International Urology and Nephrology 34: 81–86, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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The effect of nitric oxide in testicular ischemia-reperfusion injury

Meral Barlas & Celal HatibogluDepartment of Pediatric Surgery, University of Ankara, School of Medicine, Ankara, Türkiye

Abstract. This experiment was carried out to investigate the effect of endogenous nitric oxide (NO) on theischemia-reperfusion injury of testis. Testicular ischemia was achieved by twisting the right testis and spermaticcord 1080 counter-clockwise for 30 minutes and reperfusion was allowed for 30 minutes after detorsion of 33rats. Animals were treated with normal saline in controls just before detorsion, NG-nitro-L-arginine methylester (L-NAME), and L-arginine (L-arg) in others. The tissue damage was evaluated with light microscopy,malondialdehyde (MDA) level in tissue, and the blood flow measurement using 133xenon (Xe) clearencetechnique. MDA indicator of reperfusion injury increased 25% after detorsion when only normal saline was given,L-NAME further increased MDA, L-arginin decreased MDA to control level. Conclusion: L-arginin infusionduring the detorsion reduced the reperfusion injury of testis and improved the testicular blood flow after thedetorsion. Key words: Ischemi-reperfusion injury, Nitric oxide, Testis

Introduction

Testicular torsion most commonly results from devel-opmental hypermobility caused by a redundant orelongated tunica vaginalis, known as the bell clapperdeformity, with an incidence of which has been esti-mated to be as high as 1 in 158 males by the ageof 25 [14, 15]. This is a serious urologic emergencyin terms of both the need for urgent managementand potential for long term sequelae [2]. Althoughthere are various reports for and against this argu-ment; it is generally accepted that unilateral testiculartorsion may cause contralateral testicular damage aswell as the ipsilateral side, and result in diminishedfertility with abnormal spermiogram [11, 17]. Theipsilateral testicular injury due to torsion and detor-sion shares resemblances with the phenomenon ofischemia-reperfusion observed in other tissues [3,16]. However the mechanism of contralateral testi-cular injury is still controversial. Autoimmunization,the incidental occurrence of other pathologies suchas varicocele, subclinical episodes of contralateraltesticular torsion and underlying defects in spermato-genesis have been proposed to account for contrala-teral testicular injury, but none has been universally

accepted [6]. A current theory of contralateral testi-cular injury proposes a reflexive decrease in contrala-teral testicular blood flow [19]. Electromagnetic andradioisotopic studies have shown that unilateral testi-cular torsion causes a decrease in contralateral testi-cular blood flow and an increase in concentrationsof tissue hypoxia products in the contralateral testis[1, 10]. Nitric oxide is a product of the conversionof L-arginine by the enzyme nitric oxide synthase.It is involved in neurotransmission, maintenance ofvascular smooth muscle tone, and cytotoxicity [9].The pathophysiological role of nitric oxide is alsoevident in a variety of diseases, including septic shock,asthma, reperfusion injury, etc. [5, 12]. This studywas designed to determine the effect of NO in theischemia-reperfusion (I/R) injury process in rat testes.

Materials and methods

Thirty-three adult male albino Wistar rats weighingbetween 240–260 g (250,67 ± 1,24) were used intwo different sets of experiments. Rats were keptunder standardized conditions for food, water, light,and temperature with free acces to standard rat chow

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Figure 1. Three times torsion 1080◦ of the right Testis anduntwisted left testis was seen (note darkness of right testis).

and water. The experiment followed the ‘Principles ofLaboratory Animal Care’ of the NIH.

Anesthesia induced with an intramuscular injec-tion of ketamine hydrochloride (50 mg/Kg). Lowermidline laparotomy was performed to get access theboth testicle. Extravaginal torsions were created bytwisting the right testes 1080◦ in counterclockwisedirection and maintained by fixing the testes to thescrotum with a 4/0 atraumatic silk suture passingthrough the tunica albuginea and dartos (Figure 1).After 30 minutes of ischemia, the suture was removedand right testes were detorted and replaced intothe scrotum for 30 minutes of reperfusion. Duringsham operations, left testes were brought through theincisions and were then replaced without twisting,and a silk suture was placed through the tunica albu-ginea. At the end of the experiment the rats weresacrificed with an overdose of pentobarbital sodiumand bilateral orchiectomies were performed in 18 of33 rats. Testes were divided longitudinally into twohalves for histopathologic evaluation and MDA assay.Specimens for histopathologic evaluation were indi-vidually immersed in Bouin’s fixative. Three slidesprepared from the upper, lower, and midportions ofthe testes were evaluated completely for each testes.Two pathologists studied testicular biopsies blindlyand in random order. Interstitial injury was gradedon a scale from 0 to 3 as described by Mikuz [13].Grade 0: normal interstistium. Grade 1: Interstitialcapillarity edema and congestion Grade 2: Interstitialhemorrhage. Grade 3: Hemorrhagic infarct (Figure 2a,b).

Figure 2. (LNE group) a, capillary congetion(k), b, interstitialedema(e) and hemorrhage(h) was seen I.n the leydig cells oftestis(HEx40).

Specimens for MDA assay were placed in glassbottles with rubber caps, labeled and stored at the–78 C◦ until assay.

In the first set of experiments: Fifteen rats wererandomly divided into three groups and each of themconsisted of five: isotonic saline, L-NAME, and L-arginin was given into the femorale vene. Ipsilateraland contralateral testicular blood flows wese measuredbefore, during unilateral testicular torsion and detor-sion by using 133 Xenon (Xe) clearance technique.The using of 250–300 mci (0,05 ml) Xe injection ofintratesticular the testicular blood flows was measuredevery 15 minutes [5].

133 Xe in saline solution was purchased fromAmersham, England. Toshiba (GCA 601E) typegamma camera, dynamic acquisition and time-activitycurves were used for the measurements. The followingformula was used to calculate the blood flow:

Blood Flow (ml/100 g/min) : 100.λ.k;where k = 0.693/T1/2.

Testicular blood flow was expressed in milliliters per100 g tissue per minute. Here λ is the partition coef-ficient of 133 Xe between testis and whole blood andcalculated as follows:

λ = λI/gas

λE/gas.(H) + λPl/gas.(1 − H)

Partition coefficients such as λI/gas (I: Testis), λE/gas(E: erythrocytes), and λPl/gas (Pl: plasma) wereexperimentally determined and substituted in thisformula to calculate λ at different hematocrit (H)values expressed as a percentage. 133 Xe clearance

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curves were obtained for each time of period. Afterthe injection of 250–300 µci (0.05 ml) 133 Xe intothe both tests, the detector of the gamma camerawas placed as close as possible over the rats andrecording of clearance curves was started immediatelyand continued for 10 minutes. Thereafter T1/2 wascalculated using the time-activity curves. Hematocritvalues were measured before and after the operationto control the hemodynamic values during experiment.

In the second set of experiments: 18 rats wererandomly divided into three groups of six

Group 1 (Isotonic saline): Testicular ischemia wasachieved by twisting the right testis and the spermaticcord 1080◦ counter-clockwise for 30 minutes and1 ml isotonic saline was given to the femoral vene justbefore detorsion.

Group 2 (L-NAME): Right testicular torsion wasperformed in the same manner. L-NAME was givenintravenously just before the torsion of the testis forthe inhibition of NO synthesis at 10 mg/Kg singledose.

Group 3 (L-arg): After right testicular torsion, justbefore detorsion L-arg. Infusion at 100 mg/Kg/hourrate was employed during reperfusion to stimu-late NO synthesis. Bilateral orchidectomy wasperformed. MDA levels were assayed and histopath-ologic changes were evaluated in both testes of thesegroups.

Data analysis

All data were presented as the mean plus or minusthe standard error of the mean (Mean ± SEM).Within ipsilateral and contralateral sides, betweengroups, data were analyzed by Mann Whitney – Utest. Within groups, ipsilateral and contralateral datawere compared by Wilcoxon test. Differences amonggroups were considered significant when p < 0,05.

Results

The basal blood flow in the control isotonic salinegroup was 9.89 ± 0.46 ml/100 g. tissue/minute,whereas it was 9.74 ± 0.34 ml/100 g. tissue/minute forcontrol L-NAME group and 10.73 ± 0.64 ml/100 g.

Figure 3. Blood flow means into three groups of 5 (mL/100 g-tissue/min).

tissue/minute for control L-Arginin group. There wasno statistically significant difference among the bloodflow rates of the control groups.

The basal blood flow in the torsion isotonic groupwas 0.94 ± 0.26 ml/100 g. tissue/minute, where as itwas 1.25 ± 0.33 ml/100 g tissue/minute in the torsionL-NAME and 1.52 ± 0.16 ml/100 g tissue/minutein the torsion L-Arg groups. Blood flow rate in thetorsion groups were decreased when compared withthe control groups (p < 0.05).

After detorsion the blood flow rate in the isotonicsaline group was 6.23 ± 0.44 ml/100 g tissue/minute,and 5.89 ± 0.6 ml/100 g tissue/minute and 7.54 ±0.26 ml/100 g tissue/minute in L-NAME and L-arggroups respectively. In both three detorsion groups theblood flow rate of testis increased but could not reachthe control group (p < 0.05). The increase in L-arggroup was higher than both of the other groups andfound statistically significant (p < 0.05). The increasein the L-NAME group was higher than isotonic groupbut not statistically significant (p > 0.05). The bloodflow rates of three groups of five rats were shown in(Figure 3).

The MDA values of control isotonic saline groupwas 0.039 ± 0.0033 µmol/g. wet tissue, whereas thisvalue was 0.037 ± 0.0017 µmol/g. wet tissue for L-NAME control and 0.031 ± 0.0019 µmol/g. wet tissuefor L-Arg. control groups. There were no statisti-cally significant difference among the control groups(p < 0.05). The MDA values of experimental isotonicgroup was 0.039 ± 0.0033 µmol/g. wet tissue. Thevalues for L-NAME and L-arg. were 0.044 ± 0.0016µmol/g. wet tissue and 0.029 ± 0.0013 µmol/g. wettissue respectively. The MDA levels of L-arg groupwere significantly less than isotonic and L-NAMEgroups. And the results were statistically significant

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Table 1. Histopathologic evaluation in three groups of 6 rats

Animal Groups

IE IC LNE LNC LAE LAC

1 1 1 2 0 2 0

2 2 0 2 0 2 1

3 1 0 1 0 0 0

4 2 0 1 1 2 0

5 2 0 2 0 1 0

6 2 0 2 0 1 0

Mean 1,66 ± 0,21 0,16 ± 0,16 1,66 ± 0,21 0,33 ± 0,21 1,33 ± 0,33 0,16 ± 0,16

IE = Isotonic experiment, LNE = L-NAME experiment, LAE = L-arg experiment, IC = Isotonic control,LNC = L-NAME control, LAC = L-arg control; 0 = Normal, 1 = Interstitiel capillary congestion andedema, 2 = Interstitiel hemorrhage, 3 = Hemorrhagic infarct.

Figure 4. MDA values in three groups of 6 rats (µmol/g-wet tissue).

(p < 0.05). The MDA values were demonstrated in(Figure 4).

In all control groups, the testes showed normaltesticular architecture. In experimental isotonic groupscapillary congestion, interstitial edema and hemor-rhagea was observed. This group and L-NAME grouphad greater histological injury than L-arg group. Butthis was not statistically significant Histopathologicalevaluation is summarized in (Table 1).

Discussion

Testicular torsion and detorsion induces morpholo-gical and biochemical changes caused by both ischemiand reperfusion of the tissues. Several studies showedthat I/R injury is caused by oxygen free radicals.Antioxidant treatments have been used successfully todecrease reperfusion injury in multiple organ systemssuch as heart, lung, bowel, and liver. A decreasein blood flow to organs is known as ischemia andresults in elevated levels of lactic acid, hypoxanthine,

and lipid peroxides. The gradual increase following adecrease in blood flow to organs, so called reperfusion,may lead to further increase in tissue damage throughincreased lipid peroxidation following oxygen derivedfree radical production [1]. The testicular injury dueto torsion and detorsion shares resemblances withthe phenomenon of ischemia-reperfusion observed inother tissues [3, 16]. One source of free radicalsin postischemic tissue is the hypoxanthine-xanthineoxidase reaction. Ischemia causes an increase in intra-cellular hypoxanthine as a result of ATP breakdown.When oxygen is supplied during reperfusion, xanthineoxidase converts hypoxanthine to uric acid plus largequantities of superoxide radical. A second source offree radicals involves the respiratory burst by neut-rophils. The enzyme responsible for this respiratoryburst is NADPH oxidase, an enzyme bound to the cellmembranes, which converts oxygen to the superoxideanion. Calcium influx into neutrophils during ischemiacauses increased NADPH oxidase activity in the celland leads to release of the superoxide radical duringreperfusion [15].

Janetschek et al. have shown that the decrease ofperfusion and the extent of hemorrhagic infarctiondo not correlate well to the degree of an intravaginaltorsion [8]. Therefore, this type of torsion is notideal for experimental purpose. In contrast to that,the correlation is almost linear with an extravaginaltorsion. Extravaginal torsion up to 540◦ results invenous outflow obstruction where further torsionresults in arterial inflow obstruction. As experimentalmodel in rat, we therefore used extravaginal torsionthrough 720◦ to achieve testicular ischemia. In reper-fusion injury, total injury sustained is a combinationof ischemic and reperfusion components. Free radical

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scavengers are effective when the major portion ofthe total injury sustained is caused by the reperfu-sion component. In prolonged ischemia, the injurydue to ischemia itself may be so great that no furtherinjury of reperfusion could take place [1]. Bergh etal. tried to prevent reperfusion injury in testis aftersixty minutes of ischemia and found that free radicalscavengers did not influence the extent of testiculardamage [4]. Akgür et al. have speculated that sixtyminutes of ischemia might cause enough damagewithout leaving a place for further damage in thereperfusion period in testes and testis might resemblethe intestine in response to ischemia [1]. Schoen-berg has shown that all adenosine triphosphate (ATP)consumed after thirty minutes of ischemia in smallintestine, and this was a sufficient ischemic periodto see reperfusion damage in small intestine [18].Therefore we performed thirty minutes of testicularischemia in order to avoid excessive ischemic damagethat might cover reperfusion injury.

Testicular torsion and detorsion induces morpholo-gical and biochemical changes caused by both ischemiand reperfusion of the tissues. Several studies showedthat I/R injury is caused by oxygen free radicals.Antioxidant treatments have been used successfully todecrease reperfusion injury in multiple organ systemssuch as heart, lung, bowel, and liver [12, 18, 21].It has been reported that allopurinol and superoxidedismutase plus catalase treatment caused significantrescue of the testes function after testicular detorsion.Conversely, desferoxamine and diltiazem and VitaminE were used to prevent reperfusion injury in testis, butno beneficial effects have been reported [4].

Nitric oxide is a highly reactive free radical witha multitude of organ-specific regulatory functions.NO plays a major role in many organ systems, andderanged NO synthesis causes a number of patho-physiological states [7, 21].

There are conditions in which it will be benefi-cial to increase NO and other conditions in whichselective inhibition of NO formation may be desirable.In this study, L-arginine, precursor of NO, was usedto increase NO synthesis; and L-NAME, a competi-tive inhibitor of NO synthase was used to reduce NOformation.

We found that pretreatment with L-NAME, andL-arginine resulted to significant difference on MDAlevels in normal testes. But pretreatment with L-NAME before detorsion resulted the highest levelsof MDA, whereas this value was nearly equal withcontrols on L-arginine group (p < 0.05).

The severest histopathological findings were foundin L-NAME group. This concludes the decrease of NOhas negative effects on testicular ischemia-reperfusioninjury. But there is no statistically significant differ-ence from other groups. This may be because ofhistopathologic changes could not occur in hyperacute stage. In this study, L-arg infusion resulted anincrease in NO level and this concluded a decreasein acute reperfusion injury. NO inhibits the highvascular permeability on ischemia, and also adhesionof leucocytes and trombosit aggregation.

References

1. Akgur FM, Kilinc K, Tanyel FC et al. Ipsilateral and contrala-teral testicular biochemical acute changes after unilateraltesticular torsion and detorsion. Urology 1994; 44: 413.

2. Aydin S, Ugras S, Odabas O et al. Experimental testiculartorsion and its effects on the contralateral testicle. Int UrolNephrol 1997; 29: 661.

3. Becker EJ, Prillaman HM, Turner TT. Microvascular bloodflow is altered after repair of testicular torsion in the rat. J Urol1997; 157: 1493.

4. Bergh A, Damber JE, Marklund SE. Morphological changesinduced by short term ischemia in rat testis are not affectedby treatment with superoxide dismutase or catalase. J Androl1988; 9: 15.

5. Carden DL, Granger DN. Pathophysiology of ischemia-reperfusioninjury. Jpathol 2000; 190: 255.

6. Ciftci AO, Muftuoglu S, Cakar N, Tanyel FC. Histolo-gical evidence of decreased contralateral testicular blood flowduring ipsilateral testicular torsion. Br J Urol 1997; 80: 783.

7. Ferguson ND, Granton JT. Inhaled nitric oxide for hypoexemicrespiratory failure: passing bad gas? C M A J 2000; 162: 85.

8. Janetschek G, Schreckenberg F, Grimm W, Marberger M.Hemodynamic effects of experimental testicular torsion. UrolRes 1987; 15: 303.

9. Kannan MS, Guiang S, Johnson DE. Nitric oxide: biologicalrole and clinical uses. Indian J Pediatr 1998; 65: 333.

10. Kizilcan F, Bernay I, Tanyel FC et al. Ipsilateral and contrala-teral testicular blood flows during unilateral testicular torsionby 133Xe clearance technique. Int Urol Nephrol 1992; 24:515.

11. Kosar A, Sarica K, Kupeli B et al. Testicular torsion: eval-uation of contralateral testicular histology. Inti Urol Nephrol1997; 29: 351.

12. Kubes P. The role of adhesion molecules nad nitric oxide inintestinal and hepatic ischemia-reperfusion. Hepatogastroen-terology 1999; 46: 1458.

13. Mikuz G. Testicular torsion: simple grading for histologicalevaluation of tissue damage. Appl Pathol 1985; 3: 134.

14. Palmer JS, Plzak LF, Cromie WJ. Comparison of blood flowand histological changes in rat models of testicular ischemia.J Urol 1997; 158: 1138.

15. Prillaman HM, Turner TT. Rescue of testicular function afteracute experimental torsion. J Urol 1997; 157: 340.

16. Saba M, Morales CR, De Lamirande E, Gagnon C. Morpho-logical and biochemical changes following acute unilateraltesticular torsion in prepubertal rats. J Urol 1997; 157: 1149.

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17. Salman AB, Kilinc K, Tanyel FC. Torsion of only spermaticcord in the absence of testis and/epididymis results incontrala-teral testicular hypoxia. UrolRes 1997; 25: 413.

18. Schoenberg MH, Beger HG. Reperfusion injury after intestinalischemia. Crit Care Med 1993; 21: 1376.

19. Tanyel FC, Buyukpamukcu N, Hicsonmez A. Contralateraltesticular blood flow during unilateral testicular torsion. Br JUrol 1989; 63: 522.

20. Wax SH. Measurement of testicular blood flow by percu-taneous injection of 133 Xe. Invest Urol 1971; 9: 167.

21. Weinbroum AA, Kluger Y, Shapira I, Rudick V. Methyleneblue aboliches aortal tone impairment induced by liverischemia-reperfusion in a dose response manner: an isolatedperfused double-organ rat model study. Shock 2001; 15: 226.

Address for correspondence: Dr. Meral Barlas, Ankaralilar cad.499, sok No. 22, Cayyolu 06530, Ankara, Turkey