Protective Effects of Hydrogen Rich Saline Solution on Experimental Testicular Ischemia-Reperfusion Injury in Rats

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1 Protective Effects of Hydrogen Rich Saline Solution on Experimental Testicular Ischemia-Reperfusion Injury in Rats Dapeng Jiang, Dongzhen Wu, Yubo Zhang, Bo Xu, Xuejun Sun and Zhaozhu Li* From the Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin and Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University (XS), Shanghai, People s Republic of China Purpose: We examined the effectiveness of hydrogen rich saline solution on the prevention of testicular damage induced by ischemia/reperfusion in rats. Materials and Methods: Male Sprague-Dawley rats were divided randomly into 4 groups, including group 1 sham operated, group 2 torsion-detorsion, group 3 torsion-detorsion plus saline and group 4 torsion-detorsion plus hydrogen rich saline solution. Testicular torsion was performed by rotating the left testis 720 degrees clockwise for 4 hours. Reperfusion was allowed for 4 hours. Hydrogen rich saline solution (5 ml/kg) was injected intraperitoneally in rats in group 4 15 minutes before the start of detorsion. Rats were sacrificed after 4-hour initiation of detorsion. Left orchiectomy was done for histopathological examination and biochemical assay. Results: The testicular injury score in groups 2 and 3 was significantly lower than in sham operated group 1 but higher in group 4 with hydrogen rich saline than in group 2 with torsion-detorsion. The apoptosis index was significantly increased in groups 2 and 3. Hydrogen rich saline solution treatment significantly decreased the apoptosis index. A significant increase in malondialdehyde and a decrease in superoxide dismutase activity were observed in groups 2 and 3. In group 4 malondialdehyde was significantly lowered and superoxide dismutase activity was significantly improved compared with groups 2 and 3. Conclusions: Results provide a biochemical and histopathological basis for the action of hydrogen rich saline solution as a therapeutic agent for testicular damage induced by ischemia/reperfusion injury. Abbreviations and Acronyms HRSS hydrogen rich saline solution I/R ischemia/reperfusion MDA malondialdehyde ROS reactive oxygen species SOD superoxide dismutase TD torsion-detorsion Submitted for publication September 9, Study received Harbin Medical University institutional animal care and use committee approval. Supported by Postdoctoral Found of Harbin Medical University Grant BS * Correspondence: Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Rd., Harbin, People s Republic of China (telephone: ; FAX: ; zhaozhu247@163.com). Key Words: testis, spermatic cord torsion, reperfusion injury, hydrogen, reactive oxygen species TESTICULAR torsion is an urgent urological disease that usually affects children and adolescents. It can lead to biochemical and morphological changes caused by I/R injury to testicular tissue. Contralateral testicular function may also be damaged by unilateral testicular torsion during puberty. 1 TD also decreases the ability of spermatogenesis, alters the production of various hormones and induces infertility. 2 4 Testicular damage after acute spermatic cord twisting is related to the duration of ischemia and the severity of torsion. 5 The main pathophysiology of testicular torsion is testicular I/R injury. 6 A possible cause of testicular injury is the I/R injury attributable to the activation of neutrophils, inflammatory cytokines and adhesion molecules with increased thrombogenicity and generation of oxygen derived free radicals. 4,7 Testicular torsion causes a significant increase in the production of superoxide anion, hydroxyl radical, hydrogen perox /12/ /0 Vol. 187, , June 2012 THE JOURNAL OF UROLOGY Printed in U.S.A by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC. DOI: /j.juro

2 2250 PROTECTIVE EFFECTS OF HYDROGEN RICH SALINE ON TESTICULAR ISCHEMIA-REPERFUSION ide and nitric oxide during the reperfusion period. Germ cell DNA is particularly sensitive to oxidative damage. 8 ROS cause endothelial damage and germinal cell necrosis by interacting with proteins, lipids and nucleic acids, causing cellular dysfunction and even cell death, membrane lipid peroxidation, protein denaturation and DNA impairment. 4,9 Mammalian testes are extremely sensitive to oxidative free radical damage. 4 For these reasons a chemical compound, such as an antioxidant, has been used to protect the testes from I/R injury in experimental animals. 10 H 2, which could selectively reduce the hydroxyl radical, is considered a potent free radical scavenger with effective protection against tissue damage, such as the transient cerebral hypoxia-ischemia and cardiac injury induced by I/R. 11,12 A recent study revealed that HRSS has a protective effect on intestinal I/R against intestinal contractile dysfunction and damage. 13 This protective effect is largely due to its ability to limit lipid oxidation and apoptosis. 13 However, to our knowledge no group has investigated the role of HRSS in testicular TD injury. Based on these facts we hypothesized that hydrogen rich saline would be a reasonable way to protect against testicular I/R injury. We investigate the potential protective and ameliorating effect of hydrogen rich saline to treat testicular I/R injury in rats using biochemical and histopathological parameters. MATERIALS AND METHODS Animal Preparation All rat experiments were approved by the Harbin Medical University animal care and use committee. A total of 60 male Sprague-Dawley rats weighing between 100 and 120 gm were used in this study. Rats were housed with free access to food and water under a natural day/night cycle. A 1-month acclimatization period was allowed before any experimental procedures. Experimental Protocol HRSS was prepared by gassing with hydrogen, as described previously. 14 Hydrogen was dissolved in normal saline solution for 6 hours under high pressure (0.4 MPa). The saturated hydrogen saline was stored under atmospheric pressure at 4C in an aluminum foil bag with no dead volume. The hydrogen concentration was measured by gas chromatography according to the method described by Ohsawa et al. 15 Rats were fasted for 24 hours before the experiment but had free access to water. Rats were randomly divided into 4 groups, including a sham operated, a TD, a TD plus saline and a TD plus HRSS group. On the day of surgery the rats were anesthetized with xylazine and ketamine. All operations were done under sterile conditions. A mid scrotal incision was made. The tunica vaginalis was opened and the left testis was delivered to the surgical field. Testicular torsion was performed according to the method of Turner et al. 16 In the sham operated group the left testis underwent physical manipulation, a 4-zero silk suture was placed through the tunica albuginea and the testis was replaced in the scrotum. The wound was closed using a 4-zero silk suture. In the TD group the left testis was rotated 720 degrees clockwise and maintained by fixing the testis to the scrotum with a 4-zero silk suture placed through the tunica albuginea. After 4 hours of torsion the testis was counter rotated to the natural position and reinserted in the scrotum. In the TD plus saline group the same surgical procedure was done as in the TD group but saline (5 ml/kg) was injected intraperitoneally for 15 minutes before reperfusion. This dose was based on another study of this kind in the literature. 11 In TD plus HRSS group the same surgical procedure was done as in the TD group. In addition, 5 ml/kg HRSS were injected intraperitoneally in the rats 15 minutes before the start of detorsion. The animals were sacrificed by an overdose of sodium pentobarbital (200 mg/kg) after 4-hour initiation of detorsion. Left orchiectomy was done for tissue histopathological examination and biochemical assay. Histopathology Testicular specimens were individually fixed in 10% formaldehyde, embedded, sectioned and stained with hematoxylin and eosin. To minimize variation during the scoring process testicular tissue was evaluated in random order by standard light microscopy by a single histopathologist blinded to the rat experimental group. Testicular injury and histological features of the various groups were graded according to the method of Yang et al. 17 Testicular injury was determined by certain characteristics, including hemorrhage, edema and germinal cell sloughing. Each characteristic was scored on a scale of 0 absent, 1 mild, 2 moderate and 3 severe. We determined the score by evaluating 5 sites in the same histological section. A score of 0 to 9 was assigned to each tissue according to histological features. Apoptosis Assessment For cell death germ cell apoptosis was examined using the TUNEL assay. Only circular tubular cross sections cut in bold face were studied. To quantify the incidence of apoptosis we defined the apoptosis index as the mean number of TUNEL positive cells per seminiferous tubule per group. Apoptotic cells in tubules were recorded using light microscopy at 200 magnification. Biochemical Analysis To investigate lipid peroxidation we measured the lipid peroxide marker MDA in rat testes. For the MDA assay a 50 mg testicular tissue sample was homogenized in 0.15 mol/l KCl solution. After the homogenate was centrifuged at 3,000 rpm the MDA concentration in tissue homogenate supernatant was measured by colorimetric assay based on the interaction of barbituric acid with MDA. The colored reaction of 1,1,3,3-tetraethoxypropane served as the primary standard. Absorbance was measured at 532 nm. Results are shown as nmol MDA/mg protein.

3 PROTECTIVE EFFECTS OF HYDROGEN RICH SALINE ON TESTICULAR ISCHEMIA-REPERFUSION 2251 SOD activity was measured according to the method of Sun et al 18 by reducing nitroblue tetrazolium using the xanthine-xanthine oxidase system, which is a superoxide generator. One U SOD was defined as enzyme activity leading to 50% inhibition. Statistical Analysis Data are shown as the mean SD. Analysis was done with the Kruskal-Wallis and Mann-Whitney U tests using SPSS 11.0 with p 0.05 considered statistically significant. RESULTS No adverse affect of HRSS on overall rat well-being was apparent in either group. There were no significant differences among the 4 groups in water intake, food consumption, body weight or growth pattern during the experimental period. Histopathological Findings Figure 1 shows histopathological findings for each group using hematoxylin and eosin staining. Testicular tissues in the sham operated group demonstrated normal architecture of the seminiferous tubules and interstitium (fig. 1). However, interstitial edema, dilatation and germinal cell sloughing in the tubules were observed in the TD and TD plus saline groups. Testicular tissue in the TD plus HRSS group showed an improved histological appearance (fig. 1). Testicular injury scores paralleled histological findings. Figure 2 shows scores for the testes in each group. Scores in the TD and TD plus saline groups were significantly decreased compared to those in the sham operated group (p 0.01, fig. 2). Scores Figure 2. Mean SD testicular injury score and apoptosis index of testicular tissue in sham operated (sham), TD, TD plus saline and TD plus HRSS groups. Asterisk indicates significantly decreased or increased vs sham operation (p 0.01). Pound sign indicates p 0.01 vs TD and TD plus saline. were significantly higher in the TD plus HRSS group compared with the TD and TD plus saline groups (p 0.01, fig. 2). Germ Cell Apoptosis Evaluation Figure 3 shows TUNEL positive cell findings in all groups. Quantitative assessment of germ cell apopto- Figure 1. Representative photomicrographs show stained sections of rat testes with no histological abnormality in sham operated group (Sham), interstitial edema, evident hemorrhage between seminiferous tubules and germinal cell sloughing (arrows) in TD and TD plus saline groups, and improved histological appearance in TD plus HRSS group. H & E, reduced from 100. Figure 3. TUNEL staining reveals apoptotic nuclei in seminiferous tubules, including fewer apoptotic nuclei in testicular tissue of sham operated group (Sham) and significantly increased apoptotic germ cells in TD and TD plus saline groups. HRSS significantly decreased number of apoptotic nuclei. Reduced from 200.

4 2252 PROTECTIVE EFFECTS OF HYDROGEN RICH SALINE ON TESTICULAR ISCHEMIA-REPERFUSION sis index using the TUNEL assay confirmed histopathological results. Testicular tissue in the sham operated group showed fewer TUNEL positive cells (fig. 3). The TD and TD plus saline groups had a significantly increased mean number of TUNEL positive cells per seminiferous tubule (p 0.01, fig. 2). HRSS treatment of rats markedly decreased the apoptosis index compared with that in the TD and TD plus saline groups (p 0.01, fig. 2). Biochemical Results Figure 4 shows testicular MDA levels and SOD activity in all groups. The MDA concentration in the TD and TD plus saline groups was significantly increased compared to that in the sham operated group (p 0.01, fig. 4). HRSS treatment significantly decreased tissue MDA. A significant decrease was found in SOD activity in the TD and TD plus saline groups compared with the sham operated group (p 0.01, fig. 4). SOD activity was increased in the testes in the TD plus HRSS group (p 0.01, fig. 4). There was no significant difference in tissue MDA or SOD activity between the TD and the TD plus saline groups. DISCUSSION TD is an urgent surgical disease seen mostly in newborns, children and male adolescents. The pathophysiological mechanism of testicular damage due to testicular torsion is I/R injury. Although it necessitates emergency surgical detorsion to avoid a loss of germ cells, testicular injury starts during ischemia and is Figure 4. Mean SD MDA and SOD in testicular tissue of sham operated, TD, TD plus saline and TD plus HRSS groups. Asterisk indicates p 0.01 vs sham operation. Pound sign indicates p 0.01 vs TD and TD plus saline. exacerbated during reperfusion. 19 Reperfusion involves the generation of toxic ROS with the return of blood flow after a period of ischemia. Increased ROS production overwhelms the capacity of endogenous free radical scavengers. 20 ROS can cause lipid peroxidation in cellular and mitochondrial membranes. Lipid peroxidation in the membranes changes membrane permeability or disrupts membrane integrity and causes DNA damage to germ cells. 20 Thus, medical therapy directed toward the mediators responsible for reperfusion damage should be used to decrease the testicular injury. Recently investigators noted that H 2 has protective properties against free radical attack. It has been widely used in many studies of I/R injury due to its antioxidant characteristics. HRSS has a protective effect against acute pancreatitis due to its ability to inhibit oxidative stress, apoptosis and nuclear factor- B activation. 21 It can also attenuate brain, intestinal and myocardial I/R injury Although H 2 has been investigated for multiple purposes, to our knowledge its effect on I/R injury of the testes during TD has not yet been evaluated. We evaluated the effects of H 2 on testicular damage in a rat testicular I/R injury model. We measured various parameters related to testicular damage after administering HRSS. Molecular hydrogen, which is continuously produced by colonic bacteria in the body, normally circulates in blood without any side effects. 22,23 Hydrogen diffuses rapidly into tissue and could be incorporated in the body by drinking and inhalation. 24,25 Dissolving hydrogen in physiological saline makes it easy to safely deliver intraperitoneally. No adverse affect of HRSS on the rats was apparent in this study. Thus, it may have great potential for clinical use. In this study testicular TD induced germ cell degeneration and impaired spermatogenesis. The testicular injury score was decreased in the TD and TD plus saline groups compared with that in the sham operated group. HRSS ameliorated and almost normalized I/R induced histological damage in the testes in our study. All of these findings were statistically significant. We histologically confirmed the protective effect of HRSS on I/R injury to the testes. HRSS has significantly improved the post-ischemic functional recovery of rat hearts, which was paralleled by a significant attenuation of cardiac cell apoptosis. 12 Germ cell specific apoptosis causes permanent loss of spermatogenesis in TD cases. 26 On TUNEL assay rats in our TD and TD plus saline groups showed a significantly increased number of TUNEL positive cells compared to that in sham operated rats. HRSS treatment significantly decreased the germ cell apoptosis index compared with the TD group when assessed 4 hours after detorsion.

5 PROTECTIVE EFFECTS OF HYDROGEN RICH SALINE ON TESTICULAR ISCHEMIA-REPERFUSION 2253 MDA is a product of peroxidative decomposition of polyenic fatty acids in the lipid peroxidation process. It is widely used as a reliable marker of tissue damage. Intraperitoneal injection of HRSS before reperfusion significantly decreased plasma and myocardial MDA in rats exposed to I/R injury. 12 We observed that the tissue MDA concentration was significantly increased in the TD and TD plus saline groups. We also noted that HRSS treatment decreased the MDA concentration. Intraperitoneal administration of HRSS 15 minutes before detorsion normalized MDA to a level that was significantly improved compared to that of the TD group. SOD is a key component in cell growth, differentiation and protection. In I/R injury SOD cannot prevent the escape of ROS, especially in mitochondria. The antioxidant effect of H 2 is supported by the finding that hydrogen can protect myocardial degeneration from radiation induced injury via increasing myocardium endogenous SOD in vivo. 27 In our study SOD activity decreased in the TD and TD plus saline groups but it was preserved by HRSS treatment. These effects were due to the antioxidant property of hydrogen, showing that hydrogen acted as a good scavenger against free radical generation. CONCLUSIONS Although further studies are necessary to determine the mechanisms by which HRSS ameliorates testicular damage due to testicular torsion, our results indicate substantial new aspects of this field and provide a rationale for the potential therapeutic value of hydrogen for treating testicular damage induced by I/R injury. For this purpose further clinical studies are needed. ACKNOWLEDGMENTS Dr. Bo Xu provided technical assistance. REFERENCES 1. 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