Involvement of Nitric Oxide in Microcirculatory Reactions after Ischemia-Reperfusion of the Rat Urinary Bladder

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1 Originl Pper Eur Surg Res 29;42:28 34 DOI: / Received: Mrch 25, 28 Accepted fter revision: August 25, 28 Published online: November 6, 28 Involvement of Nitric Oxide in Microcircultory Rections fter Ischemi-Reperfusion of the Rt Urinry Bldder Z. Bjory, b A. Szbó c I. Király b L. Pjor b K. Messmer Institute for Surgicl Reserch, University of Munich, Munich, Germny; b Clinic of Urology, nd c Institute of Surgicl Reserch, University of Szeged, Szeged, Hungry Key Words Bldder Ischemi-reperfusion Microcircultion Nitric oxide L -NAME Abstrct Bckground: Nitric oxide (NO) plys role in inflmmtion. Our im ws to investigte the role of NO in the microcircultory chnges fter ischemi-reperfusion (I/R) of the bldder using intrvitl videomicroscopy (IVM). Methods: In rts, 6 min of bldder ischemi followed by 3 min of reperfusion ws performed in the presence of N G -nitro- L -rginine methyl ester ( L -NAME), the NO precursor L -rginine, or sline pre-tretments. Venulr red blood cell velocity (RBCV), functionl cpillry density (FCD), vessel dimeters, nd leukocyte-endothelil cell interctions in postcpillry venules were determined. Concentrtions of nitrite/nitrte in the plsm nd myeloperoxidse (MPO) levels in the lungs nd the bldder were mesured. Results: Elevtions of the numbers of rolling nd dherent leukocytes, nd of plsm nitrite/nitrte levels were found, while FCD nd RBCV decresed. L -NAME pretretment meliorted the enhnced leukocyte-endothelil cell interctions without influencing the microcircultory perfusion. In contrst, the L -rginine pretretment further incresed plsm nitrite/nitrte levels nd preserved the FCD nd RBCV, but did not ffect leukocyte-endothelil interctions. None of these tretments in- fluenced MPO ctivities. Conclusion: Our results suggest tht NO plys n enhncing role in the I/R-induced neutrophil-endothelil interctions of the bldder. Supplementtion of NO meliortes the microcircultory perfusion deficit without influencing the postischemic microcircultory inflmmtory rections. Copyright 28 S. Krger AG, Bsel Introduction In clinicl prctice, hypoperfusion of the urinry bldder cn result from globl ischemi during shock nd trum, or from different locl events cused by embolism, thrombophlebitis, ligtion, complictions of pregnncy or pelvic surgery, compression by pelvic tumors or over-distension of the bldder cused by lower urinry trct obstruction. Ischemi of the bldder is n infrequent but serious event in clinicl prctice nd, hence, the consequences cn rnge from functionl chnges to even necrosis [1]. During the reperfusion phse, reoxygention of formerly ischemic tissue induces cscde of chemicl rections nd cellulr events [2], some of which re medited by toxic oxygen-derived free rdicls with resultnt severe tissue dmge [3 5]. Among the cellulr events, neutrophils hve been shown to ct s key plyers in the Fx E-Mil krger@krger.ch 28 S. Krger AG, Bsel X/9/421 28$26./ Accessible online t: Zoltán Bjory Clinic of Urology, University of Szeged Klvri sgt. 57 HU 6725 Szeged (Hungry) Tel , Fx /28, E-Mil bjory@freemil.hu

2 pthophysiology of ischemi-reperfusion (I/R) injury [6]. Specificlly, enhnced neutrophil ctivtion nd their interction with the endothelium with consequent tissue invsion re chrcteristic inflmmtory rections in response to I/R. The present study ws conducted to exmine the pthomechnisms of these I/R-induced inflmmtory responses of the bldder microcircultion [7]. The brupt increse in blood flow t the onset of reperfusion induces the relese of endothelium-derived meditors such s nitric oxide (NO). This molecule possesses numerous physiologicl functions [8 1], but it hs lso been suggested tht NO plys n importnt role in pthophysiologicl sttes such s septic shock [11] or I/R injury [12]. Although NO serves s vsodiltor nd cn ct s n nti-inflmmtory gent [13], it hs lso been shown to possess pro-inflmmtory chrcteristics [14, 15]. Specificlly, NO hs been implicted in free rdiclmedited toxicity contributing to reperfusion injury of numerous orgns [9, 11, 12, 16]. Little is known bout the functions of NO in the urinry bldder, but it hs been demonstrted to influence the bldder smooth muscle function fter ischemi [17, 18]. As shown by Sito nd co-workers [19, 2], bldder I/R-induced contrctility dysfunction could be prevented by nitric oxide synthse inhibition. Similrly, the use of NO synthse inhibitors ppered to be beneficil nd nti-inflmmtory in cystitis models [14]. NO hs lso been shown to medite microcircultory chnges in the different lyers of filled or emptied bldder [21, 22]. Herein, we hypothesized tht NO is involved in the medition of I/R-induced microcircultory chnges in the bldder. Therefore, the consequences of the reduction of endogenous NO levels with inhibition of the NO synthse nd triggering its effects with the NO precursor L- rginine were exmined in bldder I/R model using intrvitl video microscopy. The dt show tht endogenous NO plys n enhncing role in the medition of I/R induced erly neutrophil-endothelil interctions, but plys minor role in medition of the cpillry perfusion chnges in the urinry bldder microcircultion. Mterils nd Methods Animls nd Experimentl Groups The experiments were performed fter governmentl pprovl nd in ccordnce to the Germn nd Hungrin legisltion on niml protection. 39 mle Sprgue-Dwley rts (verge weight 23 g) were housed in n environmentlly controlled room with 12-hour light-drk cycle nd were rndomly ssigned to four groups. In three experimentl groups, 6 min of bldder ischemi ws induced, nd rts were treted either with L -rginine (n = 7; 3 mg/kg; ICN, Germny), L -NAME (n = 12; 5 mg/kg; Sigm, USA) or sline (n = 13, 1 ml/kg) vehicle i.v., 1 min before ischemi nd 1 min before reperfusion in blinded mnner. Time-mtched shm operted sline treted rts served s controls (n = 7). Surgicl Procedure nd Experimentl Protocol After tropine premediction (.1 mg/kg s.c.; Brun, Germny) nd pentobrbitl (Nrcoren ; Meril GmbH, Germny) nesthesi (45 mg/kg i.p.) the nimls were plced in the supine position on heting pd. The surgicl procedures were performed under n opertion microscope (Leic M651; Germny). Polyethylene ctheters (ID.28 mm, OD.61 mm; SIMS Portex Ltd., UK) were inserted into the crotid rtery nd jugulr vein for the mesurement of the men rteril pressure (MAP) nd hert rte (HR) (Plugsys ; H. Schs Elektronik, Germny) nd for the injection of fluorescent dyes. The trche ws cnnulted (Abbocth -T; 13G; Abbott, Irelnd) to fcilitte spontneous respirtion. Following midline lprotomy, the bldder ws prepred nd the medin umbilicl ligment ws resected. The urethr ws ligted with 4- Perm-Hnd silk (Johnson & Johnson, Belgium). Both ureters were dissected in the mid-section. A polyethylene ctheter (ID.28 mm, OD.61 mm; SIMS Portex) ws inserted into the bldder dome nd fixed by drop of Histocryl (Aesculp AG & Co. KG, Germny) for filling the bldder nd to mesure nd mintin constnt intrvesicl pressure (Plugsys). To this end, the bldder ws filled with.5 ml of.9% NCl solution t body temperture. This method hs been shown to provide good qulity of visuliztion of the bldder microvsculture. Ischemi of the bldder ws induced by clmping both cystic rteries, then the bdominl wll closed. After 6 min, the bdomen ws reopened nd the clips removed llowing for 3-min reperfusion period. After 3 min reperfusion, the bldder ws exteriorized nd plced on specilly designed stge nd IVM recordings were performed. At the end of the experiments, blood smples for nitrite/nitrte mesurements nd white blood cell count determintions nd bldder nd pulmonry tissue smples for myeloperoxidse (MPO) mesurements were tken, nd the nimls were then euthnized. In t rv i t l Vi d e o m i c ro s c o p y Fluorescein isothiocynte (FITC)-lbeled lbumin (MW 7,, i.v., in.2 ml, Sigm) for the plsm stining nd rhodmine 6G (.1 ml,.2%, MW 479, Sigm) for leukocyte lbeling were used ( fig. 1, b). The microcircultion ws visulized by using high-resolution, modified Zeiss-Orthopln intrvitl microscope ttched to Ploemo-Pk illumintor with n I2/3 blue (excittion filter (Ex): 495 nm, emission filter (Em): 515 nm) nd N2 green (Ex: 525 nm, Em: 555 nm) filter block (Leitz, Wetzlr, Germny) with n epi-illumintion technique. With wter immersion objective (W 25X/.6; Leitz, Germny), the mgnifiction ws! 54 on the video screen (Sony, Jpn). The microscopic imges were recorded vi chrge-coupled device video cmer (FK 699; Pieper GmbH, Germny) connected to n S-VHS video recorder (BR-S92E; JVC, Jpn) for further computer-ssisted off-line dt evlution [23]. Video Anlysis Quntittive ssessment of microcircultory prmeters ws performed by computer-ssisted nlysis system (CAMAS, Dr. NO in Microcircultory Rections fter I/R of Rt Urinry Bldder Eur Surg Res 29;42:

3 fuged for 1 min through microfilter (MicroSpin G25 columns; Phrmci Biotech, Germny). A solution of.75 m M NADPH, 5 M flvin denine dinucleotide (Sigm) nd.4 U/ml nitrte reductse (Boehringer Mnnheim, Germny) ws then dded to 5 l plsm. After incubtion for 3 min t 37 C, solution contining 25 U/ml lctte dehydrogense nd 1 m M pyruvte (Sigm) ws dded. Upon further incubtion for 5 min, the totl concentrtion of nitrite/nitrte ws determined using the Griess regent bsorption reding t 55 nm. Absolute vlues were clculted using stndrd sodium nitrite curve. Myeloperoxidse Mesurements The MPO ctivity, n index of the neutrophil ccumultion in the tissues, ws determined by using modified method of Kuebler et l. [25]. MPO ws extrcted from the tissues by two seprtion procedures. Smples were initilly homogenized in.2 M potssium phosphte buffer t ph 7.4 contining protese inhibitor nd centrifuged t 2, g for 2 min. The pellet ws resuspended in.5 M potssium phosphte buffer t ph 6. contining.5% hexdecylmmonium bromide. The suspension ws sonicted, frozen-thwed 3 times nd centrifuged gin t 2, g for 2 min. The superntnt ws then heted t 6 C for 6 min to fcilitte the recovery of MPO. An liquot of superntnt ws mixed with solution of 1.6 m M tetrmethylbenzidine nd.2% hydrogen peroxide. The ctivity ws mesured spectrophotometriclly s the chnge in bsorbnce t 65 nm t 37 C. Results re expressed s units of MPO ctivity per grm wet tissue. S t t i s t i c s Sttisticl nlysis ws performed with sttisticl softwre pckge (SigmStt 2. for Windows, Jndel Scientific, Germny). Kruskl-Wllis nlysis of vrince nd the Student-Newmn-Keuls method were pplied for multiple comprisons between the different groups. Men vlues 8 SD were given. p!.5 ws considered significnt. Fig. 1. Intrvitl microscopic imge of the microvsculture of the urinry bldder using plsm lbeling with fluorescein isothiocynte-lbumin (MW 7,) ( ) nd rhodmine 6G for leukocyte stining ( b ). H. Zeintl, Germny) [7]. Functionl cpillry density (FCD, length of perfused cpillries per observtion re (1/cm), m = 5, m: number of the field of interest), rteriolr (AD) nd venulr (VD) dimeters ( m), nd venulr red blood cell velocity (RBCV, mm/s) were determined in the musculr lyer. Leukocyte-endothelil cell interctions were nlyzed, including the observtion of the firmly dherent (stickers) nd rolling leukocytes (rollers). Stickers were identified s cells tht did not detch from the endothelium within 3 s nd re given s number of cells/mm 2 of endothelil surfce. Rollers were defined s cells moving slower thn other blood cells nd were given s number of cells/vessel dimeter (mm)/s [23]. Nitrite/Nitrte Mesurements Totl plsm levels of nitrite nd nitrte were mesured using the Griess rection [24]. Briefly, the smples were ultrcentri- R e s u l t s Microcircultory Chnges Ischemi followed by 3 min reperfusion ws ssocited with mrked reduction in the efficcy of microcircultion s evidenced by the pproximtely 66% reduction in FCD nd by pproximtely 7% decrese in RBCV in the sline-treted nimls ( fig. 2, b). Tretment with L -rginine restored FCD nd RBCV vlues to the levels seen in shm-operted nimls, while L- NAME resulted in similr vlues to tht found nimls subjected to I/R. Arteriolr nd venulr dimeters decresed in response to I/R in the sline-treted group (AD: shm m, sline m; VD: shm m, sline m), but without reching sttisticl significnce, nd were not influenced by L - NAME or L -rginine dministrtion. 3 Eur Surg Res 29;42:28 34 Bjory /Szbó /Király /Pjor /Messmer

4 1.4 SH I/R 8 SH I/R 1.2 RBCV (mm/s) # Rolling leukocytes (1/mm/s) # FCD (1/cm) b # Adherent leukocytes (1/mm 2 ) 1,8 1,6 1,4 1,2 1, # 2 b Fig. 2. Chnges in RBCV ( ) nd FCD ( b ) fter shm opertion (SH) nd I/R of the bldder in the presence of sline, L -rginine ( L -Arg), or L -NAME tretment. Men vlues 8 SEM re indicted. p!.5 vs. the shm (SH) group, # p!.5 vs. I/R + sline group. Fig. 3. Chnges in the number of rolling ( ) nd dherent/sticking ( b ) leukocytes in the postcpillry venules fter shm opertion (SH) nd I/R of the bldder in the presence of sline, L-rginine (L- Arg), or L -NAME tretment. Men vlues 8 SEM re indicted. p!.5 vs. the SH group, # p!.5 vs. I/R + sline group. Microcircultory inflmmtion ws evidenced by the significntly higher numbers of rolling nd sticking leukocytes in the postcpillry venules in the sline- nd L- rginine-treted groups in response to I/R thn tht fter shm opertion ( fig. 3, b). In the L -NAME treted group, however, both prmeters were found similr to tht of the shm-operted group. Bio ch e mi c l C h nge s At 3 min of reperfusion, plsm nitrite/nitrte levels were significntly higher in nimls subjected to I/R thn fter shm opertion in the presence of vehicle tretment (fig. 4 ). In the L -rginine-treted nimls, nitrite/nitrte levels were even higher (p!.5), wheres in the L- NAME-treted group these vlues were similr to those seen fter shm opertion. MPO levels in the bldder were more thn 35-fold higher in response to I/R ( mu/mg) thn fter shm opertion ( mu/mg) in the slinetreted nimls. Neither the NO precursor L-rginine nor the NO synthse inhibition influenced this prmeter significntly ( fig. 5 ). Since we pplied systemic NO synthse inhibition, we wnted to exclude ny bis of the locl microcircultion mesurements resulting from polymorphonucler leukocyte deposition in other orgns such s the lungs. Therefore, we counted the systemic white blood cell numbers nd mesured MPO ctivity in lung smples. MPO ctivity did not differ significntly in NO in Microcircultory Rections fter I/R of Rt Urinry Bldder Eur Surg Res 29;42:

5 5 SH I/R 1,8 SH I/R NOX (μm) # MPO (mu/mg) 1,6 1,4 1,2 1, Fig. 4. Plsm nitrite/nitrte vlues fter shm opertion (SH) nd I/R in the presence of sline, L -rginine ( L -Arg), or L-NAME tretment. Men vlues 8 SEM re indicted. p!.5 vs. the shm (SH) group, # p!.5 vs. I/R + sline group. 5, 4, the shm-operted sline-treted nimls (2, mu/mg) nd in response to bldder I/R in the presence of ny of the pretretments ( fig. 5 b). Similrly, none of the tretment regimen pplied ffected the white blood cell counts (dt not shown). MPO (mu/mg) b 3, 2, 1, Discussion One-hour ischemi followed by 3 min of reperfusion hs been demonstrted to reduce detrusor contrctility nd to cuse leukocyte infiltrtion in the urinry bldder (s evidenced by histology) [19]. In previous studies, we hve demonstrted tht bldder I/R induces microcircultory rections shring mny of the chrcteristics of n cute inflmmtion such s ccumultion of leukocytes, mcromoleculr extrvstion nd edem formtion [7]. In the present work in the sme model, the role of NO ws exmined using L -NAME nd L -rginine. The presence of NO synthse hs lredy been demonstrted in the bldder. As shown by histochemistry, NADPH diphorse-positive nerves cn be found in the subepithelil region, longside smooth muscle bundles nd within intrmurl gngli [26]. Souz-Filho et l. [14] observed tht n incresed number of NO synthse-contining cells re present in the urothelium nd the immune cells of the lmin propri in cyclophosphmide-induced cystitis. Although I/R-induced endothelil dysfunction cn led to diminished NO synthse ctivity with consequently Fig. 5. MPO enzyme ctivity in the bldder ( ) nd the lung ( b ) fter shm opertion (SH) nd I/R in the presence of sline, L -rginine ( L -Arg), or L -NAME tretment. Men vlues 8 SEM re indicted. p!.5 vs. the shm (SH) group. reduced NO formtion [27], in the present study, n enhnced relese of NO in response to bldder I/R occurred s plsm levels of NO metbolites (nitrite/nitrte) were incresed. We think it is n evidence of n enhnced NO production in our setting, hence some of the microcircultory ltertions cn be linked to incresed intrvesicl production of NO. Furthermore, L -rginine dministrtion further incresed NO levels, s nitrite/nitrte levels were higher thn wht were induced by I/R. In most studies, protective role is ttributed to NO which mostly cts s vsodiltor nd n nti-inflmmtory gent [9, 28, 29]. As for the bldder, however, numerous dt suggest tht n enhnced relese of NO could led to detrimentl consequences. In the bove cyclophosphmide-induced cystitis, dministrtion of NOS 32 Eur Surg Res 29;42:28 34 Bjory /Szbó /Király /Pjor /Messmer

6 inhibitors meliorted plsm extrvstion, edem formtion, leukocyte infiltrtion nd vsculr congestion. As result, mucosl dmge nd hemorrhge were lso reduced [14]. In the sme study, however, excess mount of NO ws most probbly produced by the inducible NO synthse. In the present work, I/R induced mrked ctivtion of primry nd secondry neutrophil-endothelil interctions which could be meliorted by inhibition of NO production. This suggests tht even in n erly stge of reperfusion injury NO possesses pro-inflmmtory fetures. In similr study with bldder I/R, Sito et l. [19] provided direct evidence on enhnced NO relese (using NO-sensitive electrodes) nd excessive leukocyte infiltrtion in the submucos nd smooth muscle. The corresponding reduction in contrctile function of the bldder muscle lyer could lso be reserved by L-NAME. Histologicl investigtions suggest tht in erly phses of reperfusion, NO is produced by the constitutive nd neuronl NO synthse, but during lter stges, further increse in NO relese my hve been cused by inos in leukocytes infiltrting the submucos nd muscle lyer. These chnges could lso be prevented by L-NAME [2]. Although the inhibition of NO relese in the present study reduced neutrophil-endothelil interctions, these chnges could not be reversed by the NO substrte L-rginine. This suggests tht the potentil pro-inflmmtory fetures of NO re linked not only to the mount of NO produced. It could be lerned from studies trgeting the role of NO in the free-rdicl-medited toxicity tht simultneous formtion of NO nd oxygen-centered free rdicls result in n incresed production of the highly toxic metbolite peroxynitrite [1]. Similr mechnisms my operte here nd in other orgns where inflmmtory roles of NO were demonstrted [14, 3, 31]. Footprints of NO-derived toxicity (tyrosine nitrtion) were lso demonstrted in chronic model of prtil bldder outlet obstruction [32]. In this study, L-rginine resulted in mrked increse in NO synthesis with consequently incresed genertion of rective nitrogen-derived free rdicls, wheres NO synthse inhibition cused deteriortion in the obstruction-induced contrctile function. The uthors rgued tht the beneficil effects of NO on blood flow were more importnt thn the effects on free rdicl genertion. These dt re somewht contrdictory to tht of Sito et l. [19], but the differences my be explined by the different models. It is likely tht oxido-reductive stress during cute I/R syndromes hs higher impct on the bldder contrctile function thn in chronic, obstruction-relted experimentl models. MPO ctivity ws lso used s indictor of neutrophil deposition in the tissues. Interestingly, very high increses in this prmeter could be demonstrted ll I/R groups irrespectively to the pplied interventions in the bldder. As MPO mesurements cnnot differentite between intrnd extrvsculr leukocytes, we believe tht intrvitl microscopic evlutions give more pproprite nlysis of microcircultory intrvsculr processes. On the other hnd, we used these evlutions to chrcterize potentil side effect of NO synthse inhibition: the PMN deposition in the lungs which hs been lso demonstrted elsewhere [33]. By these mens, we excluded the potentil bis of deposition of ctivted PMNs in the lungs, which would hve been reduced their vilbility t the bldder. Tissue perfusion exmintions gve nother pproch for the judgment of the efficcy of the different interventions used in this study. The I/R-induced perfusion deficit (s evidenced by FCD nd RBCV mesurements) seen in the sline tretment group ws not mrkedly influenced by L -NAME dministrtion. This suggests tht, s opposed to the bove inflmmtory chnges, the I/R-induced perfusion deficit is not criticlly determined by endogenous NO. It cn be lerned from other studies tht different lyers of the urinry bldder hve different sensitivity to strtegies trgeting the NO production. Specificlly, NO donors increse, but NO inhibitors reduce perfusion of the mucos without ffecting the muscle perfusion [21]. In nother model, which did not differentite between the microcircultory chrcteristics of the different lyers of the bldder, definite role of NO in regulting the bldder perfusion ws demonstrted. Nmely, NO donor therpy incresed tissue perfusion, but its effect ws different t different filling sttes nd ntomicl loctions of the bldder [22]. The present study trgeted the chnges in the perfusion of the muscle lyer nd demonstrted no mjor effect of endogenous NO t influencing I/R-induced microcircultory disturbnces of the bldder. In our model, however, the NO substrte L-rginine completely llevited the microcircultory perfusion deficit nd the no-reflow phenomenon seen fter I/R. This effect, however, ws not ccompnied by improvement t the level of microcircultory inflmmtory rections. Conclusions Hllmrks of I/R-induced bldder injury, i.e. leukocyte recruitment nd reductions in the efficcy of tissue perfusion, cn effectively be trced by IVM which pro- NO in Microcircultory Rections fter I/R of Rt Urinry Bldder Eur Surg Res 29;42:

7 vides n excellent method for the on-line observtion of these microcircultory ltertions. Our findings suggest tht NO plys detrimentl role during I/R-induced inflmmtory rections of the bldder microcircultion. From clinicl point of view, inhibition of NO synthesis my beneficilly influence the erly microcircultory inflmmtory rections t the onset of reperfusion, without cusing mjor side effects on the regultion of locl postischemic blood flow. Acknowledgements We thnk Prof. Dr. Mihály Boros from the University of Szeged, Hungry, nd Dr. Jörg Hutter nd Prof. Fritz Krombch from the University of Munich, Germny, for their discussion of the observtions. This study ws supported by the Europen Urologicl Scholrship Progrm (EUSP) of the Europen Assocition of Urology (EAU) nd the Hungrin Reserch Fund OTKA: F References 1 Sieber PR: Bldder necrosis secondry to pelvic rtery emboliztion: cse report nd literture review. 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