immature germinal cell clustering was present in the tubular lumen. In the contralateral testis, the sham group exhibited normal morphology. Widespread germinal epithelial cells were present in the seminiferous tubular lumen in the T/D group. Normal seminiferous tubule architecture was observed in the T/D + RSV group. Rare immature epithelial cells were present in the lumen (Fig. 1). JTBS in the ipsilateral testis decreased significantly compared to the sham group. It rose significantly in the T/D + RES group compared to the T/ D group. No significant difference was determined between the groups in the contralateral testis. In the ipsilateral testis, apoptosis increased significantly in the T/D group compared to the sham group. Widespread apoptosis was observed in the seminiferous tubule germinal cells. Apoptosis decreased significantly in the T/D + RSV group and was more present in the spermatagonium cells (Fig. 2). In the contralateral testis, apoptosis increased significantly in the T/D group. It decreased significantly in the T/D + RES group compared to the T/D group.
Table 2 Apoptotic index and Johnsen's tubuler biopsy score results in bilateral testis. Groups
Sham T/D T/D + RSV a b
AI means ± SD
JTBS means ± SD
Ipsilateral
Contralateral
Ipsilateral
Contralateral
18.97 ± 2.25 69.67 ± 2.95 29.33 ± 1.09
16.43 ± 1.35 27.88 ± 4.39a 19.97 ± 1.22 a,b
9.33 ± 0.18 6.51 ± 0.56 a 8.71 ± 0.31a,b
9.41 ± 0.17 8.83 ± 0.27 9.26 ± 0.22
a a,b
P b .05 compared with sham group. P b .05 compared with T/D group.
3. Discussion Testicular torsion/detorsion (T/D) is defined as ischemia/reperfusion injury and is characterized by production of reactive oxygen radicals. Reperfusion and oxygenation are essential to salvage ischemic tissue in testicular torsion. However, reperfusion also causes additional cellular injury [12]. Previous studies have shown that 30 min–1 h torsion followed by 1–4 h detorsion is sufficient to establish oxidative stress and I/R injury [3,4]. Tissue is exposed to excessive oxygen at time of reperfusion. ROS production in the tissue rises and this leads to cell dysfunction and compromise of cell membrane integrity [4,12]. There are several antioxidant mechanisms in living organisms. Antioxidant systems include antioxidant enzymes, and non-enzymatic antioxidants. Reactive oxygen surfaces and oxidative defense capacity are in balance in healthy cells. Oxidative stress manifests with an increase in oxidants and a decrease in antioxidants. Individual measurement of antioxidant levels is time consuming, and expensive. All antioxidant levels need to be measured in order for in vivo oxidant status to be evaluated. Total oxidant status (TOS) and total antioxidant status (TAS) are therefore valuable in terms of giving the body's net stress. In this study, we used TAS and TOS, a novel technique developed by Erel in the full determination of oxidant and antioxidant status. TOS and TAS parameters are a combination of oxidant and antioxidant parameters such as MDA, Glutathione peroxidase and Catalase. Tissue and serum levels reflect total oxidant and antioxidant status. The TOS to TAS ratio gives the oxidative stress index (OSI) [25]. OSI reflects increased oxidant status or decreased antioxidant status [26]. In this study oxidative stress was calculated by measuring TAS, TOS, MDA and IMA levels in serum and MDA concentrations in tissue. In addition, we used OSI in the evaluation of oxidative stress. RSV has been emphasized as an effective scavenger of hydroxyl, superoxide and metal-induced radicals. RSV acts against DNA damage and lipid peroxidation in the cell membrane caused by ROS [27]. RSV has been reported to act by reducing oxidative stress in the seminiferous tubules and to increase sperm development [15]. In this study, we examined the probable protective effect of RSV, an antioxidant, in a testicular I/R injury model. There have been few studies evaluating TAS, TOS and OSI levels and histopathology in a testicular T/D model. Köksal et al. [28] determined no difference between groups' MDA, TAS and TOS values at the 24th hour after 1-h testicular torsion. However, they reported that it gave rise to testicular injury on the basis of Johnsen's scoring. Gökçe et al. [29] reported that TOS, OSI and MDA were significantly elevated in the torsion group compared to the control group in a 2-h torsion and 4-h detorsion model. In our study, TOS and OSI values were also significantly higher in T/D group. However, TAS values decreased significantly. Elevated TOS in the T/D group and lowered TAS values suggest that oxidative stress and antioxidant mechanisms had entered the picture. The cell possesses various antioxidants for the removal of reactive oxygen radicals. Antioxidant systems begin functioning in order to reduce the effects of ROS, resulting in a reduced antioxidant capacity. Total antioxidant capacity decreased significantly in the torsion group compared to the sham group. OSI increased significantly in our torsion group compared to the sham
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Fig. 1. A, B, C (Ipsilateral testis). A, The S group showed normal seminiferous tubule epithelium (↑) and spermatozoon (Δ). B, The T/D group showed giant cell (Δ), accumulation disordered germinel cells (↑), and vacuolization between epithelial cells (star). C, The T/D + RSV group showed normal germinal epithelial cells (↑) and immature germinal cells in the lumen (Δ). D, E, F (Contralateral testis). D, The S group showed normal seminiferous tubule epithelium (↑) and spermatozoon (Δ). E, The T/D group showed immature germinal cells in the lumen (Δ). F, The T/D + RSV group showed normal seminiferous tubule epithelium (↑) and spermatozoon (Δ).(H&E, original magnification ×200).
group, while decreasing significantly in the treatment group compared to the torsion group. Increased free radical production in oxidative injury affects membrane phospholipids and gives rise to toxic products such as MDA [30]. This is the most important and most studied biomarker showing polyunsaturated fatty acids in ischemia and oxidative stress [31]. MDA levels in our torsion group rose significantly compared to the sham group. This supports the idea of damage in membrane lipids with excessive ROS production. In the RSV treated group, plasma and tissue MDA levels decreased compared to the torsion group, but not significantly. Previous studies have emphasized that RSV protects the cell membrane by preventing lipid peroxidation in the membrane [13]. Our findings support this.
IMA is a novel and sensitive biomarker in the determination of oxidative stress and ischemia. Albumin is the most abundant plasma protein. It is capable of binding the end of the cobalt. Free oxygen radical production rises in I/R injury and an increase takes place in albumin's capacity to bind to cobalt [16]. It has recently been recommended in the early diagnosis of myocardial ischemia without infarct [32]. Clinical studies emphasize that IMA rises within minutes after ischemia, remain elevated for 6–12 h and return to normal in 24 h [33]. Recent studies have stressed that IMA is a useful short- and long-term parameter in showing oxidative stress in a testicular torsion model [34,35]. Kutlu et al. [34] reported that histopathological injury and IMA values were considerably greater compared to other groups after 4-h torsion. However, no difference was determined with
Fig. 2. A, B, C (Ipsilateral testis), D, E, F (Contralateral testis). A, D Sham group. B, E T/D group. C, F T/D + RSV group. Apoptotic spermatogenetic cells (arrow). (TUNEL technique, original magnification ×400).
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the other groups in terms of blood and tissue MDA levels. In a study of testicular torsion by Menteşe et al. [35] IMA levels remained high, compatible with histopathological injury at 2 h detorsion after 4-h torsion and 2 months after detorsion. Nonetheless, serum and tissue MDA levels fell in the long term. In our study, tissue MDA and serum MDA and IMA values 24 h after detorsion were significantly higher in the torsion than those in the sham group. In the treatment group, however, these parameters decreased compared to the torsion group. However, there was no significant difference between the torsion group and the treatment group. At histopathological examination, following 4-h torsion and 2 h detorsion in a study by Türkmen et al. [36], germinal epithelial cells were present in the lumen of the seminiferous tubule in the T/D group. In our study, the seminiferous tubule germinal epithelium was completely irregular, germinal epithelial cells were irregularly distributed in the lumen and greater damage was observed. This reveals that testis injury becomes more pronounced as the detorsion period extends after torsion. Findings regarding contralateral testis damage in previous studies using a testicular torsion model are inconsistent. While some studies report that blood flow increases in the contralateral testis during testicular torsion [37,38], the majority of studies implicate a reduction in blood flow in the contralateral testis at the time of testicular torsion [10,39]. Hypoxia associated with a decrease in blood flow may lead to contralateral testis injury. Oxygen is very important for normal spermatogenesis. The critical time in prevention of contralateral testis injury is reported to be 3 h [40], and a minimum 6–8 h torsion is reported to be needed for the contralateral testis to be affected. Although histopathological changes were detected in this study at the 24th hour of detorsion following 4-h torsion, these were not significant according to the damage score scale. Apoptosis is programmed cell death. It is important in the elimination of abnormal cells from tissue [41]. Uğuralp et al. [42] analyzed apoptosis at the 20th hour of reperfusion following 4-h torsion. Apoptosis in the ipsilateral testis in the group administered RSV treatment decreased significantly compared to the torsion group. They reported that RSV did not affect the contralateral testis. In our study we evaluated apoptosis at the 24th hour after 4-h torsion, when apoptosis is reported to be highest [43]. In our study, apoptosis increased significantly in the T/D group in both the ipsilateral and contralateral testes. Widespread apoptosis was notable in the seminiferous tubule germinal cells in this group. However, in the RSV treated group, apoptosis decreased significantly in both the ipsilateral and contralateral testes. Various treatments have been tested aimed at protecting the ipsilateral and contralateral testes [36,44,45]. We applied the antioxidant RSV in this group. This prevented histopathological injury, and particularly apoptosis, in the bilateral testis. The damage caused by I/R injury is observed in the ipsilateral and contralateral testes. RSV treatment had a positive effect in balancing the oxidant/antioxidant equilibrium, the reduction of histopathological injury and particularly in the prevention of apoptosis in experimental testicular T/D model. OSI may be a valuable parameter in showing clinical status of testicular T/D. References [1] Pentyala S, Lee J, Yalamanchili P, et al. Testicular torsion: a review. J Lower Genital Tract Dis 2001;5:38–47. [2] Cuckow PM, Frank JD. Torsion of the testis. Br J Urol Int 2000;86:349–53. [3] Wei SM, Yan ZZ, Zhou J. Protective effect of rutin on testicular ishemia–reperfusion injury. J Pediatr Surg 2011;46:1419–24. [4] Gezici A, Ozturk H, Buyukbayram H, et al. Effects of gabexate mesilate on ischemia–reperfusion-induced testicular injury in rats. Pediatr Surg Int 2006;22: 435–41. [5] Aktas BK, Bulut S, Bulut S, et al. The effects of N-acetylcysteine on testicular damage in experimental testicular ischemia/reperfusion injury. Pediatr Surg Int 2010;26:293–8.
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The short term effects of resveratrol on ischemia–reperfusion injury in rat testis Esin Yuluğ a,⁎, Sibel Türedi a, Ersagun Karagüzel b, Ömer Kutlu b, Ahmet Menteşe c, d, Ahmet Alver d a b c d
Department of Histology and Embryology, Karadeniz Technical University, Faculty of Medicine, 61080, Trabzon, Turkey Department of Urology, Karadeniz Technical University, Faculty of Medicine, 61080, Trabzon, Turkey Program of Medical Laboratory Techniques, Vocational School of Health Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey Department of Medical Biochemistry, Karadeniz Technical University, Faculty of Medicine, 61080, Trabzon, Turkey
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Article history: Received 21 May 2013 Received in revised form 15 July 2013 Accepted 19 August 2013 Key words: Resveratrol Testicular torsion–detorsion Ischemia-modified albumin Rat
a b s t r a c t Purpose: The purpose of this study was to identify changes taking place in the rat testis at the 24th hour of reperfusion following testicular torsion and to evaluate the effects of resveratrol (RSV), a powerful antioxidant, in preventing these changes using novel biochemical parameters and histopathology. Methods: Eighteen adult male rats were divided into three groups: Sham-operated (S), torsion/detorsion (T/D), and T/D + RSV groups. In the T/D group, testicular ischemia was achieved by rotating the left testis 720° clockwise for 4 h. In the T/D + RSV group, 20 mg/kg RSV was administered intraperitoneally 30 min before detorsion. All rats were sacrificed 24 h after detorsion. Serum and tissue malondialdehyde (MDA) concentrations, ischemia modified albumin (IMA), total oxidative status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), and histopathological damage score were analyzed. Results: Serum MDA, IMA, TOS, and OSI levels rose significantly in the T/D group. Serum MDA and IMA values were lower in the T/D + RES groups, but not significantly. OSI and TOS values were lower in the T/D + RES group, and the difference was significant. TAS values decreased significantly in the T/D group and rose in the T/ D + RSV group, but not significantly. Ipsilateral tissue MDA values were significantly elevated in the T/D group and decreased in the T/D + RSV group, but not significantly. Apoptosis and histopathological damage increased significantly in the T/D group and decreased significantly in the T/D + RSV group. In the contralateral testis, apoptosis increased significantly in the T/D group. It decreased significantly in the T/D + RSV group. Conclusions: Our findings show that RSV had a protective effect against oxidative damage induced with a testicular T/D model, especially at the antiapoptotic and histopathological level. OSI may be a good guide to the clinical status of testicular T/D. © 2014 Elsevier Inc. All rights reserved.
Testicular torsion is a urological emergency particularly seen in neonates, children and adolescents [1,2]. At the time of testicular torsion the germinal cells are exposed to hypoxia. If torsion is prolonged, necrosis occurs in the germinal cells. This may lead to subfertility and infertility [3,4]. Testicular tissue blood flow must therefore be re-established as soon as possible. Testicular tissue blood flow is established with reperfusion. Exposure of tissue to oxygen at the time of reperfusion leads to production of reactive oxygen radicals (ROS) [5,6]. Testicular torsion and detorsion may be regarded as an ischemia/reperfusion (I/R) injury. The mechanism involved in testicular injury is not completely clear, although oxidative stress is thought to be responsible for I/R injury. Oxidative stress is the result of imbalance between excessive ROS production and antioxidant mechanisms [7]. Ipsilateral testicular torsion has been shown to also affect the contralateral testis [8–10]. Protection of the testis in testicular torsion is therefore very important.
Antioxidants obtained from natural sources have been employed in recent studies for the protection of the testis against ischemia/ reperfusion injury [5,11,12]. Resveratrol (trans-3,5,4′-tri-hydroxystilbene) (RSV) is a natural phytoalexin with antioxidant properties. It is found in many plants, particularly grapes and peanuts. RSV protects the cell by preventing lipid peroxidation in the cell membrane and DNA damage caused by excessive ROS production [13]. Recent studies have emphasized that RSV is an effective ROS scavenger and exhibits an antioxidant property in cells producing ROS [14]. It has been reported to have a protective effect against oxidative stress in the liver, spleen [15] and heart [13]. The purpose of this study was to investigate the potential protective effects of RSV, an antioxidant, using familiar and novel oxidative stress markers and the histopathology in an experimental testicular torsion and detorsion model in rats. 1. Material and methods
⁎ Corresponding author. Tel.: +90 462 3777736; fax: +90 462 3252270. E-mail address: [email protected] (E. Yuluğ). 0022-3468/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2013.08.028
The experimental protocol was approved by the Ethical Committee on Animal research at our university. All rats were housed in a light-
E. Yuluğ et al. / Journal of Pediatric Surgery 49 (2014) 484–489
and temperature controlled room. Standard laboratory chow and water were given ad libitum. 1.1. Experimental groups Eighteen male Sprague Dawley rats weighing 250–300 g were used. Rats were divided into three groups. Surgical procedures were performed under 50 mg/kg of ketamine hydrochloride (Ketalar, Pfizer, Turkey) anesthesia. 1.1.1. Sham operated group (S) This group was established in order to determine the effect of surgical stress on spermatogenesis. A mid-line incision was made in the scrotum and the left testis gently extracted. This was then fixed to the scrotum. 1.1.2. Torsion/Detorsion group (T/D) Torsion was achieved by rotating the left testis clockwise 720°. The testis was fixed. Four hours after torsion, detorsion was performed and the testis replaced in its normal position in the scrotum [16]. 1.1.3. The torsion/detorsion and RSV treatment group (T/D + RSV) This group was subjected to exactly the same procedure as the T/D group. Thirty minutes before detorsion, a single intraperitoneal dose of 20 mg/kg RSV (R5010-500 mg, Sigma-Aldrich, St. Louis, MO, USA) was administered. All rats were sacrificed by exsanguination on the 24th hour after the surgical procedure under 50 mg/kg ketamine hydrochloride anesthesia with removal of blood and the bilateral testis. 1.2. Measurement of Ischemia-Modified Albumin (IMA) Reduced cobalt to albumin binding capacity (IMA level) was analyzed using the rapid and colorimetric method of Bar-Or et al [17]. Two hundred microliters of patient serum was placed into glass tubes and 50 μL of 0.1% cobalt chloride (CoCl2.6H2O) (Sigma, USA) in H2O was added. After gentle shaking, the solution was left for 10 min, in order to ensure sufficient cobalt albumin binding. Fifty microliters of 1.5 mg/mL dithiothreitol (DTT) (Sigma, USA) in H2O was added as a colorizing agent and the reaction was quenched 2 min later by adding 1.0 mL of 0.9% NaCl. A colorimetric control was prepared for preoperative and postoperative serum samples. For the colorimetric control samples, 50 μL of distilled water was substituted for 50 μL of 1.5 mg/mL DTT. Specimen absorbencies were analyzed at 470 nm by a spectrophotometer (Shimadzu UV1601, Ausburn, Australia). The color of the DTT containing specimens was compared with that of the colorimetric control tubes. The results were reported as absorbance units (ABSUs). 1.3. Serum MDA activity assay Lipid peroxidation in rat serum samples was determined as malondialdehyde (MDA) concentration using the method described by Yagi [18]. Tetramethoxypropane was used as a standard, and MDA levels were given as nmol/mL.
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1.5. Measurement of Total Oxidant Status (TOS) TOS levels were determined using method as previously described by Erel [20]. Serum TOS levels were calculated in μmol H2O2 equivalent/L. 1.6. Measurement of Total Antioxidant Status (TAS) The total antioxidant status level was determined using method developed by Erel [21]. Serum TAS levels were calculated in mmol Trolox equivalent/L. 1.7. Calculation of Oxidative stress index (OSI) The TOS:TAS ratio was used as the OSI. To perform the calculation, the unit of TAS, mmol Trolox equivalent/L, was converted to μmol Trolox equivalent/L, and OSI was calculated as follows: OSI = [(TOS, μmol H2O2 equivalent/L)/(TAS, μmol Trolox equivalent/L) × 100] [22]. 1.8. Histopathological evaluation Half the bilateral testis tissue was fixed in Bouin's solution. Following routine histological procedures, tissues were embedded in paraffin. Sections 5 μm in thickness were stained with hematoxylin– eosin (H&E). Slides from the groups were evaluated in terms of general testicular structural changes and spermatogenesis. A light microscope with camera attachment (Olympus DP 71; Olympus, Tokyo, Japan) was used for evaluation. Johnsen's testicular biopsy score (JTBS) was used for evaluation of spermatogenesis [23]. Mean JTBS in each section was calculated by dividing the total value by the number of seminiferous tubules. 1.9. Evaluation of germ cell apoptosis The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to analyze DNA fragmentations in the cell nuclei. TUNEL analysis was performed with an in situ cell death detection kit, POD, (ROCHE, Mannheim, Germany) in line with the manufacturer's instructions. Color was then developed with a 3,3′-diaminobenzidine including kit (DAB, Sigma, St Louis, MO, USA). Germinal cells with brown nuclei were evaluated as apoptotic. Apoptotic and normal cell numbers in 10 tubules in each slides were counted using the Analysis 5 Research program (Olympus Soft Imaging Solutions, Münster, Germany) at × 400 magnification. Apoptotic index (AI) was calculated as total TUNEL positive spermatogenetic cell number divided by total normal spermatogenetic cell number [24]. 1.10. Statistical analysis All statistical analyses were performed using the computer software Prism 5.0 (Graphpad Software Inc. San Diego, CA, USA). Data were presented as mean (±) standard deviation (SD). Kruskal Wallis analysis of variance and Mann–Whitney U-test with corrected Bonferroni test were used for statistical analysis. Statistical significance was set at P b 0.05. 2. Results
1.4. Tissue MDA activity assay Testis tissues were weighted and homogenized in ice-cold 1.15% KCl (2 and 10% w/v, respectively). The homogenate was centrifuged at 2000 g for 10 min. MDA levels in tissue samples were determined by the method of Uchiyama and Mihara [19]. Tetramethoxypropane was used as a standard and tissue MDA levels were calculated as nmol/g wet tissue.
There was no significant difference between the groups in terms of ipsilateral and contralateral testis weights. 2.1. Biochemical findings Biochemical results for the experimental groups are given in Table 1. Plasma MDA rose significantly in the T/D group. In the
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Table 1 Levels of biochemical oxidant and antioxidant parameters in all groups. Groups
Plasma MDA (nmol/mL)
Left testis tissue MDA (nmol/g)
Right testis tissue MDA (nmol/g)
TAS (mmol Trolox equivalent/L)
TOS (μmol H2O2 equivalent/L)
OSI H2O2/Trolox
IMA (ABSU)
Sham T/D T/D + RSV
0.12 ± 0.03 0.27 ± 0.05a 0.13 ± 0.03b
303 ± 54.87 521.6 ± 81.84a 502.8 ± 53.16a
297.3 ± 54 323 ± 76.58 320.7 ± 76.48
1.34 ± 0.05 1.08 ± 0.13a 1.15 ± 0.07a
11.08 ± 0.12 12.69 ± 0.89a 9.79 ± 0.62a,b
0.81 ± 0.02 1.17 ± 1.14a 0.84 ± 0.05b
0.78 ± 0.03 0.93 ± 0.06a 0.90 ± 0.02a
a b
P b .05 compared with sham group. P b .05 compared with T/D group.
T/D + RES group, it decreased compared to the T/D group, but this decrease was not significant. IMA values increased significantly in the T/D group and decreased in the T/D + RES group, this was not significant. Tissue MDA in the ipsilateral testis was significantly higher in the T/D group. It decreased in the T/D + RES group compared to the T/D group, although not significantly. There was no difference between the groups in the contralateral testis. TAS values decreased significantly in the T/D group. Although they increased in the T/D + RES group compared to the T/D group, the increase was not significant. OSİ and TOS values increased significantly in the T/D group, decreasing significantly in the T/D + RES group.
2.2. Histopathologic findings Histopathological and TUNEL analysis results for the groups are given in Table 2. Normal testicular morphology was observed in the sham group. Occasional immature germinal epithelial cells were present in the seminiferous tubule lumen. In the T/D group, degeneration in the germinal epithelium and vacuolization between epithelial cells were observed. Irregularities in the basal membranes of the seminiferous tubules and germinal epithelial cells accumulation toward the lumen were present. Multinuclear giant cells were observed in the germinal epithelium. Seminiferous tubule germinal epithelial cells in the T/D + RSV group had a close to normal morphology. No multinuclear giant cells were observed. Occasional immature germinal cell clustering was present in the tubular lumen. In the contralateral testis, the sham group exhibited normal morphology. Widespread germinal epithelial cells were present in the seminiferous tubular lumen in the T/D group. Normal seminiferous tubule architecture was observed in the T/D + RSV group. Rare immature epithelial cells were present in the lumen (Fig. 1). JTBS in the ipsilateral testis decreased significantly compared to the sham group. It rose significantly in the T/D + RES group compared to the T/ D group. No significant difference was determined between the groups in the contralateral testis. In the ipsilateral testis, apoptosis increased significantly in the T/D group compared to the sham group. Widespread apoptosis was observed in the seminiferous tubule germinal cells. Apoptosis decreased significantly in the T/D + RSV group and was more present in the spermatagonium cells (Fig. 2). In the contralateral testis, apoptosis increased significantly in the T/D group. It decreased significantly in the T/D + RES group compared to the T/D group.
Table 2 Apoptotic index and Johnsen's tubuler biopsy score results in bilateral testis. Groups
Sham T/D T/D + RSV a b
AI means ± SD
JTBS means ± SD
Ipsilateral
Contralateral
Ipsilateral
Contralateral
18.97 ± 2.25 69.67 ± 2.95 29.33 ± 1.09
16.43 ± 1.35 27.88 ± 4.39a 19.97 ± 1.22 a,b
9.33 ± 0.18 6.51 ± 0.56 a 8.71 ± 0.31a,b
9.41 ± 0.17 8.83 ± 0.27 9.26 ± 0.22
a a,b
P b .05 compared with sham group. P b .05 compared with T/D group.
3. Discussion Testicular torsion/detorsion (T/D) is defined as ischemia/reperfusion injury and is characterized by production of reactive oxygen radicals. Reperfusion and oxygenation are essential to salvage ischemic tissue in testicular torsion. However, reperfusion also causes additional cellular injury [12]. Previous studies have shown that 30 min–1 h torsion followed by 1–4 h detorsion is sufficient to establish oxidative stress and I/R injury [3,4]. Tissue is exposed to excessive oxygen at time of reperfusion. ROS production in the tissue rises and this leads to cell dysfunction and compromise of cell membrane integrity [4,12]. There are several antioxidant mechanisms in living organisms. Antioxidant systems include antioxidant enzymes, and non-enzymatic antioxidants. Reactive oxygen surfaces and oxidative defense capacity are in balance in healthy cells. Oxidative stress manifests with an increase in oxidants and a decrease in antioxidants. Individual measurement of antioxidant levels is time consuming, and expensive. All antioxidant levels need to be measured in order for in vivo oxidant status to be evaluated. Total oxidant status (TOS) and total antioxidant status (TAS) are therefore valuable in terms of giving the body's net stress. In this study, we used TAS and TOS, a novel technique developed by Erel in the full determination of oxidant and antioxidant status. TOS and TAS parameters are a combination of oxidant and antioxidant parameters such as MDA, Glutathione peroxidase and Catalase. Tissue and serum levels reflect total oxidant and antioxidant status. The TOS to TAS ratio gives the oxidative stress index (OSI) [25]. OSI reflects increased oxidant status or decreased antioxidant status [26]. In this study oxidative stress was calculated by measuring TAS, TOS, MDA and IMA levels in serum and MDA concentrations in tissue. In addition, we used OSI in the evaluation of oxidative stress. RSV has been emphasized as an effective scavenger of hydroxyl, superoxide and metal-induced radicals. RSV acts against DNA damage and lipid peroxidation in the cell membrane caused by ROS [27]. RSV has been reported to act by reducing oxidative stress in the seminiferous tubules and to increase sperm development [15]. In this study, we examined the probable protective effect of RSV, an antioxidant, in a testicular I/R injury model. There have been few studies evaluating TAS, TOS and OSI levels and histopathology in a testicular T/D model. Köksal et al. [28] determined no difference between groups' MDA, TAS and TOS values at the 24th hour after 1-h testicular torsion. However, they reported that it gave rise to testicular injury on the basis of Johnsen's scoring. Gökçe et al. [29] reported that TOS, OSI and MDA were significantly elevated in the torsion group compared to the control group in a 2-h torsion and 4-h detorsion model. In our study, TOS and OSI values were also significantly higher in T/D group. However, TAS values decreased significantly. Elevated TOS in the T/D group and lowered TAS values suggest that oxidative stress and antioxidant mechanisms had entered the picture. The cell possesses various antioxidants for the removal of reactive oxygen radicals. Antioxidant systems begin functioning in order to reduce the effects of ROS, resulting in a reduced antioxidant capacity. Total antioxidant capacity decreased significantly in the torsion group compared to the sham group. OSI increased significantly in our torsion group compared to the sham
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Fig. 1. A, B, C (Ipsilateral testis). A, The S group showed normal seminiferous tubule epithelium (↑) and spermatozoon (Δ). B, The T/D group showed giant cell (Δ), accumulation disordered germinel cells (↑), and vacuolization between epithelial cells (star). C, The T/D + RSV group showed normal germinal epithelial cells (↑) and immature germinal cells in the lumen (Δ). D, E, F (Contralateral testis). D, The S group showed normal seminiferous tubule epithelium (↑) and spermatozoon (Δ). E, The T/D group showed immature germinal cells in the lumen (Δ). F, The T/D + RSV group showed normal seminiferous tubule epithelium (↑) and spermatozoon (Δ).(H&E, original magnification ×200).
group, while decreasing significantly in the treatment group compared to the torsion group. Increased free radical production in oxidative injury affects membrane phospholipids and gives rise to toxic products such as MDA [30]. This is the most important and most studied biomarker showing polyunsaturated fatty acids in ischemia and oxidative stress [31]. MDA levels in our torsion group rose significantly compared to the sham group. This supports the idea of damage in membrane lipids with excessive ROS production. In the RSV treated group, plasma and tissue MDA levels decreased compared to the torsion group, but not significantly. Previous studies have emphasized that RSV protects the cell membrane by preventing lipid peroxidation in the membrane [13]. Our findings support this.
IMA is a novel and sensitive biomarker in the determination of oxidative stress and ischemia. Albumin is the most abundant plasma protein. It is capable of binding the end of the cobalt. Free oxygen radical production rises in I/R injury and an increase takes place in albumin's capacity to bind to cobalt [16]. It has recently been recommended in the early diagnosis of myocardial ischemia without infarct [32]. Clinical studies emphasize that IMA rises within minutes after ischemia, remain elevated for 6–12 h and return to normal in 24 h [33]. Recent studies have stressed that IMA is a useful short- and long-term parameter in showing oxidative stress in a testicular torsion model [34,35]. Kutlu et al. [34] reported that histopathological injury and IMA values were considerably greater compared to other groups after 4-h torsion. However, no difference was determined with
Fig. 2. A, B, C (Ipsilateral testis), D, E, F (Contralateral testis). A, D Sham group. B, E T/D group. C, F T/D + RSV group. Apoptotic spermatogenetic cells (arrow). (TUNEL technique, original magnification ×400).
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the other groups in terms of blood and tissue MDA levels. In a study of testicular torsion by Menteşe et al. [35] IMA levels remained high, compatible with histopathological injury at 2 h detorsion after 4-h torsion and 2 months after detorsion. Nonetheless, serum and tissue MDA levels fell in the long term. In our study, tissue MDA and serum MDA and IMA values 24 h after detorsion were significantly higher in the torsion than those in the sham group. In the treatment group, however, these parameters decreased compared to the torsion group. However, there was no significant difference between the torsion group and the treatment group. At histopathological examination, following 4-h torsion and 2 h detorsion in a study by Türkmen et al. [36], germinal epithelial cells were present in the lumen of the seminiferous tubule in the T/D group. In our study, the seminiferous tubule germinal epithelium was completely irregular, germinal epithelial cells were irregularly distributed in the lumen and greater damage was observed. This reveals that testis injury becomes more pronounced as the detorsion period extends after torsion. Findings regarding contralateral testis damage in previous studies using a testicular torsion model are inconsistent. While some studies report that blood flow increases in the contralateral testis during testicular torsion [37,38], the majority of studies implicate a reduction in blood flow in the contralateral testis at the time of testicular torsion [10,39]. Hypoxia associated with a decrease in blood flow may lead to contralateral testis injury. Oxygen is very important for normal spermatogenesis. The critical time in prevention of contralateral testis injury is reported to be 3 h [40], and a minimum 6–8 h torsion is reported to be needed for the contralateral testis to be affected. Although histopathological changes were detected in this study at the 24th hour of detorsion following 4-h torsion, these were not significant according to the damage score scale. Apoptosis is programmed cell death. It is important in the elimination of abnormal cells from tissue [41]. Uğuralp et al. [42] analyzed apoptosis at the 20th hour of reperfusion following 4-h torsion. Apoptosis in the ipsilateral testis in the group administered RSV treatment decreased significantly compared to the torsion group. They reported that RSV did not affect the contralateral testis. In our study we evaluated apoptosis at the 24th hour after 4-h torsion, when apoptosis is reported to be highest [43]. In our study, apoptosis increased significantly in the T/D group in both the ipsilateral and contralateral testes. Widespread apoptosis was notable in the seminiferous tubule germinal cells in this group. However, in the RSV treated group, apoptosis decreased significantly in both the ipsilateral and contralateral testes. Various treatments have been tested aimed at protecting the ipsilateral and contralateral testes [36,44,45]. We applied the antioxidant RSV in this group. This prevented histopathological injury, and particularly apoptosis, in the bilateral testis. The damage caused by I/R injury is observed in the ipsilateral and contralateral testes. RSV treatment had a positive effect in balancing the oxidant/antioxidant equilibrium, the reduction of histopathological injury and particularly in the prevention of apoptosis in experimental testicular T/D model. OSI may be a valuable parameter in showing clinical status of testicular T/D. References [1] Pentyala S, Lee J, Yalamanchili P, et al. Testicular torsion: a review. J Lower Genital Tract Dis 2001;5:38–47. [2] Cuckow PM, Frank JD. Torsion of the testis. Br J Urol Int 2000;86:349–53. [3] Wei SM, Yan ZZ, Zhou J. Protective effect of rutin on testicular ishemia–reperfusion injury. J Pediatr Surg 2011;46:1419–24. [4] Gezici A, Ozturk H, Buyukbayram H, et al. Effects of gabexate mesilate on ischemia–reperfusion-induced testicular injury in rats. Pediatr Surg Int 2006;22: 435–41. [5] Aktas BK, Bulut S, Bulut S, et al. The effects of N-acetylcysteine on testicular damage in experimental testicular ischemia/reperfusion injury. Pediatr Surg Int 2010;26:293–8.
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