effects of l-na and sodium nitroprusside on ischemia/reperfusion-induced leukocyte adhesion and...

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Microvascular Research 62, 128–135 (2001)doi:10.1006/mvre.2001.2324, available online at http://www.idealibrary.com on

Effects of L-NA and Sodium Nitroprussideon Ischemia/Reperfusion-Induced LeukocyteAdhesion and Macromolecular Leakagein Hamster Cheek Pouch Venules

Claudia Simoes,* Erik Svensjo,*,† and Eliete Bouskela**Laboratorio de Pesquisas em Microcirculacao, Universidade do Estado do Rio de Janeiro, Rua Sao FranciscoXavier 524, 20550-013 Rio de Janeiro; and †Instituto do Coracao, Divisao de Experimentacao,Universidade de Sao Paulo, Av. Eneas de Carvalho Aguiar 44, 05403-000 Sao Paulo, Brazil

Received November 13, 2000; published online June 20, 2001

Our objective was to study how the topical application ofa nitric oxide synthase inhibitor (L-NA, Nv-nitro-L-argi-nine) and a nitric oxide donor, sodium nitroprusside(SNP), could modulate leukocyte adhesion (sticking) andmicrovascular permeability as altered by ischemia/reper-fusion (I/R) and topically applied histamine after I/R.Golden hamsters were prepared for intravital micros-copy. Ischemia was induced by an inflatable silicon rub-ber cuff mounted around the neck of the cheek pouchprepared for intravital microscopy. Saline, L-NA, sodiumnitroprusside, and histamine were applied in the super-fusion solution. FITC–dextran was injected iv 30 minbefore initiation of ischemia as a marker of microvascularpermeability. L-NA 1025 M inhibited both the increase innumber of sticking leukocytes and the increase in vascu-lar permeability after I/R compared with the untreatedcontrol group of hamsters. SNP neutralized this effect ofL-NA on leukocytes and vascular permeability and causedarteriolar dilation at the concentration used, 1026 M.Both SNP and L-NA 1 SNP enhanced the I/R-induced

acromolecular leakage. The topical application of SNPnd SNP 1 L-NA did not modify the response to hista-
ine after I/R compared with the untreated control
roup. In hamsters not subjected to I/R, histamine-nduced macromolecular leakage was inhibited by L-NA

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and L-NA 1 SNP but was unchanged by SNP. It is con-cluded that inhibition of nitric oxide formation by L-NAreduced both leukocyte adhesion in postcapillary venulesand the increase in macromolecular leakage and that aNO donor such as SNP could enhance the macromolecu-lar leakage response to I/R. © 2001 Academic Press

Key Words: ischemia/reperfusion; plasma leakage; ni-tric oxide; postcapillary venules; nitric oxide synthase;L-NA (N-nitro-L-arginine); sodium nitroprusside.

INTRODUCTION

The accumulation of leukocytes in inflamed tissueresults from adhesive interactions between leukocytesand endothelial cells within the microcirculation andthese interactions and the excessive filtration of fluidand protein that accompanies an inflammatory re-sponse are largely confined to one region of the mi-crocirculation, the postcapillary venules, (Granger andKubes, 1994). Ischemia/reperfusion (I/R)-induced
leukocyte–endothelial interactions in postcapillaryvenules have been the subject of several recent re-views. Eppiheimer and Granger (1997) emphasized
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the critical role of leukocyte–endothelial cell adhesionin the pathobiology of I/R and showed that muchprogress has been made in defining the chemical me-diators that are responsible for the recruitment of roll-ing, adherent, and emigrating leukocytes in postcap-illary venules exposed to I/R injury (Eppiheimer andGranger, 1997). Another review emphasized the cen-tral role of oxygen-free radicals in the manifestation ofI/R injury and recommended the use of antioxidantsto counteract postischemic reperfusion damage (Mass-berg and Messmer, 1998). Grisham et al. (1998) re-viewed the evidence implicating reactive metabolitesof oxygen and nitrogen as modulators of leukocyte–endothelial cell interactions in vivo and discussed howthese mechanisms may be involved in the pathophys-iology of ischemic heart disease (Grisham et al., 1998).The chemistry of nitric oxide (NO) in biological sys-tems is extensive and complex. Wink and Mitchellhave reviewed the chemical biology of NO dividedinto two major categories, direct and indirect. Directeffects were defined as those reactions fast enough tooccur between NO and specific biological molecules.Indirect effects do not involve NO, but rather aremediated by reactive nitrogen oxide species formedfrom the reaction of NO with either oxygen or super-oxide (Wink and Mitchell, 1998).

Total ischemia of the hamster cheek pouch for 30min followed by reperfusion induces leukocyte stick-ing and increased plasma leakage from postcapillaryvenules, both of which were inhibited by a stableprostacyclin analogue, Iloprost, demonstrating the im-portance of activated leukocytes for the induction ofplasma leakage (Erlansson et al., 1991). I/R injury to

ostcapillary venules was also inhibited by superox-de dismutase (SOD) and extracellular SOD, implicat-ng a role for superoxide (Erlansson et al., 1990). Theuperoxide formed at reperfusion after ischemia mayeact with nitric oxide, resulting in peroxynitrite for-ation, which might be the injurious agent to the

ndothelium (Kooy and Royall, 1994; Wolin, 1996;urzoy-Ozdemir et al., 2000). In studies on the perme-

bility of the blood–brain barrier in rats, NO donorsere found to increase the permeability of large mol-

Leukocyte Adhesion, Plasma Leakage and NOS Inhibition

cules (FITC–dextran), which led to the suggestionhat NO and superoxide had formed peroxynitriteMayhan, 2000). Adenosine diphosphate and bradyki-

in induced plasma leakage was inhibited by NOSnhibitors such as l-NMMA and Nv-nitro-l-arginine

(l-NA) (Mayhan, 1992; Svensjo and Roempke, 1994;Feletou et al., 1996). The role of NO as a modulator of

icrovascular permeability is still somewhat contro-ersial. There are evidences that endogenous NO mayither increase or decrease the microvascular perme-bility (Kubes, 1995) and it is not clear to what extenthe observed inhibition of inflammatory leakage ex-rted by NOS inhibitors is due to hemodynamic ef-ects (arteriolar constriction) rather than direct inter-erence with the formation of venular gaps or thectivation of leukocytes which might increase vascularermeability.In order to elucidate the role of nitric oxide in the

ostischemic behavior of leukocytes and postischemichanges in microvascular permeability, we used theO synthase inhibitor l-NA and the NO donor so-

dium nitroprusside (SNP), both applied topically tothe cheek pouch, thus avoiding any interference withcentral hemodynamics outside the microcirculation ofthe preparation we used.

MATERIALS AND METHODS

Male golden hamsters (90–120 g) were anesthetizedwith 0.1–0.2 ml sodium pentobarbital (60 mg/ml ip)and supplemented with iv doses of a-chloralose (100mg/kg). Body temperature was maintained by a heat-ing pad and a rectal thermistor. To facilitate sponta-neous breathing, a tracheostomy was performed. Theright femoral vein was cannulated for injections ofFITC–dextran (fluorescein-labeled dextran, Mw 5150,000, TdB Consultancy, Uppsala, Sweden), rhoda-mine, and a-chloralose (Sigma Chemicals, St. Louis,MO). The hamster cheek pouch was prepared for in-travital microscopy according to Duling (Duling, 1973)and with our modifications (Svensjo, 1990). Thirtyminutes after completed preparation FITC–dextranwas injected as macromolecular tracer (Mw 5 150,000,25 mg/100 g bw). The cheek pouch was continuously

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superfused with a warm (36.5°) Hepes-supported bi-carbonate-buffered saline solution which was bubbledwith 95% N2 and 5% CO2 to maintain a pH of 7.4 and

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a low oxygen tension. In one part of the study micro-vascular permeability changes were measured bycounting sites of plasma leakage (leaks) in postcapil-lary venules at fixed intervals after ischemia or appli-cation of histamine (Erlansson et al., 1990). Duringintravital microscopy observations of leukocyte roll-ing and sticking, the microvascular permeabilitychanges were estimated from fluorimetric determina-tions of FITC–dextran concentrations (Perkin–ElmerLS-50) in the superfusate drained in the microscopestage and expressed as FITC–dextran efflux, (nano-grams per 30-min periods).

Observation of leukocytes. A venular segment of40-mm diameter with a length of 150 mm was selectedfor observation with a water immersion objective lensX25 (Zeiss Achroplan). Leukocytes in circulation werelabeled in vivo by iv injection of rhodamine immedi-ately prior to the observations. The weak red fluores-cence was amplified in a Hamamatsu image intensifierand images were recorded with a Sony U-matic videotape recorder. Rolling and adhering leukocytes werecounted at playback of these tape recordings. Rollingwas defined as a leukocyte in contact with the venularwall with a velocity lower than that of erythrocytes.Sticking or adhering leukocytes were those cells at-tached to the venular wall for at least 30 s.

Experimental protocols.(1) Following FITC–dextran and rhodamine injec-

tion, video recordings were made for evaluation ofleukocyte behavior during 2 min with 10-min inter-vals. In four different groups of three to seven ham-sters each, local applications of saline, 1025 M l-NA,026 M SNP, and SNP 1 l-NA (1026 1 1025 M) were

made for the entire experiment starting 30 min afterthe FITC–dextran injection. Ten minutes later, com-plete ischemia was induced for 30 min as described(Erlansson et al., (1990) and observations were madeor a further 120 min. As mentioned above, vascularermeability changes were measured simultaneouslyy fluorimetric determination of FITC–dextran con-entrations in the superfusion buffer.(2) Four groups of six animals each were subjected

o a topical application of saline (control), 1025 M

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l-NA, 1026 M SNP, and SNP 1 l-NA (1026 1 1025 M)for the entire experimental period starting 10 minprior to the ischemic period of 30 min. Histamine (1026


M for 5 min) was applied topically in all groups 1 hafter the onset of reperfusion as a test of the respon-siveness of the preparation. Vascular permeabilitychanges were quantitated by counting the number ofextravasations at postcapillary venules (leaks) at 0, 5,10, and 30 min after the start of reperfusion or thetopical application of histamine.

(3) Six other groups (six animals each) were submit-ted to the same drug concentrations as described (con-trol, l-NA, and SNP) and arteriolar and venular diam-eters were measured with (three groups of six animalseach) or without (three groups of six animals each)ischemia for 30 min.

(4) Four other groups (seven animals each) weresubjected to the same drug concentrations as de-scribed above and two sequential topical applicationsof histamine with 30-min intervals. The first histamineapplication was made before any drug application andthus served as a control for the second histamine.

Statistics. Data are presented as means 6 SEM.Statistical evaluation was performed with ANOVAand within groups a paired t test was used to evaluatechanges over time. A P value of 0.05 or less wasonsidered significant.

RESULTS

Total ischemia for 30 min caused a reversible in-crease in the number of leaks at reperfusion and thisincrease was significantly inhibited by superfusionwith l-NA (P , 0.01) and significantly (P , 0.01)enhanced by SNP (151%) as well as the combination

f l-NA and SNP (1104%, P , 0.01) (Fig. 1).Histamine stimulation at 60 min after reperfusion

caused reversible increases in the number of postcap-illary venular leaks. l-NA, SNP, and l-NA 1 SNPinhibited the histamine-induced effect (P , 0.01) by36, 39, and 52%, respectively, compared with the un-treated control group (Fig. 2).

At reperfusion there were no significant changes

Simoes, Svensjo, and Bouskela

observed in the numbers of rolling leukocytes andresults are not shown. The number of sticking leuko-cytes increased in the saline control group and the

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SNP and the l-NA 1 SNP groups, but not in the l-NAgroup (Figs. 3a and 3b).

Measurement of FITC–dextran efflux to the super-fusion solution during simultaneous observations ofleukocytes showed a reduced efflux or permeabilityonly for the l-NA group (Fig. 4).

Measurement of arteriolar diameters before and af-ter 30 min of ischemia showed that SNP augmentedthe arteriolar diameters from 40 min until the end ofexperiment (P , 0.05) but there were few changesseen in the untreated control and the l-NA groups,although there was a tendency to arteriolar constric-tion in the l-NA group which was significantly differ-ent from the control group at 15 min of reperfusion

FIG. 1. Maximal number of leaks observed during the reperfusioneriod after 30 min of total ischemia. Untreated control, superfusionith 1026 M SNP, 1025 M l-NA, and l-NA 1 SNP. Results are

expressed as means 6 SEM. **Significantly different from the con-rol group (P , 0.01).


FIG. 2. Maximal number of leaks observed after topical applica-tion of histamine at 1 h after the onset of reperfusion following 30min of ischemia. Results are expressed as means 6 SEM. **Signifi-cantly different from the control group (P , 0.01).

(P , 0.05) (Fig. 5a). Measurement of arteriolar diam-eters in hamsters not subjected to ischemia showed aslight vasodilation in the SNP group, no change in theuntreated control group, and a slight vasoconstrictionin the l-NA group (Fig. 5b).

Stimulation with 1026 M histamine (without priorI/R) prior to local application of the drugs resulted inreversible increases in the number of leaks in the fourgroups with no significant differences between the

FIG. 3. (a) Number of sticking leukocytes in postcapillary venulesbefore and after a period of total ischemia for 30 min. Effects of localapplication of normal saline (n 5 5), 1025 M l-NA (n 5 7), or 1026MSNP (n 5 3). Mean values 6 SEM of sticking leukocytes per unitrea (10,000 mm2).*Significant differences between the untreated

control and the l-NA group (P , 0.05). (b) Number of stickingleukocytes in postcapillary venules before and after a period of totalischemia for 30 min. Effects of local application of 1026 M SNP (n 5

3) and 1025 M l-NA 1 1026 M SNP (n 5 3). Mean values 6 SEM ofticking leukocytes per unit area (10,000 mm2).

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groups. Following application of normal saline, 1025

M l-NA, 1026 M SNP, and l-NA 1 SNP there was asignificant (P , 0.05) inhibition of the response of


histamine application in the l-NA and in the l-NA 1SNP groups but not in the untreated control or theSNP groups (Fig. 6).

DISCUSSION

The major finding of our study was that l-NA couldinhibit I/R-induced macromolecular leakage (numberof leaks) in postcapillary venules and that the numberof sticking leukocytes was reduced during the periodwhen vascular permeability was reduced comparedwith the saline control group. In the groups treatedwith SNP or l-NA 1 SNP the permeability increaseafter I/R measured as the number of leaks was actu-ally larger than in the saline control group, althoughthere was only a tendency to more sticking leukocytesin the l-NA and l-NA 1 SNP groups compared withthe saline control group. During observations of roll-ing and sticking it was not possible to count the num-ber of venular leaks, but measuring the concentrationof FITC–dextran in the superfusion solution showedthat there was a significantly reduced FITC–dextranefflux during the postischemic period only in thel-NA group.

Using a different model of ischemia/reperfusion in

FIG. 4. FITC–dextran efflux in nanograms per 30 min as means 6

SEM during the control period prior to ischemia for 30 min andduring reperfusion after the ischemic period during simultaneousmeasurements of sticking leukocytes as shown in Figs. 3a and 3b.

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the hamster with another NO oxide synthase inhibitor,l-NMMA, it was found that l-NMMA inhibited I/R-induced plasma leakage and that a PAF-receptor an- 1


tagonist also blocked the I/R-induced plasma leakage(Ramirez et al., 1995; Noel et al., 1996). It has also beenshown in the hamster that stimulation of protein ki-nase C with phorbol 12,13-dibutyrate increased vas-cular permeability and that l-NMMA could inhibitthis increase (Ramirez et al., 1996)). Thus, we haveconfirmed the findings by Noel et al. (1995, 1996) inI/R with another nitric oxide synthase inhibitor,l-NA.

Local application of SNP during the ischemic periodincreased the plasma leakage above compared to thatseen in the untreated control group, which could beimplicated as NO donation by SNP reacting with thesuperoxide to result in peroxynitrite. The result withthe combination of l-NA 1 SNP was not different

FIG. 5. (a) Arteriolar diameters (in micrometers) before and after aperiod of total ischemia for 30 min. Effects of local application ofnormal saline (n 5 6), 1025 M l-NA (n 5 8), or 1026 M SNP (n 5 5).


Mean values 6 SEM. *P , 0.05 compared with the control groups(normal saline). (b) Arteriolar diameters (in micrometers) duringlocal application of normal saline (n 5 6), 1025 M l-NA (n 5 8), or026 M SNP (n 5 5). Mean values 6 SEM.

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from that of SNP alone, which again could be impli-cated as an effect of peroxynitrite formed by superox-ide and NO from SNP. The results of histamine stim-ulation at 30 min after reperfusion were quite similarin the l-NA, SNP, and l-NA 1 SNP groups, suggest-ing that a mediator such as histamine which acts di-rectly on the endothelium and not via activation ofleukocytes may not be enhanced by SNP, because thatwould require superoxide from the I/R injury. Thisimplication is supported by the results of histaminestimulation in normal hamsters without previous I/Rwhere macromolecular leakage was inhibited by l-NAand l-NA 1 SNP but not enhanced by SNP (Fig. 6). Inour experience the induction and inhibition of macro-molecular leakage by histamine are very similar tothat seen with bradykinin. As bradykinin-inducedmacromolecular leakage was not modified in the pres-ence of SOD concentrations which almost completelyinhibited I/R-induced macromolecular leakage, wethink that the same is true for histamine (Erlansson etal., 1990).

The role of NO and peroxynitrite formation hasbeen studied in a mouse model of cerebral arteryocclusion. Administration of NOS inhibitors prior toreperfusion decreased tyrosine nitration, which is amarker of peroxynitrite toxicity. NOS inhibitors alsodecreased Evans blue extravasation and it was con-

FIG. 6. Maximal number of leaks at 5 min of topical application ofistamine before and 30 min after topical application of normalaline, 1026 M SNP, 1025 M l-NA, and l-NA 1 SNP. Mean values 6

SEM. *Significantly different from the control group (P , 0.05).


cluded that formation of peroxynitrite in the vascularcompartment was the cause of reperfusion injury(Gurzoy-Ozdemir et al., 2000). In a study of blood–

brain barrier permeability in the rat it was suggestedthat the increase in permeability exerted by two NOdonors, SNAP and SIN-1, was due to formation ofperoxynitrite (Mayhan, 2000). In contrast to thesestudies Okayama et al. could not observe any perme-ability effect of peroxynitrite on cultured endothelialmonolayers (Okayama et al., 1999). One importantdifference between their study and the others was theabsence of blood cells and plasma, as they used amodel of vasculature consisting of a chromatographiccell column filled with endothelial-cell-covered micro-carrier beads. The study by Okayama et al. suggeststhat we have not observed direct effects of peroxyni-trate on the endothelium, but possibly the results ofperoxynitrate action on leukocytes or plasma proteinsleading to the release or formation of other inflamma-tory molecules, e.g., IL-8 or TNFa.

A stable prostacyclin derivative, Iloprost, reducedthe number of sticking leukocytes and the plasmaleakage after I/R at doses which had no effect onarterial blood pressure or blood flow velocity in arte-rioles in the hamster cheek pouch (Erlansson et al.,1990). Antioxidants such as flavonoids and a-tocoph-erol reduced the I/R-induced sticking of leukocytesand macromolecular leakage, thus providing furthersupport for the importance of leukocytes in the induc-tion of oxidant and I/R-induced macromolecular leak-age in the hamster (Bouskela et al., 1999; Svensjo et al.,1997).

Several investigators have shown that nonspecificinhibition of nitric oxide synthase increases basal lev-els of permeability and potentiates agonist-inducedincreases in vascular permeability (Oliver, 1992; Siroisand Edelman, 1997; Kubes and Granger, 1992; Kubes,1992). However, others (Hughes et al., 1990; Mayhan1992, 1994, 1999; Yuan et al., 1992; Ialenti et al., 1992;Svensjo and Roempke, 1994; Noel et al., 1996; Ramirezet al., 1995; Wu et al., 1996) have shown that inhibitionof nitric oxide synthase does not influence basal levelsof vascular permeability in normal animals, but inhib-its increases in venular permeability in response toactivation of constitutive (endothelial) nitric oxidesynthase stimulated by ADP, histamine, substance P,

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bradykinin, platelet activating factor, and vascular en-dothelial growth factor. At the present time there is noexplanation for these discrepancies.


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Johnston et al. (1999) found that a NO donor, sper-mine-NO, blocked histamine and CINC/gro-inducedvascular permeability in the rat mesentery. They sug-gested that there may be organ- or tissue-specific dif-ferences in the effects of NO and also that NO mayexert different effects in different species (Johnston etal., 1999). Grisham et al. discussed these discrepanciesin detail and concluded that “a definitive explanationfor the apparent contradictory role of NO in differentmodels of I/R does not yet exist” (Grisham et al., 1998)

Feletou et al. observed that the vasodilator and po-tassium channel opener cromakalim could neutralizethe constricting effect of l-NOARG and suggested thatNO was only involved in the arteriolar dilation part ofthe bradykinin effect on vascular permeability (Fele-tou et al., 1996). In our study we observed a slightarteriolar constriction in the l-NA groups both withand without I/R and therefore it cannot be excludedthat at least part of the inhibition by l-NA in our studywas due to this effect which should reduce the bloodflow, the transmural pressure in venules, and possiblyalso the number of inflowing leukocytes. We observedsignificantly increased arteriolar diameters in SNP-treated animals which could be implicated as a hemo-dynamic contribution to the increased macromolecu-lar leakage in this group.

CONCLUSION

We have shown that a NO synthase inhibitor, l-NA,nhibited the macromolecular leakage evoked by I/Rnd histamine and that the number of adherent leu-ocytes was reduced in parallel with the reduction inlasma leakage. SNP and l-NA 1 SNP enhanced the

/R-induced macromolecular leakage compared withhe saline control group, possibly because of enhancedormation of peroxynitrite due to more available NOffered by SNP, which then could induce formation ofther inflammatory molecules, e.g., IL-8 or TNFa.

ACKNOWLEDGMENTS

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This work was supported by grants from the National ResearchCouncil (CNPq), the Foundation to Support Research of Rio de

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Janeiro State (FAPERJ), and the State University of Rio de Janeiro(UERJ).

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effects of l-na and sodium nitroprusside on ischemia/reperfusion-induced leukocyte adhesion and...

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