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Abstract

Testicular ischemia-reperfusion injury is an urgent situation which needs a timely and precise diagnosis for prevention of testis damages. Here in we investigated ozone/oxygen therapy in Testicular Ischemia/Reperfusion Injury. For this purpose, animals (rats) were divided to four groups; control, torsion/detortion, torsion/detortion + ozone/oxygen (30 µg/ml) and only ozone/oxygen. Four hours after detorsion, in all groups orchiectomy was done -- for measuring the oxidative stress and mitochondrial toxicity parameters. Also, we preformed analysis of testicular spermatogenesis after 90 days. Our data showed that testicular torsion-detorsion induced significant increase in mitochondrial toxicity and decrease of spermatogenesis, malondialdehyde and GSSG levels were shown. Also, spermatogenesis, a remarkable decrease in malondialdehyde GSSG levels and mitochondrial toxicities were observed when compared with torsion-detorsion group. Obtained results for this research showed that ozone/oxygen therapy enhance antioxidant properties in the spermatogenic cells and protects testes from ischemia-reperfusion injury.

Introduction

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.

Results

Results Determination of Semen Collection

We isolated the sperm from the left epididymis and analyzed. We showed that semen characteristic parameters such as sperm number (×10⁶/ejaculate), volume (ml), sperm concentration(M/ml) showed no changes in all groups but sperm viability (%) and normal sperm morphology (%) parameters significantly decreased in compared to control and torsion/detorsion+ozone/oxygen group. The results are presented in Table 1, Table 2.

Table 1. Analysis of semen collection

Parameter	C	C+30µg/ml	C.SCI	SCI.30 µg/ml
Sperm number (×10 ⁶ /ejaculate)	21±1	22±1	22±1.09	21±1
Volume(ml)	1.1±0.5	1.1±1	1.2±1	1.1±1
Sperm viability (%)	100±1.2	98±1	48±1.3***	100±1
Sperm concentration(M/ml)	40.11±1	41.22±1	38.66±1***	41.22±1.2
Normal sperm morphology (%)	100±1	97±1	78±1***	92±1

Semen collection and semen characteristic. Values represented as mean ± SD (n=4). ns= nonsignificant; *** P> 0.001 compared to control

Table 2. Semen parameters and sperm vitality

Parameters	C	C+30 µg/ml	C.SCI	SCI.30 µg/ml
Fields	4±1.1	4±1	4±1	4±1.5
Quantity	288±1.2	288±1.1	280±1	288±1
Concentration (M/ml)	15±1.3	15±1	15±1	15±1
Class: A+B(PR)%	77±1.4	67±1.2	32±1.1***	88±1
Class: C(NP) %	6±1	5±1	9±18***	3±1
Class: D(IM) %	14±1.7	11±1	65±1***	15±1
Round Cell	0	0	0	0

CASA result for the semen parameters and sperm vitality of the 4 motility classes of 30 µg/ml of ozone/oxygen (O2/O3). Values represented as mean ± SD (n=4). ns= no significant; *** P> 0.001 compared to control

Determination of Sperm Motility and Kinetic Parameters

Sperm motility and kinetic parameter results are shown in Table 3. The results indicated remarkable changes in sperm motility and kinetic parameter in torsion/detorsion group compared to control and torsion/detorsion+ozone/oxygen group. There was a significant decrease in Linearity (LIN) = VSL/VCL × 100, Velocity of Straight Line (VSL), Velocity of Curved Line (VCL), Velocity of Average Path (VAP) and Straightness (STR) = VSL/VAP x 100. Compared to the control and torsion/detorsion+ozone/oxygen group, there was no significant statistical difference in Beat Frequency (BCF) and Lateral Amplitude (ALH) in torsion/detorsion group.

Table 3. Mean percentages kinematic parameters

Parameters	C	C+30 µg/ml	C.SCI	SCI.30 µg/ml
Class Name	A+B(PR)	A+B(PR)	Non motile	A+B(PR)
LIN, rel. units	85.47±1	77.39±1	45.91±1***	87.71±1
VSL, µm/s	100±0.00	89±1.09	45±1.08***	100±1.2
VCL, µm/s	117±1.1	115±0.7	98±1.5**	114±1.5
VAP, µm/s	107±1.5	115±1	41±1.11***	121±1
ALH, µm	1±1.2	1±1.1	1.8±1	1±1
STR, rel. units	0.99±1	0.87±1	0.55±1**	0.84±1.3
BCF, Hz	4.11±1	4±1.5	4±1.2	4.1±1

CASA result for the semen kinematic parameters of 30 µg/ml of ozone/oxygen (O2/O3). Values represented as mean ± SD (n=4). ns= no significant; ** P> 0.01, *** P> 0.001 compared to control

Histopathological Evaluation

The results of the histopathological examination for each group are displayed in Figure 1, Figure 2. The presence of uniform seminiferous tubular morphology and there was normal testicular structure seen in the control and ozone/oxygen groups. In torsion/detorsion group, there was a severe distortion of tubules and a significant decrease in the diameter of the seminiferous tubular. Administration of ozone/oxygen protected the seminiferous tubular from damage after torsion/detorsion.

Figure 1. Torsion/detorsion surgical process: (A) before surgical, (B) after surgical. Figure 2. Light microscope observations of H&E stained sections (×100). (A) positive control group: normal testicular architecture was seen. (B) Health group+ ozone/oxygen (30µg/ml). (C) torsion/detorsion. (D) torsion/detorsion + ozone/oxygen (30 µg/ml) therapy group. Cell Viability

Figure 3 showed that injection ozone/oxygen with concentration of 5, 10, 20, 30, 40, 60 and 80 µg/ml in testicular cells. Otherwise of 5, 10, 20 and 40, 60 and 80 µg/ml concentration in torsion/detorsion group significantly decreased viability of cell compared to untreated control group but 30 µg/ml , we did not see any change level in viability testis cells in animal model.

Figure 3. Effect of Torsion /detorsion and ozone/oxygen on Cell viability in rat testis (A, B, C and D). Cytochrome c release in testis cells. As shown in graph all of concentration except 30 µg/ml induced decreased viability. ns indicates not significant difference with control group. ***indicates significant difference with control group. Cellular ROS Level

Figure 4, we showed that the rate of ROS formation in cells in torsion/detorsion group significantly raised compared to untreated control group.

Figure 4. Flowcytometric analysis of ROS formation in testis cell. Analysis were measured using BD flowcytometry. Fluoresce intensity mean of DA-DCF significantly increased in animal ischemic group in compared to control group that led to transporting pick from left to right of the histogram. Fluoresce intensity mean of DA-DCF. Significantly decreased in ischemic group + ozone/oxygen group treated group in compared to ischemic group group that led to transporting pick from right to left of the histogram. GSH and GSSG of Testicular Tissue

GHS level of testicular tissue has a significant decrease in 4 hours torsion/detorsion group comparing to control group (***p<0.001). Also, GSSG level of testicular tissue had a significant increase in torsion/detorsion group comparing to control group. These effects inhibited by oxygen/ozone therapy for both measured parameters (Figure 5 A and B).

Malondialdehyde Assay

Based on the results of Figure 5 C, MDA level of testicular tissue in torsion/detorsion group has significant increase comparing to control group (***p<0.001). MDA level of testicular tissue has a significant decrease ozone/oxygen-torsion/detorsion group comparing to torsion/detorsion group.

ADP/ATP Ratio Assay

As shown in Figure 5 D, ischemia reperfusion injury in torsion/detorsion group significantly decreased ATP content in testicular tissue. Animals treated with oxygen/ozone and torsion/detorsion showed a significant increase in the ATP content in comparison with the torsion/detorsion group.

Figure 5. Effect of Torsion /detorsion and ozone/oxygen on GSH, GSSG , MDA, ATP and cytochrome c release in rat testis (A, B, C ,D and E). MDA, GSH, GSSG ATP and cytochrome c release were measured in testicular tissues after treatment with Torsion /detorsion and ozone/oxygen. Graph A shows that content of GSH significantly (p<0.001) decreased in Torsion /detorsion group in compared to control group. The content of GSH not show any significant changes in Torsion /detorsion + ozone/oxygen and ozone/oxygen treated groups in compared to control group. Also, B graph indicates that content of GSSG significantly increased in Torsion /detorsion group in compared to control group. While there are not any significant changes in the content of GSSG in Torsion /detorsion + ozone/oxygen and ozone/oxygen treated groups in compared to control group. Similarly, Graphs C shows that lipid peroxidation significantly increased in Torsion /detorsion group in compared to control group while this effect inhibited in Torsion /detorsion + ozone / oxygen group. Graph D, ATP content decrease significantly in Torsion /detorsion group in compared to control group while this effect reflected in Torsion /detorsion + ozone/oxygen group. Ozone/oxygen alone did not show a significant effect. Graph E, Cytochrome c release in testis cells. As shown in graph Torsion /detorsion induced cytochrome c release and ozone/oxygen therapy significantly reflected this endpoint in cells. ns indicates not significant difference with control group. ***indicatessignificant difference with control group. Cytochrome c Release

We examined cytochrome c release in the isolated cell obtained from testis in each group. Cytochrome C release was significantly raised in the isolated mitochondria in torsion/detorsion group in compared to control group. Moreover, cytochrome c release was inhibited with oxygen/ozone therapy (Figure 5E).

Succinate Dehydrogenase (Complex II) Activity

We examined Complex II in the isolated cell obtained from testis in each group. Complex II was significantly decreased in the isolated mitochondria in torsion/detorsion group in compared to control group. (Figure 6A)

Figure 6. Effect of Torsion /detorsion and ozone/oxygen on succinate activity and MMP in rat testis (A, B). these toxicity factors were measured in testicular tissues after treatment with Torsion /detorsion and ozone/oxygen. Graphs A shows that succinate activity significantly decreased in ischemic group in compared to control group while this effect inhibited in TD + ozone / oxygen group. Graph B shows that MMP significantly (p<0.001) increased in ischemic group in compared to control group. Mitochondrial Membrane Potential Collapse

The redistribution of the rhodamine 123 into the cytosol has been utilized for assessment of the MMP collapse as the known indicator of mitochondrial damage which subsequent leads to mitochondrial membrane permeability transition (MPT). As shown in Figure 6B, there was a remarkable increase in the rhodamine 123 redistributions in torsion/detorsion group compared to control group.

Discussion

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.

Conclusion

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.

Journal of Antioxidant Activity