One of the essential mechanism for testicular torsion and detorsion (TTD) have been known the testicular ischemia reperfusion injury (tIRI), which is a desperately serious urological emergency that can cause severe damage to testes, the main reproductive organ, and result in infertility 12. Two important time periods have been expected for the occurrence of the disease, the first appearing before puberty, the second most commonly happens in the puberty. The timely and accurate diagnosis may prevent disease progression and deterioration 3. In Global statistics, it is estimated that its incidence would be around 1 in 4,000 young men population4. If the disease is identified in time, it will be possible to treat it to some extent with surgery and leading to blood reperfusion into testes. On the other hand, It is also possible that even the both testis often becomes completely dysfunctional with surgery 5. During Testicular torsion, the spermatic cord that conveys blood to the scrotum would be twisted. Eventually, the reduced blood flow initiates ischemia and following – detorsion --, the reperfusion generates reactive oxygen species (ROS)13. Because of increase in generation of ROS and antioxidant defense to degrade excess ROS, the balance between ROS production and elimination is disturbed that all cellular components such as nucleic acids and proteins can be deeply affected by oxidative stress. As shown in many previous studies, it is obviously concluded to cause reduction sperm motility, plus DNA damage in the genome of spermatozoa and induces germ cell apoptosis (GCA)67. It is well documented that ischemia reperfusion (IR) injury can strongly influence mitochondrial dysfunction , also the excessive production of free radicals can cause increase mitochondrial permeability transition pore (MPTP) opening, resulting in damage of membrane potential and extra free radical production, disorders in the mitochondrial respiratory chain and ATP production89. The first study of Ozone therapy was reported by Wolff in 19741.  Ozone therapy, a gas mixture of oxygen and ozone, has been referred to the process of managing ozone gas into your body to treat numerous of disease including wound infections, ischemic diseases, and joint problems 12. As demonstrated in some studies, production of ATP has been stimulated by overproduction of oxidative carboxylation of pyruvate. Moreover, it can cause substantial -- decline in NADH level and oxidation of cytochrome C 3. Ozone acts as an alleviation of damage of oxidative stress -- by increasing GSH, SOD, CAT, and decreasing MDA, stimulating the antioxidant system of the cell and mitochondria 124.Our study suggested that the ozone-oxygen therapy exert the significant influence on testicular I/R injury in a rat after exposure to testicular torsion-detorsion.

All chemicals and reagents were purchased from Sigma-Aldrich (Taufkrichen, Germany) in the best commercial grade.

We used of animal and all protocols by Animal Experimentation Committee of Baquatollah University of Medical Sciences. The 40 male Sprague-Dawley rats -- weighing 250–300 g and 8 weeks old were purchased from Pasteur Institute (Tehran, Iran). Animals were homed in cages with a 12 hr light: 12 hr dark cycle at 25 ± 3 ^∘C under normal environmental conditions and had free access to tap water and pellet diet. Animal’s anesthetization was performed by intra-peritoneal injection of 100 mg/kg ketamine hydrochloride combined with 20 mg/kg xylazine. Full experiments were done under aseptic environment. In the torsion/detorsion group, via a left-sided ilioinguinal cutting the left testicles were scooped. The left testis were whirled 720° in an anticlockwise direction and retained in this position through fixation by 11–0 silk suture to the scrotum and was reversed to the scrotum, and the cutting point was sealed. Testicular ischemia reperfusion injury is caused by testicular torsion-detorsion. In the treated group (control), the left testis was subjected with the same cutting mentioned above. The incised point was opened after 2 hours, and the testicles were rotated to the normal position. The testis were still viable and was inserted into the scrotum again 2. Orchiectomy operation in both testes was done on half of the animals after the recovery of torsion at 4 hours in each group. After one month, the animals were euthanized by cervical decapitation for collection of tissues. Animals were randomly assigned into 4 groups, including control, torsion/detortion, torsion/detortion + ozone/oxygen (30 µg/ml) and only ozone/oxygen, with the same surgical method-- done as in the torsion-detorsion group. Ozone-oxygen therapy (30 µg/ml) was syringed intravenously (IV) via the tail vein. The animal number in each group were 10.

Findings are expressed as mean ± SED. Each experiment was done in triple, and the mean was utilized for statistical analysis. Statistical significance was defined using the one-way ANOVA tests, followed by the post hoc Tukey. Statistical significance was set at P < 0.05.

This study was designed to evaluate the protective effects of ozone-oxygen therapy on testicular ischemiareperfusion injury (IRI) in a rat after induction of testicular torsion (TT). Research has shown that oxidative stress plays a main role in the pathologic mechanism underlying IRI of the testis 13. Testicles due to high levels of unsaturated fatty acids and also ROS are sensitive to oxidative stress process 3. Oxidative stress is a condition in which the level of ROS increases compared to the level of cellular antioxidants 46. Research has shown that reperfusion injury (RI) occurs in two phases. The first phase is associated with mitochondrial dysfunction and an excessive increase in the generation of ROS7. High levels of ROS are associated with irreversible damage to macromolecules including proteins, lipids and DNA 817. IRI involves the generation of ROS, apoptosis and lipid peroxidation (LPO). Antioxidants are known as one of the most important lines of protection of the organism against side effect of IRI in the testicular cell environment. Also, various antioxidants have been used to prevent damage caused by oxidative stress in IRI 3. Ozone therapy is one of the most important methods used to prevent IRI. Furthermore, the positive effects of ozone therapy on IRI have been studied in different tissues and organs 11819. Studies have shown that ozone therapy is associated with a decrease in the level of ROS and subsequent oxidative stress, a decrease in the level of LPO and ATP depletion and an increase in the level of activity of cellular antioxidants, including glutathione (GSH) 1271920. The results showed that ozone therapy was able to reduce the level of ROS in testicular mitochondria in the testicular torsion group. The results of our study are in agreement with previous studies that have shown that ozone therapy has the ability to reduce the ROS generation and oxidative stress 81718. ROS as one of the mitochondrial products can affect the mitochondria by collapsing in the MMP. Collapse in the MMP is considered to be one of the events during which caspase cascade activation occurs, resulting in irreversible cell death 2122. Ozone therapy has been able to reduce the collapse of the MMP in the mitochondria of testicular tissue in the testicular torsion group. The results suggest that ozone therapy reduces mitochondrial dysfunction by reducing the level of ROS and subsequently improving the MMP collapse. In biological systems, the evaluation of GSH changes is considered as one of the important indicators of oxidative stress. Our results showed that ozone therapy causes an increase in GSH levels and other antioxidant enzymes and a decrease in GSSG levels in the testicular torsion group 79. These results are in agreement with the results of another study that showed that ozone therapy is associated with an increase in the level of intracellular antioxidant enzymes 19. LPO is considered as one of the oxidation products and indicators of cellular toxicity 918. Reduction in MDA levels in the testicular torsion group treated with ozone compared to the testicular torsion group indicates the therapeutic effects of ozone in reduction of LPO. Research has shown that there is a direct relationship between the level of ROS and LPO 2324. The results show that ozone therapy has reduced LPO levels by decreasing the level of ROS and increasing the level of cellular antioxidants in testicular tissue in the testicular torsion. Mitochondrial dysfunction is known as a consequence of oxidative stress during IRI. In addition, oxidative damage in mitochondria is associated with changes in its structure and function, including mitochondrial swelling and cytochrome c release and eventually cell death 13. Furthermore, ozone therapy has been able to reduce mitochondrial damage and cell death in the heart IRI by increasing the level of the antioxidant system as well as inhibiting the release of cytochrome c, caspase cascade and apoptotic signaling 125. In this study, a reduction in mitochondrial damage, mitochondrial swelling and cytochrome c release was observed in the in the testicular torsion group treated with ozone. These results are in agreement with previous studies 1. During the process of apoptosis, the release of cytochrome c is considered as one of the most important events 2627.

In this study, we injected ozone/oxygen to the testicular ischemiareperfusion (IRI) rat animal model. However , our results showed that a higher concentration of ozone/oxygen caused electron transfer chain impairment, which leads to decreased cell viability , but we injected 30 μg to animal model and ozone/oxygen decreased ROS production, lipid peroxidation ,GSSG and ADP and cytochrome c release compared with IRI animal group. Our study suggests that mitochondrial dysfunction and the uncoupling of oxidative phosphorylation may play key roles in the etiology of testicular ischemia. In general, the present study results confirmed that a moderate concentration of ozone (30 μg) can be considered as a protective in animal rat model.