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In this study, we investigate the effect of montelukast on histologic damage induced by testicular torsion-detorsion in rats.
Twenty-one male Sprague-Dawley rats were separated into 3 groups, each containing 7 rats. A sham operation was performed in group 1 (control). In group 2 (ischemia-reperfusion [IR]/untreated), 1-hour detorsion of the testis was performed after 6 hours of unilateral testicular torsion. In group 3 (I-R/dextroamphetamine), after performing the same surgical procedures as in group 2, montelukast was given intraperitoneally. In all experimental rats, ipsilateral orchiectomies were performed for histological examination and tissue malondialdehyde (MDA), glutathione and myeloperoxidase assays.
Montelukast treatment significantly decreased the I-R-induced elevation in testes tissue MDA and glutathione levels were found to be preserved. The level of myeloperoxidase (MPO) activity was significantly increased in the testes tissue of the IR/untreated group. However, in I-R/montelukast treatment group significantly decreased testes tissue MPO level. Histopathologically, the in the group 2 rats, edema, congestion, hemorrhage between seminiferous tubules and necrosis of the germinal cells were predominant features in sections. However, most of the specimens in the montelukast treated group 3 showed grades-I and II injury. Additionally, the testicular injury score was lower in group 3 rats compared with group 2.
The current findings demonstrate that the montelukast decreased the severity of testicular injury by reversing the oxidative effects of testes I-R.
Dans cette étude, nous examinons l’effet du montélukast sur les lésions tissulaires provoquées par torsion-détorsion testiculaire chez le rat.
Vingt-et-un rats Sprague-Dawley mâles ont été répartis en trois groupes de 7 rats. Une opération fictive a été réalisée dans le groupe 1 (groupe témoin). Dans le groupe 2 (ischémie-reperfusion / non traité), une détorsion testiculaire d’une heure a été réalisée après 6 heures de torsion testiculaire unilatérale. Dans le groupe 3 (ischémie-reperfusion / dextroamphétamine), on a administré du montélukast par voie intrapéritonéale après les mêmes interventions chirurgicales que pour le groupe 2. Chez tous les rats, une orchidectomie homolatérale a été effectuée en vue d’un examen histologique et de mesures du malonaldéhyde, du glutathion et de la myéloperoxidase dans les tissus.
Le traitement par montélukast a significativement réduit l’élévation dans les tissus testiculaires provoquée par l’ischémie-reperfusion, mais les taux de malonaldéhyde et de glutathion sont demeurés les mêmes. Le niveau d’activité de la myéloperoxidase (MPO) a significativement augmenté dans les tissus testiculaires du groupe ayant subi l’ischémie-reperfusion sans traitement sub-séquent. Cependant, dans le groupe ayant subi l’ischémie-reperfusion avec traitement par montélukast, le taux de MPO dans les tissus testiculaires avait significativement diminué. Selon les examens d’histopathologie, dans le groupe 2, de l’œdème, de la congestion, des hémorragies entre les tubules séminifères et une nécrose des cellules germinales étaient les caractéristiques prédominantes observées dans les coupes. Par comparaison, la plupart des échantillons du groupe 3 (traité par montélukast) présentaient des lésions de stade I et II. En outre, le score de lésions testiculaires était plus faible dans le groupe 3 que dans le groupe 2.
Les observations actuelles montrent que l’administration de montélukast diminue la gravité des lésions testiculaires en annulant les effets oxydatifs de l’ischémie-reperfusion des testicules.
Testicular torsion is a common pediatric urologic emergency.1,2 Late presentation or failure to diagnose can lead to decreased spermatogenesis, altered hormone production and subfertility.3,4 Mammalian testes are extremely sensitive to oxidative-free radical damage.5,6 Reperfusion is one of the most important factors in further injury.7 Ischemia-reperfusion (I-R) injury in the testis induces germ cell-specific apoptosis attributable to increases in neutrophil infiltration and reactive oxygen species (ROS).8,9 These ROS can lead to lipid peroxidation and loss of cell viability by disrupting membrane integrity and DNA damage.5,6 Once neutrophils migrate into the ischemic area, they release proteases, elastase, myeloperoxidase (MPO), cytokines and various other mediators,10 all of which are involved in tissue injury. The ROS are difficult to quantify directly in tissue because of their high reactivity and short half-life. In our experiment, we determined the malondialdehyde (MDA) level, an end product of lipid peroxidation;11 the glutathione (GSH) level, a key antioxidant;12 and the tissue-associated MPO activity, as indirect evidence of neutrophil infiltration.13
Anti-leukotriene agents have been shown to be effective to protect against injury in several inflammatory models in rats, such as in ethanol-induced gastric mucosal damage,14 colitis15,16 burn- and sepsis-induced multiorgan damage17,18 and renal I-R injury.19 Based on these findings, in the present study, we examined the protective effect of the selective LTD4 receptor antagonist, montelukast (MK-0476), on histologic damage induced by testes I-R in rats.
Twenty-one prepubertal (35 days old) male Sprague-Dawley rats, weighing 120 to 140 g, were randomly divided into 3 groups, with each group containing 7 rats. All the animals were housed at a temperature- and light-controlled room with ad libitum access to water and rat food. All the experimental protocols were approved by the Abant Izzet Baysal University School of Medicine Animal Care and Use Committee. Rats were anesthetized during all the surgical procedures.
The surgical procedures were performed under 50 mg/kg ketamine and 20 mg/kg xylazine body weight, intraperitoneal injection (IP). The right femoral vein was cannulated to administer drugs and saline. The testes were exposed through identically opened and closed right-sided ilioin-guinal incision. Torsions were created by rotating the ipsilateral testes 720° clockwise for 6 hours and were maintained by fixing the testes medially and laterally to the scrotum using a 5/0 silk suture. The detorsion procedure was performed again under sterile conditions, after 6 hours of testicular torsion. The rats were divided into 3 groups according to the procedure performed.
In all 3 groups, ipsilateral orchiectomies were performed for the biochemical and histological examinations.
Samples of testis were frozen at −70°C until the assay. Tissue samples were homogenized with ice-cold solution (150 mM KCl) to determine MDA and GSH levels. The MDA levels were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as previously described.11 Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1.56 × 105 M−1 cm−1 and the results were expressed as nmol MDA/g tissue.
The GSH measurements were performed using a modification of the Ellman procedure.12 Briefly after centrifugation at 2000 g for 10 minutes, 0.5 mL of supernatant was added to 2 mL of 0.3 mol/L Na2HPO4·2H2O solution. A 0.2-mL solution of dithiobisnitrobenzoate (0.4 mg/mL, 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. The GSH levels were calculated using an extinction coefficient of 1.36 × 105 M−1 cm−1. Results were expressed in mol GSH/g tissue.
Activity of tissue MPO, an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes, correlates with the number of polymorphonuclear neutrophils determined histochemically in the inflamed tissues; it is therefore used as an indication of tissue neutrophil accumulation.13 Testes MPO activity was measured using a procedure similar to that documented previously.20 Testes samples were homogenized in 50 mM potassium phosphate buffer ([PB], pH 6.0), and centrifuged at 41 400 g (10 minutes); pellets were suspended in 50 mM PB containing 0.5% hexadecyl trimethyl ammonium bromide (HETAB). After 3 freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41 400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of the MPO present that caused a change in absorbance measured at 460 nm for 3 minutes. The MPO activity was expressed as U/g tissue.
The extracted testes were immediately placed into 10% formalin solution. The tissue specimens were placed in paraffin blocks, sectioned at 5 μm, and stained with hematoxylene & eosine. The sections were blindly examined under light microscope by 2 investigators. The histological parameters was scored according to Cosentino and colleagues’s classification (Table 1).21
All values were expressed as mean standard deviation. The significance of the data obtained was evaluated by using analysis of variance (ANOVA). Differences between means were analyzed by using the post-ANOVA (Tukey’s b) test. Significance was determined as p values less than 0.05.
The MDA, GSH and MPO values for the different groups are shown in Fig. 1. The testes MDA levels in the control group were elevated by I-R injury (p < 0.05); however, mon-telukast treatment significantly decreased the I-R-induced elevation in the testes MDA level (p < 0.05). The I-R caused a significant decrease in testes GSH level (p < 0.05) when compared with the control group, while in the montelukast-treated I-R group, the testes GSH level was found to be preserved (p < 0.05) and not significantly different from that of the control group. The level of MPO activity was significantly increased in the testes tissue of the I-R/untreated group (p < 0.001) compared with the control group. However, in the I-R/montelukast treatment group, there was significantly decreased testes tissue MPO level (p < 0.5); this was found to be similar to the control group.
The testicular injury score increased significantly in the I-R/untreated and I-R/montelukast groups compared with the control group (p < 0.05, p < 0.05, respectively). On the other hand, this score was decreased in the I-R/montelukast group compared with the I-R/untreated (p < 0.05) (Fig. 2).
Histopathologically, the rats in the control group had essentially normal seminiferous tubule morphology (Fig. 3, part a). In the I-R/untreated group (group 2), the lesions varied between grade-III and grade-IV. In this group, edema, congestion, hemorrhage between seminiferous tubules and necrosis of the germinal cells were predominant features in sections (Fig. 3b, part b). However, in the montelukast-treated group (group 3), 6 rats had grade-I and II injury and histopathological features which were significantly lower than the I-R/untreated group. In group 3, interstitial edema, congestion and hemorrhage between seminiferous tubules were reduced (Fig. 3c, part c).
The underlying pathophysiologic mechanisms in testicular I-R damage are most likely multifactorial, and interdependent involving hypoxia, inflammatory responses and oxidative stress, which is characterized by an imbalance between ROS and the antioxidative defense system.22,23 The present study demonstrates that unilateral testicular I-R causes testicular damage in testes, as evidenced by biochemical and histologic changes. Montelukast prevented the biochemical changes and protected the morphology in ipsilateral testes after unilateral testicular I-R.
The pathophysiologic mechanism in testicular damage owing to testicular torsion is an ischemic process for the testis. The ROS can oxidize cell membrane lipids, proteins and DNA, which leads to cellular dysfunction and, sometimes, cell death. This cascade of events is known as reperfusion injury.23,24 In addition, more neutrophils accumulated in the testis after testicular torsion-detorsion and generated excess ROS; this caused spermatogenic injury in the ipsilateral testis. The elimination of ROS has been shown to be beneficial in treating ischemia-reperfusion injury.16,25,26
The ROS are difficult to quantify directly in tissue because of their high reactivity and short half-life. Malondialdehyde, a stable end-product of lipid peroxidation generated by ROS, is usually used as a good indicator of the degree of lipid peroxidation.27 In our study, we demonstrated that the levels of tissue MDA were significantly increased in the I-R/untreated group. The increase of MDA levels indicated the presence of oxidative damage I-R injury induced on ipsilateral testes. Furthermore, MDA activity was preserved by montelukast treatment. Glutathione is a key component in cell growth, differentiation and protection.25 It is a tripeptide, L-g-glutamyl-L-cysteinyl-glycine, with a molecular weight of 307,28 and is an important cellular thiol “redox buffer” participating actively in the maintenance of the thiol/disulfide redox potential.29 As reported by Ross and colleagues,30 cell injury is related to the efflux of GSH precursors and decreases GSH biosynthesis. In this sense, GSH and other antioxidants play a critical role in limiting the propagation of free-radical reactions, which would otherwise result in extensive lipid peroxidation. In the present study, we found that the concentration of GSH decreased in the I-R/untreated group compared to control group. Decreased tissue GSH activities might have occurred as a result of consumption of the enzyme by severe oxidative stress. However, treatment with montelukast enhanced higher antioxidant capacity because the concentrations of testicular GSH were significantly higher in the montelukast-treated animals when compared with the GSH testicular concentrations found in I-R/untreated rats. The infiltration of polymorphonuclear leukocytes in a tissue is characteristic of acute inflammation and indicates the collective action of chemotactic mediators.31 Various chemokines and a lipid mediator, cysteinyl leukotrienes ([CysLTs], the 5-lipoxygenase metabolites of arachidonic acid) are potent inflammatory mediators that are associated with I-R-induced tissue injury.10 Testicular neutrophil content was determined by MPO assay in the present study. Myeloperoxidase is stored in the primary granules of neutrophils and the enzyme activity is a common measure of neutrophil accumulation.32 We have found a significant decrease in testis tissue MPO activity after montelukast administration. These data suggest that the anti-inflammatory effects of Montelukast contribute to its protective role in testicular torsion. Although the results of our study are promising, there are several limitations. We did not study the effect of montelukast on testicular I-R injury at different doses or different administration times. Additional studies are required to examine these factors on the effect of montelukast.
These data collectively support that testes I-R injury causes oxidative response as evidenced by alterations in testes MPO, MDA and GSH levels. The results also demonstrate that montelukast attenuates the I-R-induced testes damage through the mechanisms that involve an inhibitory action on tissue neutrophil infiltration, release of reactive oxygen species and activation of inflammatory cytokines. This may provide a potential therapeutic approach to post-ischemic testicular damage. Further studies will clarify the role of the selective LTD4 receptor antagonist, montelukast (MK-0476), in decreasing long-term sequelae responsible for subfertility.
Competing interests: None declared.
This paper has been peer-reviewed.