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Journal of Pediatric Urology (2016) xx, 1e8

Mannitol has a protective effect on testicular torsion: An experimental rat model

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Namik Kemal University, School of Medicine, Department of Urology, Tekirdag, Turkey

Omer Kurt a, Cenk Murat Yazici a, Mustafa Erboga b, Cuneyt Turan c, Yeliz Bozdemir b, Alpaslan Akbas d, Polat Turker a, Cevat Aktas b, Murat Aydin e, Ebru Yesildag f Summary

b

Namik Kemal University, School of Medicine, Department of Histology and Embryology, Tekirdag, Turkey

c Namik Kemal University, School of Medicine, Department of Anesthesiology and Reanimation, Tekirdag, Turkey

d

18 Mart University, School of Medicine, Department of Urology, Canakkale, Turkey

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Namik Kemal University, School of Medicine, Department of Biochemistry, Tekirdag, Turkey

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Namik Kemal University, School of Medicine, Department Pediatric Urology, Tekirdag, Turkey Correspondence to: O. Kurt, Namik Kemal University School of Medicine, Department of Urology, Tekirdag, Turkey

Objective Testicular torsion is an emergency condition that causes testicular injury. Any treatment opportunity reducing the destructive effect of testicular torsion is important for the future life of patients. In this experimental study we investigated the protective effect of mannitol on ischemiaereperfusion (I/R) injury in a rat testes torsion model. Method In total, 32 male Sprague Dawley rats were included. Four experimental groups included eight rats each. Group A was a sham group in which the right testis was brought out through a scrotal incision and then replaced in the scrotum without torsion. In Group B, the right testis was torsioned, by rotating 720 clockwise and fixed to the scrotum with no treatment. In Group C, the same testicular torsion process was performed with saline infusion just after testicular torsion. In group D, mannitol infusion was used just after testicular torsion. Testicles were detorsioned after 3 h and left inside for more than 2 h before orchiectomy. Histopathological, immunohistochemical, and biochemical analyses were performed.

Results Testicular architecture was disturbed significantly in the torsion groups without mannitol infusion. However, testicular tissue structure was significantly better in the mannitol-treated group, demonstrating a protective effect. Similar findings were also shown for the proliferating cell nuclear antigen (PCNA) index and antioxidant activity; both were higher in the mannitol group than in the no-treatment and saline groups (p < 0.01). The apoptotic index was also significantly lower in the mannitol-treated group compared with the no treatment and saline groups (p < 0.01). Conclusions The seminiferous tubule structure in testicular torsion without mannitol treatment was significantly disturbed, whereas the structural disruption was considerably less in the mannitol group. Mannitol treatment also decreased reactive oxygen radical levels significantly and was able to decrease apoptosis. These results were consistent with other organ model studies that evaluated the protective effects of mannitol treatment in I/R injury. Mannitol infusion had a protective effect against I/R injury in testicular torsion in rats. This experimental study may guide clinicians to evaluate the effectiveness of mannitol in human testicular torsion.

[email protected] (O. Kurt) Keywords Testicular torsion; Mannitol; Ischemia reperfusion injury; Treatment; Rat model Received 28 July 2015 Accepted 4 January 2016 Available online xxx

Table

Histopathological evaluation of study groups. Apoptotic index

Group Group Group Group

A B C D

3.88 25.88 25.52 15.25

   

b

0.83 2.91a,b 2.87a,b 1.48a

PCNA index 36.47 22.22 22.51 29.28

   

MTBS b

1.36 2.34a,b 2.99a,b 1.66a

9.23 4.57 4.76 6.72

   

MSTD b

0.17 0.22a,b 0.14a,b 0.25a

273.25 209 210.63 229.63

   

5.14b 6.11a,b 5.09a,b 6.18a

MSTD Z mean seminiferous tubular diameter; MTBS Z mean testicular biopsy score; PCNA Z proliferating cell nuclear antigen; I/R Z ischemia-reperfusion. Group A was a sham operation group, Group B (I/R) had 3 h ischemia and 2 h reperfusion, Group C (I/ R þ Saline) had 3 h ischemia and 2 h reperfusion and saline bolus treatment, Group D (I/R þ Mannitol) had 3 h ischemia and 2 h reperfusion and mannitol bolus treatment. a p < 0.01 compared with group A. b p < 0.01 compared with group D. http://dx.doi.org/10.1016/j.jpurol.2016.01.004 1477-5131/ª 2016 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

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O. Kurt et al.

Introduction Testicular torsion is an emergency condition with an incidence of 1/4000 in males under 25 years of age [1]. It causes testicular injury, leading to potential infertility and subfertility; thus, immediate diagnosis and intervention are important [2]. Although the main pathological mechanisms of testicular injury in torsion are only partially understood, overproduction of reactive oxygen species (ROS) has been implicated as one of the main factors in cellular and tissue damage [3]. Several antioxidant molecules, such as alphalipoic acid, quercetine, and melatonin, were found to be effective against ischemiaereperfusion (I/R) injury, but these molecules have not been used widely in clinical practice because of their toxic side effects [4e6]. Mannitol has traditionally been administered before partial nephrectomy to reduce ischemic renal damage as an intravascular volume expander with free-radical scavenging properties, as well as being an osmotic diuretic [7]. It reduces oxidant-derived injury in kidneys, heart, and lungs [8e10]. In this experimental study, we hypothesized that mannitol may have antioxidant protective effects against I/ R injury in testicular torsion and sought to investigate this protective effect with histopathological and biochemical analyses in rat testes.

Materials and methods With the approval of the local animal care and use committee, in total, 32, 6-month-old (mature) male Sprague Dawley rats, weighing 240e280 g, were used in the study. We randomly divided them into four experimental groups, each with eight rats (Table 1). All animals were housed in a temperature- and light-controlled room, with ad libitum access to water and rat chow. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals.

Experimental design Surgical procedures were performed under ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia

Table 1

and sterile conditions. A scrotal midline incision was made and torsion was induced by rotating the right testis 720 clockwise and maintained by fixing the testis. The same surgical procedure was performed in the sham group except there was no testicular torsion. Saline (NaCl, 0.09%, 10 mL/kg/min) was administered during the procedure to all groups for hydration. After a 3-h torsion period, the suture was removed with a detorsioning procedure. The testis was replaced in the scrotum for an additional 2-h period. At the end of study, the rats were decapitated, and a right orchiectomy was performed for biochemical and histopathological examinations (Table 1). Testicular specimens were individually immersed in Bouin’s fixative, dehydrated in alcohols, and embedded in paraffin wax. Sections of 5 mm were obtained, deparaffinized, and stained with hematoxylin and eosin (H&E) for evaluation by a histologist in a random order under blinded conditions with standard light microscopy. Three slides, prepared from the upper, lower, and midportions of the testes were examined. Mean seminiferous tubule diameter (MSTD) was measured, in micrometers. Spermatogenesis was assessed histopathologically using Johnsen’s mean testicular biopsy score (MTBS) [3,4]. A score of 0e10 was given to each tubule according to epithelial maturation. Preparations were evaluated with a bright-field microscope (Olympus CX41, Japan) and photographed. We determined testicular tissue antioxidant enzyme activities, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Testicular tissue levels of lipid peroxidation products, malondialdehyde (MDA), and xanthine oxidase (XO) activity, were also determined. Immunohistochemical reactions were performed according to the avidin-biotin complex technique described by Hsu et al. [11]. Sections were incubated with a specific monoclonal antibody to proliferating cell nuclear antigen (PCNA; Cat. # MS-106-B, Thermo LabVision, USA). To quantify the incidence of PCNA, 10 seminiferous tubules were counted in each slide. Both stained and non-stained germ cells were counted, and the ratio of stained cells to the total number of germ cells, the “PCNA index,” was calculated for each seminiferous tubule.

Experimental groups.

Sham group (Group A): A sham procedure was performed to determine biochemical and histopathological basal values. The right testis was brought out through the incision and then returned to the scrotum without torsion. A 4/0 silk suture was used to fix the testis in the scrotum. After a 2-h period, the right testis was removed for evaluation. Ischemia-reperfusion (I-R)/untreated group (Group B): After 3 h of unilateral testicular torsion, detorsion was performed and the testis was replaced in the scrotum and fixed. The rats in this group received only saline solution (NaCl at 0.02%, 10 mL/ kg/min) during the procedure, and did not receive any treatment after the detorsion process. After 2 h of detorsion, the right testis was removed for evaluation. Ischemia-reperfusion (I-R)/saline bolus treated group (Group C): The same surgical procedure (torsion and detorsion) was performed as in Group B. The rats were given a bolus of saline solution (1 mg/kg; i.v.) immediately after detorsion. The bolus injection was given intravenously in a 2-min period, and the testis was removed 2 h after detorsion. Ischemia-reperfusion (I-R)/mannitol bolus treated group (Group D): The same surgical procedure (torsion and detorsion) was performed as in other groups. The rats were treated with a bolus of mannitol (1 mg/kg; i.v.) immediately after detorsion. The bolus injection was given intravenously in a 2-min period that started immediately after reperfusion, and the testis was removed 2 h after detorsion.

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Mannitol has a protective effect on testicular torsion

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Figure 1 Light microscopy of testicular tissue in different groups. H&E: (A) In the sham group, normal testicular architecture was seen. (B) After I/R with the saline group, severe testicular damage was noted. (C) Mannitol group; treatment prevented testicular damage (H&E, scale bar, 50 mm). Values are expressed as means  SD, n Z 8, for each group. H&E Z hematoxylin and eosin; I/ R Z ischemiaereperfusion; MTBS Z mean testicular biopsy score; MSTD Z mean seminiferous tubule diameter. a p < 0.01, vs. Group A. b p < 0.01, vs. Group D.

Germ cell apoptosis was evaluated by the terminal dUTP nick end-labeling (TUNEL) assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was used with an apoptosis detection kit (TdT-Fragel DNA Fragmentation Detection Kit, Cat. No. QIA33, Calbiochem, USA). Quantitative analysis of testicular apoptosis was estimated. The incidence of apoptosis was evaluated in 100 tubules of each testis. The numbers of seminiferous tubules containing three or more apoptotic cells by TUNEL staining were calculated. The apoptosis percentage was calculated as the ratio of the seminiferous tubules positive for apoptosis to the total number of seminiferous tubules in a cross-section. Frozen testis tissue was homogenized. The homogenates were filtered and centrifuged using a refrigerated centrifuge (4  C), and supernatants were frozen at 20  C in aliquots until use for biochemical assays. The protein content of the supernatant was determined using the Lowry method

[12]. Catalase, SOD, GSH-Px, and MDA levels were determined using a rat ELISA kit (SRB/Shanghai). Serum XO activity was measured spectrophotometrically by the formation of uric acid from xanthine through an increase in absorbance at 293 nm [13]. The results are expressed in units per liter of plasma (U/L).

Statistical analysis All data were analyzed with the SPSS software (ver. 17.0 for Windows; SPSS Inc., Chicago, IL, USA). Data are presented as means and standard deviations or percentages. Data in independent groups were analyzed for a normal distribution with the KolmogoroveSmirnov test and further evaluated with the ManneWhitney U test. Data in dependent groups were analyzed with the Wilcoxon signed ranks test after evaluation of a normal distribution with the KolmogoroveSmirnov test.

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Figure 2 Light microscopy of testicular tissue in different groups. PCNA: (A) In the sham group, PCNA-positive cells were detected in the spermatogonia and early-stage spermatocytes. (B) After I/R, the number of PCNA-positive germinal cells was significantly lower in the I/R group than in the sham group. (C) Treatment with mannitol markedly increased the number of PCNApositive germinal cells (arrowhead: spermatogonia, arrow: germinal cells). TUNEL: (D) In the sham group, a few TUNEL-positive germ cells were observed in the seminiferous epithelium. (E) Positive cells for TUNEL staining were increased in I/R rats. (F) Treatment with mannitol markedly reduced the degree of germ cell apoptosis in the seminiferous epithelium (arrow: TUNELpositive germ cells), (immunoperoxidase, hematoxylin counterstaining, and TUNEL, scale bar, 50 mm). Values are expressed as means  SD, n Z 8, for each group. PCNA Z proliferating cell nuclear antigen; TUNEL Z terminal dUTP nick end-labeling. a p < 0.01, vs. Group A. b p < 0.01, vs. Group D.

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Mannitol has a protective effect on testicular torsion

Results The mean MSTD and Johnsen’s MTBS values in each group are shown in the table in the summary section. Mean MSTD and MTBS of Group B (I/R) and Group C (I/R þ saline) were significantly lower than those of Group D (I/R þ mannitol) and the sham group (p < 0.01). Testicular architecture was normal in the sham group. Seminiferous tubular morphology was regular, with normal spermatogenesis (Fig. 1A). Microscopic findings of testicular tissue were similar in Groups B (I/R) and C (I/R þ saline). These groups showed a marked decrease in the seminiferous tubular diameter, with severe distortion, extensive disorganization, sloughing, and a loss of maturation of germ cells (Fig. 1B). The tissue appearance was significantly better in the mannitol group with improved histological findings compared with Groups B and C (Fig. 1C). Under light microscopy, the I/R and I/R þ saline groups showed similar immunohistochemical findings. There were significant numbers of PCNA-positive cells in the sham

5 group (Fig. 2A). The number of PCNA-positive germinal cells was lower in the I/R and I/R þ saline groups (Fig. 2B). PCNA-positive germinal cells were also lower in the mannitol-treated group, compared with the sham group (Fig. 2C), but the mean PCNA index of this group was significantly higher than those of Groups B and C (p < 0.01) (Fig. 3). The degree of germ cell apoptosis was higher in the I/R and I/R þ saline groups (Fig. 2E) than in the sham group (Fig. 2D). Mannitol infusion markedly reduced the amount of germ cell apoptosis (Fig. 2F). The apoptotic index decreased significantly in the mannitol-treated I/R group versus the I/R and I/R þ saline groups (p < 0.01). Oxidant (MDA, XO) and antioxidant (GPx, CAT, and SOD) levels in the experimental groups are shown in Fig. 2. The SOD, CAT, and GPx activities in the I/R þ mannitol group were significantly higher than those in the I/R group (p < 0.01). Malondialdehyde levels in testes were significantly higher in the I/R group than the sham and I/ R þ mannitol groups (p < 0.01). Xanthine oxidase activity in

Figure 3 Biochemical evaluation of antioxidant enzymes. Group A: sham operation group, Group B (I/R): 3 h of ischemia and 2 h of reperfusion, Group C (I/R þ saline): 3 h of ischemia and 2 h of reperfusion and saline bolus-treated group, Group D (I/ R þ mannitol): 3 h of ischemia and 2 h of reperfusion and mannitol bolus-treated group. Values are expressed as means  SD, n Z 8, for each group. MDA Z malondialdehyde; GPx Z glutathione peroxidase; SOD Z superoxide dismutase; CAT Z catalase; XO Z xanthine oxidase. a p < 0.01, vs. Group A. b p < 0.01, vs. Group D. c p < 0.05, vs. Group A. d p < 0.05, vs. Group D.

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6 the I/R þ mannitol group was significantly lower than that of the I/R group, but was higher than that of the control group (p < 0.01).

Discussion The pathogenic mechanism of I/R injury is mostly attributed to overproduction of ROS in various organ models [2e6]. During the ischemic phase of the I/R process, hypoxic conditions develop because of disruption of the blood flow. Hypoxic conditions lead to conversion of hypoxanthine deoxygenase to XO, a superoxide enzyme generator [2,14]. With restoration of the oxygen supply to the tissues, the mitochondrial respiratory system causes overproduction of free radicals. The enzymatic antioxidant defense system, which includes SOD, CAT, and GSH-Px, reacts to scavenge the free radicals to protect tissues from I/R injury [2,3]. Ischemia stimulates chemotactic factors and leads to migration of polymorphonuclear leukocytes to the ischemic region, which also generates ROS [14]. Excessive amounts of ROS react with membrane lipids, resulting in lipid peroxidation and loss of cellular components [15]. This lipid peroxidation reaction can indirectly be shown by MDA, which indicates cellular damage caused by ROS effects [3,6]. Changes in tissue antioxidant enzyme activities (CAT, SOD, and GSH-Px) and changes in the tissue levels of MDA and XO in the I/R group demonstrated a significant I/R injury in testicular torsion. The most important indicator of tissue injury from the I/R effect was the MDA level [3,6]. Malondialdehyde was significantly higher in Groups B (I/R) and C (I/R þ saline) compared with the sham group, but not significantly different in Group D (I/R þ mannitol). This finding may be related to the ability of mannitol to act as a scavenger of hydroxyl radicals, which decrease the devastating effects of the I/R reaction. Tissue levels of XO are also an indicator of I/R reactions that increase rapidly after ischemia. This is probably caused by degradation of xanthine dehydrogenase to XO, as an effect of the ischemia [16]. During this degradation, highly toxic oxygen species, such as superoxide anions and hydrogen peroxide, are generated. Compared with the sham group, XO levels were significantly higher in Groups B and C. However, XO levels in the mannitol group were comparable with those of the sham group. Similar to our findings, XO levels increased significantly after reperfusion in double organ models, but the rise of XO levels was limited by mannitol [17]. Mannitol usage was able to decrease unfavorable effects of the I/R reaction in testicular torsion. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, are generated from oxygen species by partial de-oxygenation [18]. Enzymatic antioxidant defense systems, such as SOD, catalase, and GSH-Px, protect tissues from ROS. Superoxide dismutase is a strong antioxidant that can selectively and rapidly reduce superoxide anion radicals to hydrogen peroxide. It can be used for measuring oxidative stress. Glutathione peroxidase is also effective against cellular damage caused by H2O2 and constitutes the first step in the antioxidant defense system against I/R damage [19]. Catalase is another enzyme that protects tissues from I/R

O. Kurt et al. damage. We demonstrated a significant decrease of these antioxidant enzymes in Groups B and C versus the sham group. Infusion of mannitol had protective effects against tissue damage; the reduction in antioxidant enzyme levels was lower for the mannitol group than Groups B and C. Previous studies showed that MSTD and MTBS were good markers for evaluating tissue damage in early periods of testicular detorsion [3e6]. We also used them to investigate the preservative effect of mannitol administration in testicular torsion. The seminiferous tubule structure in testicular torsion without mannitol treatment was significantly disturbed, whereas the structural disruption was considerably less in the mannitol group. The MTBS and MSTD values were lowest in the non-mannitol torsion group. The differences between non-mannitol and mannitoltreated groups were significant, in terms of MTBS and MSTD values. These differences predicted the preservative function of mannitol in the testicular torsion model in rats. Turner et al. demonstrated an increase in testicular ROS levels after the repair of torsion and suggested that ROS were responsible for germ cell apoptosis after detorsion [20]. With this proposal, antioxidant treatments have been applied to ROS level reduction and were able to decrease apoptosis in testicular torsion models [4e6,20]. The number and signal density of TUNEL-positive germ cells were also significantly higher in the non-mannitol torsion group. Mannitol treatment reduced the reactivity and the number of apoptotic germ cells. Another predictive factor determining the germ cell damage is PCNA in testicular germ cells, which indicates the reduction of cellular proliferative activity and spermatogenesis. We were able to detect PCNA-positive cells in spermatogonia and early-stage spermatocytes in control rats. However, the signal density of positive cells was significantly lower in the non-mannitoltreated group. The rate of PCNA expression in the mannitoltreated group was also lower than in the control group, but it was significantly higher than in the non-mannitol-treated group. This finding was similar to that of Kanter [5]. Besides the free radical-scavenging effect of mannitol, its osmotic properties also provide a protective effect against I/R injury. As mannitol has hyperosmolar properties, it does not penetrate cellular membranes, and thus causes hemodilution and dehydration of tissues [21]. Mannitol was shown to decrease blood viscosity and systemic vascular resistance [22]. In a rabbit model, mannitol was able to restore cardiac microcirculatory flow and decreased myocardial damage and edema [23]. We also observed a marked decrease in testicular edema with mannitol infusion. The protective effect of mannitol against I/R injury may also be related to the restoration of microvascular circulation in the early phase of the I/R period, thus decreasing the possible damage to testicular tissue in this situation. However, it has been documented that hypo-osmolar mannitol solution was also effective against organ damage in a pancreatic ischemia model; thus, the protective effects of mannitol against I/R injury appear to extend beyond its osmolar properties [17]. Indeed, the antioxidant properties of mannitol alone were able to decrease tissue damage in I/R injury models. There are also several studies documenting a lack of an effect of mannitol on I/R injury. Although mannitol was reported to be effective in I/R injury in renal

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Mannitol has a protective effect on testicular torsion transplantation, it did not show any difference in renal function in partial nephrectomy models [24,25]. Mannitol was also shown to decrease muscular edema in a low-flow ischemia model, but it caused no difference in no-flow ischemia of the gracilis muscle in a dog model [26,27]. Differences between models and treatment doses could be an explanation for the different results. In a study by Weinbroum, mannitol was found to be effective at a specific dose and any increase or decrease reduced the antioxidant activity of the regimen [17]. To eliminate this possible bias, we used a dose that has been shown to have antioxidant activity in I/R injury models, and we were able to observe a protective effect of mannitol infusion against testicular torsion in rats. Evaluating the proper dosage of mannitol for testicular torsion may be a meaningful idea for future research. We did not evaluate the contralateral testis, which was a limitation of our study. The effect of testicular torsion on the contralateral testis is a debatable subject. We were not able to make a conclusion about this issue, because of our study design. Mannitol infusion may also be given prior to detorsion in clinical use. Most patients with testicular torsion present to emergency units, and there is a time delay between diagnosis and treatment; mannitol infusion during this period may have a protective effect on testicular structure. As we did not have a group for this, it is not possible to reach any conclusion regarding the protective effect of mannitol infusion prior to detorsion. This subject can be examined in future research.

Conclusions In conclusion, mannitol infusion had a protective effect against I/R injury in testicular torsion in rats. Unlike other antioxidant molecules that have toxic side effects, mannitol usage may be a solution to decreasing I/R injury in testicular torsion.

Conflict of interest None.

Funding This study was financed by a scientific research project of Namik Kemal University, Tekirdag, Turkey.

References [1] Williamson RC. Torsion of the testis and allied conditions. Br J Surg 1976;63(6):465e76. [2] C ¸ ay A, Alver A, Ku cu ¨¸ ¨k M, Is‚ik O, Eminagaoglu MS, Karahan SC, et al. The effects of N-Acetylcysteine on antioxidant enzyme activities in experimental testicular torsion. J Surg Res 2006; 131:199e203. [3] Dokmeci D, Inan M, Basaran UN, Yalcin O, Aydogdu N, Turan FN, et al. Protective effect of L-carnitine on testicular ischemia-reperfusion injury in rats. Cell Biochem Funct 2006; 25:611e8. [4] Aktoz T, Kanter M, Aktas C. Protective effects of quercetin on testicular torsion/detorsion-induced ischaemia-reperfusion injury in rats. Andrologia 2010;42(6):376e83.

7 [5] Kanter M. Protective effects of melatonin on testicular torsion/detorsion-induced ischemia-reperfusion injury in rats. Exp Mol Pathol 2010;89(3):314e20. [6] Ozbal S, Ergur BU, Erbil G, Tekmen I, Bagrıyanık A, Cavdar Z. The effects of a-lipoic acid against testicular ischemiareperfusion injury in Rats. Sci World J 2012;2012:489248. http://dx.doi.org/10.1100/2012/489248. [7] Zager RA, Mahan J, Merola AJ. Effects of mannitol on the postischemic kidney. Biochemical, functional, and morphologic assessments. Lab Invest 1985;53:433e42. [8] England MD, Cavarocchi NC, O’Brien JF, Solis E, Pluth JR, Orszulak TA, et al. Influence of antioxidants (mannitol and allopurinol) on oxygen free radical generation during and after cardiopulmonary bypass. Circulation 1986;74(III):134e7. [9] Haraldsson G, So ¨rensen V, Nilsson U, Pettersson S, Rashid M, Scherste ´n T, et al. Effect of pre-treatment with desferrioxamine and mannitol on radical production and kidney function after ischemia-reperfusion. A study on rabbit kidneys. Acta Physiol Scand 1995;154:461e8. [10] Weinbroum AA, Hochhauser E, Rudick V, Kluger Y, Karchevsky E, Graf E, et al. Multiple organ dysfunction after remote circulatory arrest: common pathway of radical oxygen species? J Trauma 1999;47:691e8. [11] Hsu SM, Raine L, Fanger H. Use of avidin-biotinperoxidase complex (ABC) in immunperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577e80. [12] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193(1):265e75. [13] Prajda N, Weber G. Malign transformation-linked imbalance: decreased XO activity in hepatomas. FEBS Lett 1975;59: 245e9. [14] Adivarekar PK, Bhagwat SS, Raghavan V, Bandivdekar AH. Effect of lomodex-MgSO4 in the prevention of reperfusion injury following unilateral testicular torsion: an experimental study in rats. Pediatr Surg Int 2005;21:184e90. [15] Barlas M, Hatiboglu C. The effect of nitric oxide in testicular ischemia-reperfusion injury. Int Urol Nephrol 2002;34: 81e6. [16] Bulkley GB. Free radical-mediated reperfusion injury: a selective review. Br J Cancer 1987;55(Suppl. 8):66e73. [17] Weinbroum A. Mannitol prevents acute lung injury after pancreas ischemia-reperfusion: a dose-response, ex vivo study. Lung 2009;187:215e24. [18] Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012;24:981e90. [19] Filho DW, Torres MA, Bordin AL, Crezcynski-Pasa TB, Boveris A. Spermatic cord torsion, reactive oxygen and nitrogen species and ischemia-reperfusion injury. Mol Asp Med 2004;25: 199e210. [20] Turner TT, Tung KSK, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997;57:1267e74. [21] Rudehill A, Gordon E, Ohman G, Lindqvist C, Andersson P. Pharmacokinetics and effects of mannitol on hemodynamics, blood and cerebrospinal fluid electrolytes, and osmolality during intracranial surgery. J Neurosurg Anesthesiol 1993;5: 4e12. [22] Burke AM, Quest DO, Chien S, Cerri C. The effects of mannitol on blood viscosity. J Neurosurg 1981;55:550e3. [23] Magovern Jr GJ, Bolling SF, Casale AS, Bulkley BH, Gardner TJ. The mechanism of mannitol in reducing ischemic injury: hyperosmolarity or hydroxyl scavenger? Circulation 1984;70(3 Pt 2):I91e5. [24] Porras I, Gonzalez-Posada JM, Losada M, Jordano A, Lorenzo V, Gonzalez-Miranda F, et al. A multivariate analysis

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8 of the risk factors for post-transplant renal failure. Transpl Proc 1992;24:52e3. [25] Omae K, Kondo T, Takagi T, Iizuka J, Kobayashi H, Hashimoto Y, et al. Mannitol has no impact on renal function after open partial nephrectomy in solitary kidneys. Int J Urol 2014;21(2):200e3.

O. Kurt et al. [26] Shah DM, Powers Jr SR, Stratton HH, Newell JC. Effects of hypertonic mannitol on oxygen utilization in canine hind limbs following shock. J Surg Res 1981;30:593e601. [27] Faust KB, Chiantella V, Vinten-Johansen J, Meredith JH. Oxygen derived free radical scavengers and skeletal muscle ischemic/reperfusion injury. Am Surg 1988;54:709e19.

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Mannitol has a protective effect on testicular torsion: An experimental rat model.

Testicular torsion is an emergency condition that causes testicular injury. Any treatment opportunity reducing the destructive effect of testicular to...
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