Journal of Pediatric Surgery xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model Erdal Türk a,⁎, İrfan Karaca a, Emine Ozcinar b, Aydan Celebiler c, Hülya Aybek d, Ragıp Ortac e, Ahmet Güven f a
Department of Pediatric Surgery, Izmir University, Faculty of Medicine, Izmir, Turkey Department of Embriology and Histology, Izmir University, Faculty of Medicine, Izmir, Turkey Department of Biochemistry, Izmir University, Faculty of Medicine, Izmir, Turkey d Department of Biochemistry, Pamukkale University, Faculty of Medicine, Denizli, Turkey e Department of Pathology, Izmir University, Faculty of Medicine, Izmir, Turkey f Department of Pediatric Surgery, Gulhane Military Medical Faculty, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 9 September 2014 Received in revised form 5 January 2015 Accepted 13 January 2015 Available online xxxx Key words: Ovarian tissue Hypothermia Ischemia Adnexal torsion/detorsion injury
a b s t r a c t Purpose: Much attention has been given to hypothermia as it is effective in inhibiting inflammatory responses and also ischemia/reperfusion injury. Therefore, the aim of this study was to evaluate the effect of hypothermia on torsion/detorsion injury in rats. Methods: Twenty-eight rats were randomly divided into four groups of sham-operated (SG), adnexal torsion/ detorsion group (TG), adnexal torsion/detorsion + hypothermia group (THG) and hypothermia group (HG). In the SG group, right ovaries were excised after 3-h fixation to abdominal wall. In the TG, right adnexal underwent 720° torsion in a counterclockwise direction for 3 h and then excised after 3-h detorsion period. In the THG, after 3-h torsion period, ovaries were immediately subjected to hypothermia (4 °C) for 30-min and they were excised after 3-h detorsioned period. In the HG, the right ovaries were subjected to hypothermia for 30-min and excised after 3-h fixation period. One half of each ovary was immediately stored for antioxidant enzyme activity and tissue lipid peroxidation. The remainder was fixed for histopathological examination. Results: Adnexal torsion and detorsion significantly increased the tissue level of Malondialdehyde, Superoxide dismutase and Reduced glutathione. On the other hand, hypothermia significantly reduced these oxidative stress parameters. The histopathological changes were less in the THG group; these changes were not statistically different from the other groups. Conclusion: The results of this study suggested that hypothermia inhibited the production of oxidative stress in the ovaries subjected to torsion/detorsion injury. © 2015 Elsevier Inc. All rights reserved.
Ovarian torsion in childhood and adolescence is a rare entity; approximately, 15% of cases occur during infancy and childhood [1]. Early diagnosis and treatment are required to preserve fertility [2]. Traditionally, it is managed through oophorectomy due to the concern for thromboembolic phenomena and missing malignancy, and mostly due to misdiagnosing that the ovary is dead and needs to be removed. But multiple studies have found that many women retain ovarian function following detorsion, even if the ovary is blue or black. It has been reported that the rate of follicular development after detorsion is 80% or higher [3]. Detorsion of the torsioned adnexes has some local and systemic consequences due to reperfusion of ovaries. During the detorsion process, reperfusion of ovary causes the activation and adhesion of neutrophils, with the release of proinflammatory substances and the formation of free radicals, which are nitrogen-derived reactive nitrogen species ⁎ Corresponding author at: Yeni Girne Bulvarı 1825 Sok., No:12 Karsiyaka/Izmir. Tel.: +90 232 399 50 50; fax: +90 232 367 05 59. E-mail address: [email protected] (E. Türk).
(RNS) or oxygen-derived reactive oxygen species (ROS) [4,5]. These radicals are formed continuously in a particular amount and neutralized by endogenous antioxidant enzymes. During reperfusion, however, an excessive concentration of RNS and ROS and their toxic products causes DNA and cellular damage and also lipid peroxidation in the cellular and mitochondrial membranes [6,7]. Many therapeutic modalities have been evaluated to prevent ischemia reperfusion injury (IRI) in ovarian torsion [1,8–17]; however, none are effective in routine clinical practice. Although the protective effect of hypothermia on ischemic tissues has been recognized for some 65 years, recently, therapeutic hypothermia has been accepted as a novel treatment which increased survival chances and quality for patients suffering from ischemic insults such as cardiac arrest, stroke, and neurogenic fever following brain trauma [18–20]. Several studies have also demonstrated that hypothermia can inhibit IR-induced oxidative stress and apoptosis in different organs subjected to IRI [21–24]. Although it is reported that hypothermia reduces the degree of testicular damage and restrains the production of oxygen free radicals, there is no study evaluating the effect of hypothermia on post-torsion
http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011 0022-3468/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
2
E. Türk et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
ovarian damage. Therefore, this study was designed to investigate whether hypothermia has protective effects on the ovary in an experimental model of ovarian tortion/detorsion injury in rats. 1. Material and methods
1.3.2. Determination of superoxide dismutase (SOD) The SOD activity was determined as described by Fridovich [28]. This method involved using xanthine and xanthine oxidase to generate superoxide radicals, which reacted with 2-(4-iodophenyl)-3-(4-nitro phonol-sphenyl tetrazolium chloride) to form a red formazon dye. The SOD activity was then measured by the degree of inhibition of this reaction.
1.1. Animals and experimental design The experiments were performed in adherence to the Declaration of Helsinki and by approval of the Ethics Committee of Ege University (Ethical Committee no: 2013-031). Twenty-eight Wistar Albino female rats were randomly divided into 4 groups; sham group (SG), torsion/ detorsion group (TG), torsion/detorsion group and hypothermia group (THG) and hypothermia group without torsion/detorsion group (HG). After fasting overnight, rats were anesthetized with intramuscular ketamine hydrochloride (80 mg/kg, Ketalar; Eczacibasi, İstanbul, Turkey) and 5 mg/kg Xylazine (Rhompun, Abdi İbrahim, Istanbul, Turkey). In the SG, right ovaries were fixed to abdominal wall for 3 h and excised. In the TG, right adnexes underwent 720° torsion in a counterclockwise direction and fixed to abdominal wall for 3 h, then right ovaries were excised after a 3-h detorsion period. In the THG, ovaries were subjected to 3-h torsion period as in the TG group. At the end of torsion period, ovaries were placed in a bag and the ovary targettemperature (4 °C) was achieved through superfusion of iced saline solution for 30 min. The ovary temperature was assessed with an intraparenchymal probe connected to a specific thermometer (ThermoJack, Probe Thermometer, TFA Dostmann GmbH&Co. Germany). After the hypothermia period, ovaries were excised at the end of 3-h detorsioned period. In the HG, right ovaries were fixed to abdominal wall for 3 h, then subjected to hypothermia for 30 min and excised after 3 h. The rats were placed on a heating pad kept at 39 °C to maintain constant body temperature throughout the experimental period. Excised ovaries were immediately bisected sagittally. One half of each ovaries was immediately stored at −80 °C for antioxidant enzyme activity and tissue lipid peroxidation levels; the remainder was fixed in 10% formaldehyde for histopathological examination. 1.2. Histopathological evaluations The ovarian samples were preserved and fixed with 10% formalin. After tissue processing, all segments were embedded in paraffin. Tissues were sectioned in 4–5-μm pieces and stained with routine hematoxylin and eosin stain. The specimens were examined under a light microscope by the same pathologist who was blinded to the study. Histopathological examination of ovarian and periovarian sections was performed for the presence of Congestion (C), hemorrhage (H), interstitial edema (IE), and polymorphonuclear neutrophilic (PMN) infiltrations. Histopathological findings were graded semiquantitively for each parameter separately as follows: 0 represented no pathologic findings, and 1, 2, and 3 represented pathologic findings of less than 25%, 25% to 75%, and greater than 75% of the ovarian section, respectively [25]. 1.3. Biochemical evaluations 1.3.1. Determination of malondialdehyde (MDA) levels All samples were preserved in a deep freezer (−80 °C). After washing with 0.9% NaCl, tissue was homogenized with 1-mL 0.9% NaCl solution in ice. Homogenized tissue was centrifuged at 1500 ×g for 10 min at 4 °C. Supernatants were used for protein and MDA examinations. Protein level was measured using the method of Lowry [26]. Malondialdehyde levels, indicating lipid peroxidation, were measured by the method described by Armstrong and Al-Awadi [27], which was modified from the Yagi method. The calibration curve was prepared with 1,1,3,3tetraethoxypropane standards of 1 to 25 nmol/L dilutions. The results were expressed in nanomoles per milligram protein.
1.3.3. Determination of reduced glutathione (GSH) Reduced glutathione levels in tissue homogenates were determined with the sulfhydryl group analysis as defined by Ellman. Following sample deproteinization, the absorbance of the colored complex that developed as a result of the supernatant's reaction with Ellman's color reactive (40% DTNB in 1% w/v sodium citrate) was measured against the reactive blank by spectrophotometry at 412 nm. The GSH concentrations were calculated using the GSH curve. The results were expressed as nmol GSH/mg protein. 1.3.4. Statistical analysis The data obtained from the experiments were analyzed with Kruskal–Wallis test and Mann–Whitney U test (SPSS 15.0; SPSS, Chicago, IL). P b .05 was considered as significant. 2. Results All animals survived throughout the experiment period. The outcome of oxidative stress parameters is shown in Table 1. Adnexal torsion/ detorsion significantly increased MDA levels, suggesting increased lipid peroxidation, and increased SOD and GSH levels, which also suggest increased production of free radicals in the ovarian tissue (p b 0.05; SG vs. TG). On the other hand, hypothermia significantly reduced the lipid peroxidation and restrained the antioxidant enzyme activities in the THG group (p b 0.05; THG vs. TG). There were no significant differences between SG and HG group in terms of biochemical values. The table shows total tissue damage scores while Fig. 1 to 4 shows morphological findings of the ovaries in the 4 experimental groups. The ovaries from the control operation group showed no pathologic changes (Fig. 1A). The ovaries of the TG and THG groups showed moderate congestion, hemorrhage, and mild edema on histopathological examination (Fig. 1B, C and D). Although the ovaries of the animals in the TGH group showed less morphological changes of hemorrhage and congestion than the TG, there were no statistically differences in the groups in terms of tissue damage scores. 3. Discussion To the best of our knowledge, this is the first report investigating the effect of hypothermia on adnexal torsion/detorsion injury in an experimental rat ovary model. We determined by biochemical evaluations that exposing an ovary that had undergone ovarian torsion to hypothermia before the detorsion procedure decreased oxidative stress markers. It showed that hypothermia decreased histopathological changes in Table 1 Median values of oxidative markers and total tissue damage score in groups. Groups
MDA (nmol/mg prot)
GSH (μmol/mg prot)
SOD (U/mg prot)
Total tissue damage score
SG TG THG SHG
0.891 1.667 0.928 0.884
0.977 0.557 0.822 0.961
7.214 5.357 6.500 7.142
0.285 2.285 2.142 0.428
± ± ± ±
0.313 0.145a 0.598c 0.639
± ± ± ±
0.570 0.590a 0.386d 0.247
± ± ± ±
0.859 0.852b 0.595 0.748
± ± ± ±
0.487 0.755 0.377 0.534
Control and IRI: IRI and hypothermia: a p b 0.001. b p b 0.01. c p b 0.001. d p b 0.01.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
E. Türk et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
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Fig. 1. A: Score 0, ovarian tissue with no congestion, edema or bleeding B: Score 1, edema and congestion in less than 25% of subjects, C: Score 2, edema, bleeding and congestion in about 40% of the area, D: Score 3, Interstitial bleeding more than 75% at the subject (×100, HE).
ovaries that had undergone ovary torsion, although this decrease was not statistically different from that of the untreated group. MDA as an oxidative stress index in the ovary tissue was measured. The result showed that adnexal torsion/detorsion caused a considerable increase in the ovarian tissue levels of MDA. It is known that torsion of adnexa decreased the blood flow in the ovary and resulted in elevated lactic acid and hypoxanthine as a result of adenosine triphosphate breakdown and lipid peroxidation. Ovarian detorsion causes formation of free oxygen radicals peroxidizing membrane lipids, oxidizing DNA, and denaturing enzyme proteins, which may lead to cellular injury. Various studies on lipid peroxidation report that MDA level is increased in ovarian torsion/detorsion injury. The results of this study are also consistent with those of previous studies. SOD and GSH enzyme activities measured to evaluate the tissue antioxidant system showed that both enzyme activities increased in the ovaries subjected to torsion/ detorsion injury. Under physiological conditions, antioxidant defense mechanism protects the ovary cells against ischemia/reperfusion injury. However, during torsion/detorsion of ovary, these natural defenses may respond to the excessive generation of free oxygen radicals by increasing their activities as in our study. The ovary-sparing management approach increased the need for new modalities to decrease adnexal torsion/detorsion injury. Few chemicals and drugs such as melatonin, vitamin C, mannitol, verapamil, and pentoxiphylline have been studied to prevent IRI of the ovary [22,29]. However, none of them have been effective in clinical practice. Although the demonstration that hypothermia is sufficient to offer liver, brain and kidney of rats protection against the deleterious actions of IRI, there are no studies concerning rats’ ovary [22,29,30]. In this study, we found that hypothermia decreased lipid peroxidation and retained antioxidant enzyme activities. There are a few explanations on how hypothermia protects the ovary from tortion/detortion injury. The benefits of using hypothermia before and after reperfusion in ischemia–reperfusion injury have been debated in previous studies. It is claimed that hypothermia applied during reperfusion in extreme injury could cause vasoconstriction [31]. It has also been reported in liver preservation studies that the adhesion of leukocytes and especially neutrophils to endothelial cells during cold ischemia period could cause reperfusion damage [32]. However, some experimental studies have shown
beneficial effects of hypothermia applied during reperfusion in cerebrovascular occlusion or cardiac bypass [33–35]. Locally applied hypothermia decreases the stimulating amino acid level and lactate concentrations and therefore decreases free radical production and increases apoptosis severity during ischemia reperfusion injury. It also suppresses inflammatory mechanisms causing late damage [36]. Hypothermia slows down metabolism and leads to decreased oxygen presentation and carbon dioxide production. We therefore believe that although the venous circulation is blocked together with the arterial circulation in children with ovarian torsion, the hypothermia to be applied to the ovary before detorsion might protect the tissue from reperfusion damage as in other ischemia reperfusion studies. The effect of hypothermia on adnexal torsion/detorsion injury of the ovary was also histologically evaluated. Ovarian injury criteria such as congestion, hemorrhage, interstitial edema, and PMN infiltrations were assessed. Ipsilateral histopathological changes occurred 3–4 h later in the rat ovarian torsion models as in many studies previously conducted [11]. We also found that torsion/detorsion injury for 3 h of ipsilateral ovaries led to histopathological changes in the ovaries. Although there was no statistically difference between treated and untreated groups in terms of total tissue damage scores, we saw that some injury criteria of subjects in the hypothermia group were decreased. But, we assume that long-term follow-up is needed to make further conclusions. This study is inadequate in terms of showing the relationship between the duration of ischemia and the protective effect of hypothermia against adnexal torsion/detorsion injury and in terms of demonstrating the long-term tissue protective effects of hypothermia. However, this study may be a precursor to further studies aiming to show the longterm results of hypothermia in the ovary or other organs with longer periods of ischemia and hypothermia. The other concern is how the hypothermia can be applied to a patient presenting with ovarian torsion in clinical practice. We think that hypothermia therapy can be adapted to both open and laparoscopic surgical techniques. It may also provide some information for studies investigating the decrease of the preparation time to reperfusion with the use of special liquids that will reduce the tissue temperature to + 4° in a shorter time before the torsioned ovary is detorsioned.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
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E. Türk et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
In conclusion, it is evidenced from this study that local hypothermia implemented in rats where ovarian torsion had been performed restricted the levels of oxidative stress products that appeared in the ovary tissue after detorsion and also reduced the tissue damage, but did not result in histopathological changes. This is a pioneering study in terms of showing the effects of hypothermia on the damage in ovarian tissue after torsion, as well as the clinical use of local cooling in the surgical treatment of ovarian torsion.
Conflict of Interest We declare that we have no conflict of interest.
Acknowledgements Many thanks to Gülin Kavakalan, Özgecan Engür, Tulu Batı, Izmir University, Faculty of Medicine, Izmir/TURKEY, for good assistance in the practice of the experimental study.
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[12] Halici Z, Karaca M, Keles ON, et al. Protective effects of amlodipine on ischemia–reperfusion injury of rat ovary: biochemical and histopathologic evaluation. Fertil Steril 2008;90:2408–15. [13] Kara M, Daglioglu YK, Kuyucu Y, et al. The effect of edaravone on ischemia–reperfusion injury in rat ovary. Eur J Obstet Gynecol Reprod Biol 2012;162:197–202. [14] Karaca M, Odabasoglu F, Kumtepe Y, et al. Protective effects of erythropoietin on ischemia/reperfusion injury of rat ovary. Eur J Obstet Gynecol Reprod Biol 2009;144:157–62. [15] Kumtepe Y, Odabasoglu F, Karaca M, et al. Protective effects of telmisartan on ischemia/reperfusion injury of rat ovary: biochemical and histopathologic evaluation. Fertil Steril 2009;93:1299–307. [16] Sahin FK, Cosar E, Koken G, et al. Protective effect of aprotinin on ischemia–reperfusion injury in rat ovary. J Obstet Gynaecol Res 2008;34:794–800. [17] Sak ME, Soydinc HE, Sak S, et al. The protective effect of curcumin on ischemia–reperfusion injury in rat ovary. Int J Surg 2013;11(9):967–70. [18] Bernard SA, Gray TW, Buist MD, et al. Treatment of comatose survivors of out-ofhospital cardiac arrest with induced hypothermia. N Engl J Med 2002;346:557–63. [19] Nagel S, Papadakis M, Hoyte L, et al. Therapeutic hypothermia in experimental models of focal and global cerebral ischemia and intracerebral hemorrhage. Expert Rev Neurother 2008;8:1255–68. [20] Shankaran S, Laptook AR, Ehrenkranz RA, et al. Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J Med 2005;353:1574–84. [21] Almodhen F, He X, Loutochin O, et al. Protective role of hypothermia on ischemia of prepubertal rodent testicle. Urology 2011;77:762.e8-12. [22] dos Santos EB, Koff WJ, Grezzana Filho Tde J, et al. Oxidative stress evaluation of ischemia and reperfusion in kidneys under various degrees of hypothermia in rats. Acta Cir Bras 2013;28:568–73. [23] Belzer FO, Southard JH. Principles of solid-organ preservation by cold storage. Transplantation 1988;45:673–6. [24] Haj M, Shasha SM, Loberant N, et al. Effect of external scrotal cooling on the viability of the testis with torsion in rats. Eur Surg Res 2007;39:160–9. [25] Sagsoz N, Kisa U, Apan A. Ischaemia–reperfusion injury of rat ovary and the effects of vitamin C, mannitol and verapamil. Hum Reprod 2002;17:2972–6. [26] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [27] Armstrong D, al-Awadi F. Lipid peroxidation and retinopathy in streptozotocininduced diabetes. Free Radic Biol Med 1991;11:433–6. [28] Fridovich I. Superoxide dismutases. Adv Enzymol Relat Areas Mol Biol 1974;41:35–97. [29] Biberthaler P, Luchting B, Massberg S, et al. The influence of organ temperature on hepatic ischemia–reperfusion injury: a systematic analysis. Transplantation 2001; 72:1486–90. [30] Behrends M, Hirose R, Serkova NJ, et al. Mild hypothermia reduces the inflammatory response and hepatic ischemia/reperfusion injury in rats. Liver Int 2006;26:734–41. [31] Mowlavi A, Neumeister MW, Wilhelmi BJ, et al. Local hypothermia during early reperfusion protects skeletal muscle from ischemia–reperfusion injury. Plast Reconstr Surg 2003;111:242–50. [32] Monden K, Arii S, Ishiguro S, et al. Involvement of ICAM-1 expression on sinusoidal endothelial cell and neutrophil adherence in the reperfusion injury of coldpreserved livers. Transplant Proc 1995;27:759–61. [33] Canevari L, Console A, Tendi EA, et al. Effect of postischaemic hypothermia on the mitochondrial damage induced by ischaemia and reperfusion in the gerbil. Brain Res 1999;817:241–5. [34] Carroll M, Beek O. Protection against hippocampal CA1 cell loss by post-ischemic hypothermia is dependent on delay of initiation and duration. Metab Brain Dis 1992;7:45–50. [35] Kim JH, Seo M, Suk K. Effects of therapeutic hypothermia on the glial proteome and phenotype. Curr Protein Pept Sci 2013;14:51–60. [36] Alzaga AG, Cerdan M, Varon J. Therapeutic hypothermia. Resuscitation 2006;70: 369–80.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model Erdal Türk a,⁎, İrfan Karaca a, Emine Ozcinar b, Aydan Celebiler c, Hülya Aybek d, Ragıp Ortac e, Ahmet Güven f a
Department of Pediatric Surgery, Izmir University, Faculty of Medicine, Izmir, Turkey Department of Embriology and Histology, Izmir University, Faculty of Medicine, Izmir, Turkey Department of Biochemistry, Izmir University, Faculty of Medicine, Izmir, Turkey d Department of Biochemistry, Pamukkale University, Faculty of Medicine, Denizli, Turkey e Department of Pathology, Izmir University, Faculty of Medicine, Izmir, Turkey f Department of Pediatric Surgery, Gulhane Military Medical Faculty, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 9 September 2014 Received in revised form 5 January 2015 Accepted 13 January 2015 Available online xxxx Key words: Ovarian tissue Hypothermia Ischemia Adnexal torsion/detorsion injury
a b s t r a c t Purpose: Much attention has been given to hypothermia as it is effective in inhibiting inflammatory responses and also ischemia/reperfusion injury. Therefore, the aim of this study was to evaluate the effect of hypothermia on torsion/detorsion injury in rats. Methods: Twenty-eight rats were randomly divided into four groups of sham-operated (SG), adnexal torsion/ detorsion group (TG), adnexal torsion/detorsion + hypothermia group (THG) and hypothermia group (HG). In the SG group, right ovaries were excised after 3-h fixation to abdominal wall. In the TG, right adnexal underwent 720° torsion in a counterclockwise direction for 3 h and then excised after 3-h detorsion period. In the THG, after 3-h torsion period, ovaries were immediately subjected to hypothermia (4 °C) for 30-min and they were excised after 3-h detorsioned period. In the HG, the right ovaries were subjected to hypothermia for 30-min and excised after 3-h fixation period. One half of each ovary was immediately stored for antioxidant enzyme activity and tissue lipid peroxidation. The remainder was fixed for histopathological examination. Results: Adnexal torsion and detorsion significantly increased the tissue level of Malondialdehyde, Superoxide dismutase and Reduced glutathione. On the other hand, hypothermia significantly reduced these oxidative stress parameters. The histopathological changes were less in the THG group; these changes were not statistically different from the other groups. Conclusion: The results of this study suggested that hypothermia inhibited the production of oxidative stress in the ovaries subjected to torsion/detorsion injury. © 2015 Elsevier Inc. All rights reserved.
Ovarian torsion in childhood and adolescence is a rare entity; approximately, 15% of cases occur during infancy and childhood [1]. Early diagnosis and treatment are required to preserve fertility [2]. Traditionally, it is managed through oophorectomy due to the concern for thromboembolic phenomena and missing malignancy, and mostly due to misdiagnosing that the ovary is dead and needs to be removed. But multiple studies have found that many women retain ovarian function following detorsion, even if the ovary is blue or black. It has been reported that the rate of follicular development after detorsion is 80% or higher [3]. Detorsion of the torsioned adnexes has some local and systemic consequences due to reperfusion of ovaries. During the detorsion process, reperfusion of ovary causes the activation and adhesion of neutrophils, with the release of proinflammatory substances and the formation of free radicals, which are nitrogen-derived reactive nitrogen species ⁎ Corresponding author at: Yeni Girne Bulvarı 1825 Sok., No:12 Karsiyaka/Izmir. Tel.: +90 232 399 50 50; fax: +90 232 367 05 59. E-mail address: [email protected] (E. Türk).
(RNS) or oxygen-derived reactive oxygen species (ROS) [4,5]. These radicals are formed continuously in a particular amount and neutralized by endogenous antioxidant enzymes. During reperfusion, however, an excessive concentration of RNS and ROS and their toxic products causes DNA and cellular damage and also lipid peroxidation in the cellular and mitochondrial membranes [6,7]. Many therapeutic modalities have been evaluated to prevent ischemia reperfusion injury (IRI) in ovarian torsion [1,8–17]; however, none are effective in routine clinical practice. Although the protective effect of hypothermia on ischemic tissues has been recognized for some 65 years, recently, therapeutic hypothermia has been accepted as a novel treatment which increased survival chances and quality for patients suffering from ischemic insults such as cardiac arrest, stroke, and neurogenic fever following brain trauma [18–20]. Several studies have also demonstrated that hypothermia can inhibit IR-induced oxidative stress and apoptosis in different organs subjected to IRI [21–24]. Although it is reported that hypothermia reduces the degree of testicular damage and restrains the production of oxygen free radicals, there is no study evaluating the effect of hypothermia on post-torsion
http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011 0022-3468/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
2
E. Türk et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
ovarian damage. Therefore, this study was designed to investigate whether hypothermia has protective effects on the ovary in an experimental model of ovarian tortion/detorsion injury in rats. 1. Material and methods
1.3.2. Determination of superoxide dismutase (SOD) The SOD activity was determined as described by Fridovich [28]. This method involved using xanthine and xanthine oxidase to generate superoxide radicals, which reacted with 2-(4-iodophenyl)-3-(4-nitro phonol-sphenyl tetrazolium chloride) to form a red formazon dye. The SOD activity was then measured by the degree of inhibition of this reaction.
1.1. Animals and experimental design The experiments were performed in adherence to the Declaration of Helsinki and by approval of the Ethics Committee of Ege University (Ethical Committee no: 2013-031). Twenty-eight Wistar Albino female rats were randomly divided into 4 groups; sham group (SG), torsion/ detorsion group (TG), torsion/detorsion group and hypothermia group (THG) and hypothermia group without torsion/detorsion group (HG). After fasting overnight, rats were anesthetized with intramuscular ketamine hydrochloride (80 mg/kg, Ketalar; Eczacibasi, İstanbul, Turkey) and 5 mg/kg Xylazine (Rhompun, Abdi İbrahim, Istanbul, Turkey). In the SG, right ovaries were fixed to abdominal wall for 3 h and excised. In the TG, right adnexes underwent 720° torsion in a counterclockwise direction and fixed to abdominal wall for 3 h, then right ovaries were excised after a 3-h detorsion period. In the THG, ovaries were subjected to 3-h torsion period as in the TG group. At the end of torsion period, ovaries were placed in a bag and the ovary targettemperature (4 °C) was achieved through superfusion of iced saline solution for 30 min. The ovary temperature was assessed with an intraparenchymal probe connected to a specific thermometer (ThermoJack, Probe Thermometer, TFA Dostmann GmbH&Co. Germany). After the hypothermia period, ovaries were excised at the end of 3-h detorsioned period. In the HG, right ovaries were fixed to abdominal wall for 3 h, then subjected to hypothermia for 30 min and excised after 3 h. The rats were placed on a heating pad kept at 39 °C to maintain constant body temperature throughout the experimental period. Excised ovaries were immediately bisected sagittally. One half of each ovaries was immediately stored at −80 °C for antioxidant enzyme activity and tissue lipid peroxidation levels; the remainder was fixed in 10% formaldehyde for histopathological examination. 1.2. Histopathological evaluations The ovarian samples were preserved and fixed with 10% formalin. After tissue processing, all segments were embedded in paraffin. Tissues were sectioned in 4–5-μm pieces and stained with routine hematoxylin and eosin stain. The specimens were examined under a light microscope by the same pathologist who was blinded to the study. Histopathological examination of ovarian and periovarian sections was performed for the presence of Congestion (C), hemorrhage (H), interstitial edema (IE), and polymorphonuclear neutrophilic (PMN) infiltrations. Histopathological findings were graded semiquantitively for each parameter separately as follows: 0 represented no pathologic findings, and 1, 2, and 3 represented pathologic findings of less than 25%, 25% to 75%, and greater than 75% of the ovarian section, respectively [25]. 1.3. Biochemical evaluations 1.3.1. Determination of malondialdehyde (MDA) levels All samples were preserved in a deep freezer (−80 °C). After washing with 0.9% NaCl, tissue was homogenized with 1-mL 0.9% NaCl solution in ice. Homogenized tissue was centrifuged at 1500 ×g for 10 min at 4 °C. Supernatants were used for protein and MDA examinations. Protein level was measured using the method of Lowry [26]. Malondialdehyde levels, indicating lipid peroxidation, were measured by the method described by Armstrong and Al-Awadi [27], which was modified from the Yagi method. The calibration curve was prepared with 1,1,3,3tetraethoxypropane standards of 1 to 25 nmol/L dilutions. The results were expressed in nanomoles per milligram protein.
1.3.3. Determination of reduced glutathione (GSH) Reduced glutathione levels in tissue homogenates were determined with the sulfhydryl group analysis as defined by Ellman. Following sample deproteinization, the absorbance of the colored complex that developed as a result of the supernatant's reaction with Ellman's color reactive (40% DTNB in 1% w/v sodium citrate) was measured against the reactive blank by spectrophotometry at 412 nm. The GSH concentrations were calculated using the GSH curve. The results were expressed as nmol GSH/mg protein. 1.3.4. Statistical analysis The data obtained from the experiments were analyzed with Kruskal–Wallis test and Mann–Whitney U test (SPSS 15.0; SPSS, Chicago, IL). P b .05 was considered as significant. 2. Results All animals survived throughout the experiment period. The outcome of oxidative stress parameters is shown in Table 1. Adnexal torsion/ detorsion significantly increased MDA levels, suggesting increased lipid peroxidation, and increased SOD and GSH levels, which also suggest increased production of free radicals in the ovarian tissue (p b 0.05; SG vs. TG). On the other hand, hypothermia significantly reduced the lipid peroxidation and restrained the antioxidant enzyme activities in the THG group (p b 0.05; THG vs. TG). There were no significant differences between SG and HG group in terms of biochemical values. The table shows total tissue damage scores while Fig. 1 to 4 shows morphological findings of the ovaries in the 4 experimental groups. The ovaries from the control operation group showed no pathologic changes (Fig. 1A). The ovaries of the TG and THG groups showed moderate congestion, hemorrhage, and mild edema on histopathological examination (Fig. 1B, C and D). Although the ovaries of the animals in the TGH group showed less morphological changes of hemorrhage and congestion than the TG, there were no statistically differences in the groups in terms of tissue damage scores. 3. Discussion To the best of our knowledge, this is the first report investigating the effect of hypothermia on adnexal torsion/detorsion injury in an experimental rat ovary model. We determined by biochemical evaluations that exposing an ovary that had undergone ovarian torsion to hypothermia before the detorsion procedure decreased oxidative stress markers. It showed that hypothermia decreased histopathological changes in Table 1 Median values of oxidative markers and total tissue damage score in groups. Groups
MDA (nmol/mg prot)
GSH (μmol/mg prot)
SOD (U/mg prot)
Total tissue damage score
SG TG THG SHG
0.891 1.667 0.928 0.884
0.977 0.557 0.822 0.961
7.214 5.357 6.500 7.142
0.285 2.285 2.142 0.428
± ± ± ±
0.313 0.145a 0.598c 0.639
± ± ± ±
0.570 0.590a 0.386d 0.247
± ± ± ±
0.859 0.852b 0.595 0.748
± ± ± ±
0.487 0.755 0.377 0.534
Control and IRI: IRI and hypothermia: a p b 0.001. b p b 0.01. c p b 0.001. d p b 0.01.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
E. Türk et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
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Fig. 1. A: Score 0, ovarian tissue with no congestion, edema or bleeding B: Score 1, edema and congestion in less than 25% of subjects, C: Score 2, edema, bleeding and congestion in about 40% of the area, D: Score 3, Interstitial bleeding more than 75% at the subject (×100, HE).
ovaries that had undergone ovary torsion, although this decrease was not statistically different from that of the untreated group. MDA as an oxidative stress index in the ovary tissue was measured. The result showed that adnexal torsion/detorsion caused a considerable increase in the ovarian tissue levels of MDA. It is known that torsion of adnexa decreased the blood flow in the ovary and resulted in elevated lactic acid and hypoxanthine as a result of adenosine triphosphate breakdown and lipid peroxidation. Ovarian detorsion causes formation of free oxygen radicals peroxidizing membrane lipids, oxidizing DNA, and denaturing enzyme proteins, which may lead to cellular injury. Various studies on lipid peroxidation report that MDA level is increased in ovarian torsion/detorsion injury. The results of this study are also consistent with those of previous studies. SOD and GSH enzyme activities measured to evaluate the tissue antioxidant system showed that both enzyme activities increased in the ovaries subjected to torsion/ detorsion injury. Under physiological conditions, antioxidant defense mechanism protects the ovary cells against ischemia/reperfusion injury. However, during torsion/detorsion of ovary, these natural defenses may respond to the excessive generation of free oxygen radicals by increasing their activities as in our study. The ovary-sparing management approach increased the need for new modalities to decrease adnexal torsion/detorsion injury. Few chemicals and drugs such as melatonin, vitamin C, mannitol, verapamil, and pentoxiphylline have been studied to prevent IRI of the ovary [22,29]. However, none of them have been effective in clinical practice. Although the demonstration that hypothermia is sufficient to offer liver, brain and kidney of rats protection against the deleterious actions of IRI, there are no studies concerning rats’ ovary [22,29,30]. In this study, we found that hypothermia decreased lipid peroxidation and retained antioxidant enzyme activities. There are a few explanations on how hypothermia protects the ovary from tortion/detortion injury. The benefits of using hypothermia before and after reperfusion in ischemia–reperfusion injury have been debated in previous studies. It is claimed that hypothermia applied during reperfusion in extreme injury could cause vasoconstriction [31]. It has also been reported in liver preservation studies that the adhesion of leukocytes and especially neutrophils to endothelial cells during cold ischemia period could cause reperfusion damage [32]. However, some experimental studies have shown
beneficial effects of hypothermia applied during reperfusion in cerebrovascular occlusion or cardiac bypass [33–35]. Locally applied hypothermia decreases the stimulating amino acid level and lactate concentrations and therefore decreases free radical production and increases apoptosis severity during ischemia reperfusion injury. It also suppresses inflammatory mechanisms causing late damage [36]. Hypothermia slows down metabolism and leads to decreased oxygen presentation and carbon dioxide production. We therefore believe that although the venous circulation is blocked together with the arterial circulation in children with ovarian torsion, the hypothermia to be applied to the ovary before detorsion might protect the tissue from reperfusion damage as in other ischemia reperfusion studies. The effect of hypothermia on adnexal torsion/detorsion injury of the ovary was also histologically evaluated. Ovarian injury criteria such as congestion, hemorrhage, interstitial edema, and PMN infiltrations were assessed. Ipsilateral histopathological changes occurred 3–4 h later in the rat ovarian torsion models as in many studies previously conducted [11]. We also found that torsion/detorsion injury for 3 h of ipsilateral ovaries led to histopathological changes in the ovaries. Although there was no statistically difference between treated and untreated groups in terms of total tissue damage scores, we saw that some injury criteria of subjects in the hypothermia group were decreased. But, we assume that long-term follow-up is needed to make further conclusions. This study is inadequate in terms of showing the relationship between the duration of ischemia and the protective effect of hypothermia against adnexal torsion/detorsion injury and in terms of demonstrating the long-term tissue protective effects of hypothermia. However, this study may be a precursor to further studies aiming to show the longterm results of hypothermia in the ovary or other organs with longer periods of ischemia and hypothermia. The other concern is how the hypothermia can be applied to a patient presenting with ovarian torsion in clinical practice. We think that hypothermia therapy can be adapted to both open and laparoscopic surgical techniques. It may also provide some information for studies investigating the decrease of the preparation time to reperfusion with the use of special liquids that will reduce the tissue temperature to + 4° in a shorter time before the torsioned ovary is detorsioned.
Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
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E. Türk et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
In conclusion, it is evidenced from this study that local hypothermia implemented in rats where ovarian torsion had been performed restricted the levels of oxidative stress products that appeared in the ovary tissue after detorsion and also reduced the tissue damage, but did not result in histopathological changes. This is a pioneering study in terms of showing the effects of hypothermia on the damage in ovarian tissue after torsion, as well as the clinical use of local cooling in the surgical treatment of ovarian torsion.
Conflict of Interest We declare that we have no conflict of interest.
Acknowledgements Many thanks to Gülin Kavakalan, Özgecan Engür, Tulu Batı, Izmir University, Faculty of Medicine, Izmir/TURKEY, for good assistance in the practice of the experimental study.
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Please cite this article as: Türk E, et al, The effect of hypothermia on adnexal torsion/detorsion injury in a rat ovary model, J Pediatr Surg (2015), http://dx.doi.org/10.1016/j.jpedsurg.2015.01.011
