The Role of Nitric Oxide in Testicular Ischemia-Reperfusion Injury

By B. Hayri bzokutan, Mustafa Ktiplikaydln, Sabahattin MuhtaroQlu, and Yiicel Tekin Ka yseri, Turkey

Purpose: This study was designed to determine the role of nitric oxide (NO) in the ischemia-reperfusion (I/R) injury process in testes.

Methods: Fifty prepubertal male rats were divided into 5 groups each containing 10 rats. After 4-hour torsion and 4-hour detorsion, bilateral orchiectomies were performed for measurement of tissue malondialdehyde (MDA) level and histopathologic examination. The results were compared statistically. The groups were labeled as group 1, basal values of biochemical parameters in testes; group 2 (control group), torsion plus detorsion; group 3, torsion plus N-monomethyl- L-arginine (L-NMMA) plus detorsion; group 4, torsion plus L-arginine plus detorsion; group 5, sham operation.

Results: The highest MDA values were determined in the L-arginin group in ipsilateral testes. Group 3 and group 4 were statistically different from control group. Histological

examination showed that specimens from group 4 had a significantly (PC .05) greater histological injury than group 3, and contralateral testes showed normal testicular architec- ture in all groups.

Conclusions: These results suggest that NO plays an impor- tant role in damaging the testis with I/R. Although inhibition of NO synthesis with L-NMMA significantly improves I/R injury in testes, enhancing NO production by providing excess of L-arginine increases such damage. In the early periods of detorsion, there is no damage to contralateral testes after unilateral testicular torsion. J Pediatr Surg 35:101-103. Copyright o 2000 by W.B. Saun- ders Company.

INDEX WORDS: Testis, testicular torsion, reperfusion injury, nitric oxide.

T ESTICULAR TORSION is a surgical emergency that requires immediate intervention to untwist the

affected testis. Treatment of testicular torsion by detor- sion may further damage the testis.‘,” Reperfusion of ischemic tissue leads to a sequence of events that, paradoxically,. injure tissues. The injury produced by reperfusion can be more severe than the injury induced by ischemia. A possible cause of the injury is the ischemia- reperfusion (I/R) injury attributed to oxygen free radi- cals.3.4 Several enzymes and drugs have been studied to prevent such injury in various organs including testis.

Recently, nitric oxide (NO) has received a tremendous amount of attention in the medical literature. NO is a highly reactive free radical with a multitude of organ- specific regulatory functions. It remains controversial whether enhancing or blocking of NO synthesis is beneficial in many conditions.

This study was designed to determine the role of NO in testicular I/R injury.

MATERIALS AND METHODS

This study was approved by Erciyes University Animal Research Committee. Fifty prepubertal (35 to 40 days old) male Sprague-Dawley rats weighing 180 to 200 g were used in this study. The animals were housed in a temperature- and light-controlled room and maintained on pellet food and water ad libitum.

Rats were divided into 5 groups each containing 10 rats. Surgery was conducted under intraperitoneal one-

Journal ofpediatric Surgery, Vol35, No 1 (January), 2000: pp 101-103

shot ketamine (50 mg/kg) anesthesia. Torsion, detorsion, and sham operations all were performed on the left testes through the midscrotal vertical incision. Torsion was created by rotating the left testis 720” clockwise and maintained by fixing the testis to the scrotum with a Prolene suture. During the sham operations the testes were brought through the incision, and a Prolene suture was placed through the tunica albuginea, and the testes were replaced. After each surgical intervention the inci- sions were closed. At the end of the experiments, bilateral orchiectomies were performed for measurement of tissue malondialdehyde (MDA) level and histological examina- tion.

In group 1, bilateral orchiectomies were performed to determine basal values of biochemical parameters in testes. In group 2, after 4-hour torsion and 4-hour detorsion, bilateral orchiectomies were performed (con- trol group). In group 3, detorsion was carried out after torsion lasting 4 hours. N-monomethyl+arginine (L-

NMMA; Sigma Chemical Company, St Louis, MO; 30

From the Departments of Pediatric Surgery, Biochemistry. and Pathology, Erciyes University Medical Faculty, Kayseri, Turkey.

Presented at the 46th Annual International Congress of the British Association of Paediatric Surgeons, Liverpool, England, July 21-24, 1999.

Address reprint requests to Mustafa K@Ikaydtn, Departments of Pediatric Surgery, Erciyes University Medical Faculfy. Kayseri, Turkey.

Copyright Q 2000 by U!B. Saunders Company 0022-3468/00/3501-0022$03.00/o

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mg/kg) was injected intraperitoneally 5 minutes before detorsion. Both testes were harvested 4 hours after detorsion (L-NMMA group). In group 4, detorsion was carried out after torsion lasting 4 hours. L-arginine (Sigma Chemical Company; 200 mg/kg) was injected intraperitoneally 5 minutes before detorsion. Both testes were harvested 4 hours after detorsion (L-arginine group). In group 5, after the sham operation, bilateral orchiecto- mies were performed.

Biochemical Analyses

Tissues were homogenized with 1.15% KC1 to make a 10% homogenate, using a glass teflon homogenizer. The degree of lipid peroxidation in tissue homogenates was assessed by the method of Ohkawa et aL5 measuring MDA levels. The principle of the method is based on measuring the concentration of the pink chromogen compound that forms when MDA couples to thiobarbitu- ric acid. The protein content of homogenates was deter- mined according to the procedure of Lowry et aL6 and values were expressed as nanomoles of MDA per gram of protein.

Histological Examination

The testicles were removed, placed in paraffin blocks, sectioned at 5 pm, and stained with H&E. The light microscope histological examination was done by a pathologist in a blinded fashion. A Clevel grading scale of Cosentino et al’ was used to quantify histological injury. Grade 1 showed normal testicular architecture with an orderly arrangement of germinal cells. Grade 2 injury showed less orderly, noncohesive germinal cells, and closely packed seminiferous tubules. Grade 3 injury exhibited disordered, sloughed germinal cells with shrunken pyknotic nuclei and less distinct seminiferous tubule borders. Grade 4 injury defined seminiferous tubules that were closely packed with coagulative necro- sis of the germinal cells.

Statistical Analysis

After the measurement of tissue MDA level, results are expressed as mean 2 SEM. Data were analyzed by analysis of variance followed by Tukey’s b test. A value of P < .05 was considered to be significant. Histological findings were compared using a x2 test.

RESULTS

The results of testicular MDA values in all groups are shown in Table 1. There were no statistically significant differences within groups in contralateral (right) testes. In ipsilateral testes, the highest MDA values were deter- mined in the L-arginin group. Group 3 and 4 were statistically different from control group.

Histological evaluation is summarized in Table 2. In all

~Z~KUTAN ET AL

Table 1. Tissue MDA Levels

Groups Right Testis MDA Left Testis MDA lnmollg protein) (nmollg protein)

1 171.61 + 17.99 171.61 ? 17.99 2 153.91 + 30.48 771.56 -c 150.11 3 146.25 -c 21.04 403.78 z 54.15’ 4 135.89 + 15.70 1108.47 + 76.35’

5 181.71 + 40.71 222.50 + 44.71

NOTE. P > .05.

*PC .05 compared with control group.

groups, right testes showed normal testicular architecture. Specimens from group 4 had a significantly (P < .05) greater histological injury than group 3. Most of the specimens in group 4 showed grade-II injury.

DISCUSSION

Testicular torsion and detorsion induces morphological and biochemical changes caused by both ischemia and reperfusion of the tissues. ‘v2.* Several studies showed that I/R injury is caused by oxygen free radicals.3J Antioxi- dant treatments have been used successfully to decrease reperfusion injury in multiple organ systems such as heart,g lung, ‘O bowel ” and liver.‘*

It has been reported that allopurinol’3 and superoxide dismutase plus catalase’ treatment caused significant rescue of testes function after testicular torsion. Con- versely, deferoxamine and diltiazem’ and vitamin El4 were used to prevent reperfusion injury in testis, but no beneficial effects have been reported.

Nitric oxide is a highly reactive free radical with a multitude of organ-specific regulatory functions. NO plays a major role in many organ systems, and deranged NO synthesis causes a number of pathophysiological states.15

There are conditions in which it will be beneficial to increase NO and other conditions in which selective inhibition of NO formation may be desirable.16 In this study, L-arginine, precursor of NO, was used to increase NO synthesis; and L-NMMA, a competitive inhibitor of NO synthase, was used to reduce NO formation. We found that pretreatment with L-NMMA resulted in signifi- cantly decreased MDA levels, compared with control group. Enhancing the NO production by using L-arginine caused the highest levels of MDA within all the groups.

Table 2. Histological Evaluation

Groups

Right Testis

Grade I Grade II

Left Testis

Grade I Grade II

1 10 0 10 0 2 10 0 5 5 3 10 0 9 1* 4 10 0 3 7 5 10 0 10 0

NOTE. P> .05. l f < .05 compared with L-arginin group.

ROLE OF NO IN TESTICULAR ISCHEMIA

The L-arginine pretreated group showed the most severe histological damage. These data suggest that NO acts as a free radical and inhibition of NO production could prevent reperfusion injury.

There are many reports about enhancing or blocking the NO synthesis as a therapeutic maneuver. Findings have suggested that NO production is significantly en- hanced during endotoxemia and sepsis and that inhibition of NO synthesis may be beneficial.” In contrast, other studies have indicated that NO production is reduced in endotoxemia and sepsis and that inhibition of NO syntbe- sis may be detrimental under such conditions.i8 NO also has been studied in myocardial I/R injury. There are contradictory reports about effects of the administration of L-arginine in myocardial ischemia.i9

The effect of unilateral torsion on contralateral testis is

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controversial. Although there have been reports to the contrary,*O many investigators have reported that unilat- eral testicular torsion or ischemia causes contralateral damage, and this injury was attributed to an immunologic mechanism.“.*’ In our study, testes were harvested after 4-hour torsion and 4-hour detorsion. Histological exami- nation and biochemical analyses showed no damage in contralateral testes. We suggest that an 8-hour period is not sufficient for immunologic response.

Nitric oxide plays an important role in damaging the testis with I/R. Although inhibition of NO synthesis with L-NMMA significantly improves I/R injury in testes, enhancing NO production by providing excess of L-arginine increases such damage. In the early periods of detorsion, there is no damage to contralateral testes after unilateral testicular torsion.

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