Amelioration of Postischemic ReperfusionInjury by Antiarrhythmic Drugs in IsolatedPerfused Rat LungKumuda C. Das and Hara R MisraDepartment of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine,Virginia Polytechnic Institute, and State University, Blacksburg, Virginia

Antiarrhythmic drugs, such as lidocaine, quinidine, and procainamide, have been shown to be effective against postischemic reperfusion injury inisolated rat lungs. Rat lungs were perfused at a constant flow with Krebs-Henseilet buffer supplemented with 4% bovine serum albumin and venti-lated with air containing 5% CO2. The lungs were subjected to ischemia by stopping perfusion and ventilation for 60 min followed by 30 min ofreperfusion. Lung injury was determined by measuring the increase in wet-to-dry lung weight ratio, while pulmonary arterial pressure and peak air-way pressure were calculated from the pre- and postischemic differences. Lidocaine, quinidine, and procainamide at doses of 5, 10, and 20 mg/kgbody weight, respectively, were found to attenuate the postischemic lung injury significantly (p<0.0001). The formation of cyclooxygenase prod-ucts, which were elevated during reperfusion, was also significantly (p<0.0001) inhibited by these drugs. Since these antiarrhythmic agents arefound to be powerful scavengers of hydroxyl radicals and can prevent membrane lipid peroxidation, these findings suggest that the antioxidant prop-erties of these drugs may, in part, be responsible for protecting the lungs against reperfusion injury. - Environ Health Perspect 102(Suppl 10):117-122 (1994)

Key words: lung, ischemia, reperfusion, lidocaine, quinidine, procainamide, local anesthetics, free radical, antiarrhythmic

IntroductionLung transplant is rapidly becoming a clin-ical alternative for patients with end-stagelung disease. Preservation of the cadaverlung during transplant has been a majorimpediment to the wide clinical use oftransplant procedures. Reperfusion oftransplanted lung previously subjected to

brief period of ischemia causes irreversibletissue injury (1). Ischemia-reperfusioninjury has been extensively studied in manyorgans, including brain, heart, intestine,and kidney (2-7). Little work has beendone to elucidate the mechanism of reper-

fusion injury in the lung. Reactive oxygen

species has been implicated in the etiologyof ischemia-reperfusion injury (3,8-10).Antiarrhythmic drugs have been used as

membrane stabilizers and were found to

prevent microvascular permeability result-ing from acute lung injury (11). Themechanism by which these antiarrhythmicagents diminish lung edema, however, is

This paper was presented at the Conference onOxygen Radicals and Lung Injury held 30 August-2September 1993 in Morgantown, West Virginia.

The work reported herein was supported, in part,by grant HL42009 from the National Institutes ofHealth, Bethesda, MD.

Address correspondence to Dr. Hara P. Misra,Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine,Virginia Tech, Blacksburg, VA 24061. Telephone (703)231-7174. Fax (703) 231-7367

unclear and their ability to reduce post-ischemic reperfusion injury of the lung hasnot been tested. Recently we reported thatantiarrhythmic agents such as lidocaine,quinidine, and procainamide are potenthydroxyl radical scavengers and were foundto inhibit lipid peroxidation (12). Sincefree radicals of oxygen are important in ini-tiating lipid peroxidation (13,14), andlipid peroxides are known to be producedduring postischemic reperfusion of lung(15), we developed the hypothesis thatantiarrhythmic drugs may protect lungsfrom reperfusion injury. Here we presentevidence that antiarrhythmic drugs such aslidocaine, quinidine, and procainamideattenuate postischemic reperfusion injuryin isolated perfused rat lung and thesedrugs were effective in preventing the accu-mulation of cyclooxygenase products dur-ing reperfusion of ischemic lung.

Materials and MethodsEa Vivo Lung PreparationMale Sprague-Dawley rats (Harlan'sSprague-Dawley, Dublin, VA) weighing300 to 500 g were anesthetized with 64.8mg/kg, ip, pentobarbital sodium (AnthonyProducts Co., Arcadia, CA). A tra-cheostomy was performed that permittedventilation with a Harvard rodent ventilator(model 683) at 62 strokes per min, a tidalvolume of 2.3 to 3 ml, and positive end

expiratory pressure of 2.5 cm H20. Theinspired gas mixture was air mixed with 5%CO2 (Analyzed, Industrial Gas and SupplyCo., Radford, VA). Subsequently a mediansternotomy was performed, heparin (200IU) was injected into the right ventricle, andcannulas were placed in the pulmonaryartery and left ventricle. The heart, lungs,and mediastinal structures were removed enbloc and suspended from a Fort-250 rigidlinear force transducer (World PrecisionInstruments, New Haven, CT) to monitorany weight change and placed in ahumidified chamber. The lungs were per-fused by a masterflex pump (Cole ParmerInstruments, Chicago, IL) with Krebs-Hanseilet buffer at a constant flow of0.05ml/min/g bw. The Krebs-Hanseiletbuffer contained 118 mM NaCl, 4.7 mMKCl, 1.17mM MgSO4, 25 mM NaHCO3,1.18 mM KH2PO4, 1.90 mM CaCl2, 11.1mM glucose and 4% bovine serum albumin(66,000 mw, Sigma Chemical Co., St.Louis, MO). The pH of the perfusate wasmaintained between 7.35 and 7.45 by peri-odic addition ofsodium bicarbonate.

The first 50 ml of lung effluents werediscarded to eliminate circulating bloodelements from the vascular space of thelung. Subsequently a recirculating modewas established with 50 ml of perfusate.Pulmonary artery pressure (Pa) was con-stantly monitored with a transducer bloodpressure BPLR-0111 (WPI). Peak airway

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pressure (Paw) was constantly monitoredby a PNEU-01 (WPI) pressure transducer.The pressure transducers were calibratedwith a blood pressure measurement instru-ment (The Lumiscope Co.,[S-NelkinHome Care, APCC] Edison, NJ).

Mean pulmonary Pa, change in lungweight, and mean peak Paw were con-stantly monitored through the pressure andlinear force transducers connected to anamplifier bridge (WPI). The bridge wasconnected to a MacLab-4 (WPI) that inturn was connected to a Macintosh SEcomputer. The data were recorded byScope version 3.1 software for the MacLabsystem (WPI).

Inducion ofLung IschemiaIsolated lungs were perfused for 10 min toensure a stable preparation and were thensubjected to ischemic injury for 60 min bystopping ventilation and perfusion. Thelungs were inflated by instillating 2 ml ofgas mixture into tracheal cannula beforeocclusion at the start of the ischemic period.Lung inflation was done to facilitate reper-fusion after ischemia. Throughout the 60-min ischemia the lungs and perfusate werekept at 37°C.

Lung ReperfisionReperfusion after ischemic interval wasstarted slowly and the flow rate wasincreased such that a mean pulmonary Paof 14 mm Hg was never exceeded. Within5 min of the onset of the reperfusion theperfusate flow was increased to the originalflow rate present before the ischemic period(0.05 ml/min/g, bw). During reperfusionthe perfusate reservoir and the lungs weremaintained at 37 to 38°C. Lungs werereperfused for 30 min while they were ven-tilated with the same gas mixture.

Experimental GroupsFive experimental groups were studied. Thefirst group of six lungs underwent 60 min ofischemia followed by 30 min of reperfusion.The same protocol was maintained forgroups 2, 3, and 4 (n = 6) with the exceptionthat lidocaine, quinidine, and procainamideat 5, 10, and 20 mg/kg bw, respectively,were added to the perfusate. This dose wascalculated taking the blood volume at 8% ofthe body weight. Group 5 served as controland underwent no ischemia. The drugs wereadded to the lung perfusate at the onset oflung perfusion prior to ischemia.

Measurement ofLung InjuryWet-to-Dry Lung Weight Ratio. At theend of each experiment the left main stem

bronchus was transected and the left lungwas isolated for the determination of thewet-to-dry lung weight ratio. Lungs wereweighed and placed in a convection oven(Model 605, Precision Scientific Inc.,Chicago, IL) at 1200C and weighed dailyfor 3 days. Lung weight at 72 hr wasreported as dry weight because no furtherweight loss occurred after that time.

Pulmonary Artery Pressure. Meanpulmonary Pa was measured for 10 minduring the preischemic period and entirepostischemic period after the full flow wasresumed. Percentage change was calculatedtaking the difference of the mean pre- andpostischemic pulmonary artery pressures.

Peak Airway Pressure. Mean peakPaw was monitored for 10 min of pre-ischemic period and 30 min during post-ischemic period. Percentage change wascalculated taking the difference of pre- andpostischemic pressures.

Measurement ofCydooxygenaseMetabolitesTxB2 and 6-keto-PGFia, the stablemetabolites of TxA2 and prostacyclin,respectively, were measured as indicators ofcyclooxygenase metabolite production.Samples (1.8 ml) of pulmonary venouseffluent were collected immediately beforethe onset of ischemia, and at 5, 10, and 20min of reperfusion. Time-matched sampleswere also obtained in the uninjured con-trols. Measurements of TxB2 and 6-keto-PGFIa were made in thromboxane B2[3H] and 6-keto-PGF1J[3H] scintillationproximity assay (SPA) systems [(16);Amersham International Plc, Amersham,UK] on methyl formate extracted samplesin duplicate (Bakerbond C18; J. T. Baker,Inc., Phillipsburg, NJ).

Statistical AnalysisValues were expressed as mean ± SEM.Groups were compared using one-wayanalysis of variance and the Tukey's multi-ple comparison test using SAS statisticalsoftware (SAS Institute, Cary, NC). Datafor PGFIa and TXB2 were analyzed bytwo-way analysis of variance taking preis-chemia and ischemia as factors. A p valueof less than 0.05 was considered significant.

ResultsEffect ofAntiarrhythmic Drugs onWet-to-Dry LungWeight RatioLung weight remained stable in uninjuredcontrol lungs during the 100 min of perfu-sion. Lungs subjected to ischemia reperfu-sion had increased lung weight at the end

20-

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Figure 1. Effects of antiarrhythmic drugs on wet-to-drylung weight ratio of ischemic-reperfused rat lung. Wet-to-dry lung weight ratios obtained after 100 min oflung perfusion in experiments exposed to ischemiareperfusion (I/R) and I/R with lidocaine, quinidine, andprocainamide at doses of 5, 10 and 20 mg/kg bw,respectively (equivalent of 170, 320 and 900 pM,respectively, in the perfusate). Values are mean ± SE.*p<0.0001 compared with ischemia-reperfused lungs.** p<0.0001 compared to the control. For each treat-ment group, n = 6.

of 30 min of reperfusion as recorded by aMacLab-4 connected to a linear forcetransducer (data not shown). Wet-to-drylung weight ratio, a measure of edema for-mation, was significantly higher(p< 0.000 1) in ischemia-reperfused lungscompared to uninjured controls (Figure 1).The antiarrhythmic drugs lidocaine, quini-dine, and procainamide at single doses of5, 10, and 20 mg/kg bw, respectively,significantly (p < 0.000 1) reduced the wet-to-dry weight ratios compared to ischemia-reperfused lungs (Figure 1).

Effect on Pulmonary PressurePulmonary Pa remained stable over the100 min of perfusion in the control lungsnot subjected to ischemia. Lungs subjectedto 60 min of ischemia were allowed toreach a stable Pa 10 min after the onset ofreperfusion. The Pa was found to besignificantly higher (p< 0.008) 10 min afterthe onset of reperfusion compared to thepressures present in the same lung duringpreischemia or compared to the Pa mea-sured in the time-matched control lungs(Figure 2). The Pa remained elevated com-pared to preischemic values in the samelung at 10 and 30 min after the onset ofreperfusion. Addition of lidocaine, quini-

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U Control40 r 40 a

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*U Quinidine

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Figure 2. Effects of antiarrhythmic drugs on pulmonaryarterial pressure of ischemic-reperfused rat lung.Uninjured control lungs were perfused for 100 min.Lungs subjected to ischemia reperfusion underwent 10min of preischemia followed by 60 min of ischemia and30 min of reperfusion. Lidocaine, quinidine, and pro-cainamide at doses of 5, 10 and 20 mg/kg bw, respec-tively, were added to the perfusion buffer at thebeginning of perfusion. Results were expressed as per-cent change in mean pulmonary arterial pressurebetween preischemia and postischemic reperfusion.*p<0.0001 and **p<0.008 compared to the control.For each treatment group, n=6.

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Figure 3. Effects of antiarrhythmic drugs oway pressure of ischemic-reperfusedUninjured control lungs were perfused foLungs subjected to ischemia reperfusion un(

min of preischemia followed by 60 min of is(30 min of reperfusion. Lidocaine, quinidinEcainamide at doses of 5, 10 and 20 mg/kg ttively, were added to the perfusion bulbeginning of perfusion. Results were exprescent change in mean peak airway pressurpreischemia and postischemic reperfusion.compared to control. **p<0.0001 corischemia-reperfused lung. For each treatrn= 6.

Table 1. Effect of antiarrhythmic drugs on cyclooxygenase product formation in isolated rat lung siischemia reperfusion.

6-Keto-PGFia, pg/ml TxB2, pg/mlPreischemiaa Postischemiab Preischemia Postisch

Controlc 220± 10 200± 20 58± 1.95 64±1I/Rd 163 ± 15 1428 ± 315* 68 ± 1.28 132 ± 9I/R + lidocaine 205 ± 11 312 ± 63** 62 ± 1.68 85 ± 2I/R + quinidine 250 ± 36 209 ± 73** 55 ± 1.75 63 ± 1I/R + procainamide 251 ± 15 282 ± 74** 55 ± 2.80 65 ± 1

aFirst 10 min of perfusion. bSamples taken 5 min after reperfusion. CControl lungs not subjected to isperfused for 100 min. d60-min ischemia followed by 30 min of reperfusion. Lidocaine, quinidine and prat doses of 5, 10 and 20 mg/kg bw, respectively, were added to the perfusate at the begining of*p< 0001, compared to control; **p< 0.001, compared to ischemia reperfusion.

no significant differences (Table 1). In theischemia-induced lungs, 5 min of reperfu-sion resulted in 7- and 2-fold increases inthe production of the cyclooxygenasemetabolites, 6-keto-PGFia and TxB2,respectively (Table 1). These increases were

ide significantly (p < 0.05) higher than eithercontrol preischemic lung or preischemiclung with addition of drugs in the per-

fusate (Table 1). The time course of accu-

mulation of these metabolites in lungeffluents was studied at 5, 10, and 20 minpostischemia. Both the 6-keto-PGFIa andTxB2 levels were found to remain elevated(p<0.0001) in the lung effluent collectedup to 20 min after reperfusion comparedto effluent collected from the same lungprior to ischemia and when compared to

time-matched lung effluent samples fromcontrol lungs not subjected to ischemia

In peak air- (Figures 4, 5). Effluents collected at 5, 10,r 100 min. and 20 min after reperfusion from lungsderwent 10 subjected to ischemia reperfusion andchemia and treated with lidocaine, quinidine, and pro-e, and pro- cainamide at 5, 10, and 20 mg/kg bw,bw, respec- respectively, had significantly lessffer at the (p<0.0001) 6-keto-PGFta and TxB2 com-;sed as per- pared to untreated lungs that were sub-e between jected to similar ischemia reperfusion

mp<0.aOdl conditions (Figures 4, 5). When the levelsmpared to of PGF1I and TxB2 were compared inent group, lungs subjected ischemia and reperfu-

sion, the levels of both cyclooxygenasemetabolites were found to be significantly(p<0.05) higher 5 mmn after reperfusion,when compared to effluents collected at 10or 20 min after reperfusion. The levels of

emia PGFIa and TxB2 in effluents from of lungs

.47 treated with the antiarrythimic drugs were1.68* also compared, with no significant change..62** observed in levels of PGFia and TxB2

48** between quinidine and procainamide ineffluents collected after 5, 10, or 20 min of

chemia but reperfusion. However, the lungs treatedocainamide with lidocaine had significantly higher

perfusion. (p< 0.05) levels of PGFIa when compared

with control lungs or lungs treated withquinidine or procainamide.

dine, and procainamide at 5, 10 and 20mg/kg bw, respectively, significantlyreduced the Pa respectively, of ischemia-reperfused lungs compared to untreatedreperfused lungs (Figure 2).

Effect on Peak Airway PressurePeak Paw remained stable over the 100min of perfusion in the uninjured controllungs. Lungs subjected to ischemia andreperfusion had a significantly higher(p< 0.000 1) Paw after 30 min of reperfu-sion. Lungs subjected to ischemia reperfu-

sion but treated with lidocaine (5 mg/kgbw), quinidine (10 mg/kg bw), and pro-

cainamide (20 mg/kg bw) had significantly(p< 0.000 1) reduced Paw compared to theuntreated ischemia-reperfused lungs. Thesedata are presented in Figure 3.

Effect ofAntiarrhythmic Drugs onCydooxygenase Product FormationMeasurements of 6-keto-PGFia and TxB2in lung effluents collected prior to ischemiaand in the time-matched lung effluentsfrom the uninjured control lungs showed

DiscussionA major source of damage in postischemicreperfusion injury is believed to be the gen-

eration of oxygen-free radicals and othertoxic oxygen metabolites (9,17,18). Theseradicals and metabolites have been impli-cated in postischemic reperfusion injury in

the heart, kidney, intestine, brain, andother organs (14,18-20). Studies in thepostischemic reperfusion injury of thelungs also implicate toxic oxygen metabo-lite as a source of damage (8,17,21,22). Inour present study, antiarrhythmic drugs,

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Figure 4. Effects of antiarrhythmic drugs on 6-keto-PGFia on ischemic-reper-fused rat lung. Samples for 6-keto-PGFa, measurements were obtained priorto ischemia as well as 5, 10, and 20 min after reperfusion or at time-matchedpoints in control lungs not subjected to ischemia. Lidocaine, quinidine, andprocainamide at 5, 10, and 20 mg/kg bw, respectively, were added to the per-fusate at the beginning of the perfusion period. 6-keto-PGF1a was measuredby the scintillation proximity assay method on extracted samples in duplicate.*p<0.0001 compared to control and drug-treated postischemic-reperfusedlungs. **p<0.05 when compared with control lungs or lungs treated withquinidine or procainamide but significantly lower than the ischemia-reperfusedlungs. For each treatment group, n= 6.

Figure 5. Effects of antiarrhythmic drugs on TxB2 on ischemic-reperfused rat lung.Samples for TxB2 measurements were obtained prior to ischemia as well as 5, 10, and20 min after reperfusion or at time-matched points in control lungs not subjected toischemia. Lidocaine, quinidine, and procainamide at 5, 10, and 20 mg/kg bw, respec-tively, were added to the perfusate at the beginning of the perfusion period. TxB2 wasmeasured by the scintillation proximity assay method on extracted samples in dupli-cate. *p<0.0001 compared to control and drug-treated ischemia-reperfused lungs. Foreach treatment group, n= 6.

such as lidocaine, quinidine, and pro-

cainamide, significantly reduced pul-monary edema, pulmonary Pa and Paw.These drugs also inhibited the formation ofcyclooxygenase metabolites known to beproduced during reperfusion of ischemiclungs (23). Although these agents havenumerous systemic and local effects on var-

ious biological tissues, both in vivo and invitro (24,25), their ability to amelioratepostischemic reperfusion injury in thelungs has not previously been recognized.

The mechanism by which the antiar-rhythmic agents are effective against reper-

fusion injury may be explained, in part, bytheir antioxidant properties. In a recent

study, Stelzner et al. (11) found that thesedrugs protect lungs against thiourea-induced injury in rats. As oxygen radicals are

likely to be produced during metabolism ofthiourea (11), these authors speculated an

antioxidant action of antiarrhythmic drugs.However, they found that these drugs are

not scavengers of O°- or H202. Recently,we have demonstrated that these drugs are

powerful hydroxyl radical scavengers

(k=1.8xloil, 1.61x10tl and 1.45x 101M- sec-1 for quinidine, lidocaine and pro-

cainmide, respectively) and inhibitors oflipid peroxidation (12). Therefore, it islikely that these drugs protect pulmonarytissue against postischemic reperfusioninjury by preventing lipid peroxide forma-tion and/or scavenging -OH.

The concentration of three antiarrhyth-mic drugs in the perfusat were 40, 120,and 250 pg/ml for lidocaine, quinidine,and procainamide, respectively. Howeverthese drugs are also known to bind to

plasma albumin, thereby reducing theavailability of free drug for therapeuticaction (26). Since our perfusate contained4% bovine serum albumin, the actual con-

centrations of free drugs may be much lessthan indicated. The LD50 and pharmacoki-netics of these drugs have not yet been elu-

cidated in rats. Taking into account thedoses of 4, 20, and 30 mg/kg bw for lido-caine, quinidine, and procainamide in thestudy by Stelzner et al. (11), our doses of40, 120, and 250 pg/ml for rats are

significantly smaller. Detailed pharmacoki-netic studies of these drugs in rats are

needed to determine the typical nontoxicplasma levels of these drugs in rats.

Because toxic oxygen metabolites can

directly cause cell injury or lead to the pro-duction of other mediators, such as arachi-donate metabolites, we measured the effectof antiarrhythmic agents on the cyclooxyge-nase metabolites thromboxane and prosta-cyclin during the reperfusion of ischemiclungs. Postischemic reperfusion caused a

significant elevation of both the thrombox-ane and prostacyclin levels as measured bytheir stable metabolites TxB2 and 6-keto-PGFia. Similar increases of these metabo-lites have been observed by Ljungman et al.(16). It is believed that cyclooxygenase

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metabolites, produced secondary to theproduction of oxygen radicals, may in turn,cause tissue damage (16). However, theseauthors have concluded that cyclooxygenasemetabolites may not be the sole source ofinjury because protection by inhibitors ofcyclooxygenase product formation was notcomplete. Burghuber et al. (27) have alsoreported similar findings and concludedthat cyclooxygenase products are not pri-marily responsible for lung damage. It hasbeen shown that lidocaine is not an

inhibitor of prostaglandin biosynthesis invitro (28), but rather increases the produc-tion of prostacyclin (29). Our data demon-strate that all these antiarrhythmic drugsinhibit cyclooxygenase product formation.Since -OH are known to be responsible forthe release of arachidonic acid from mem-brane phospholipids (30), it is possible thatthese drugs may not inhibit prostaglandinformation per se but might inhibit the lib-eration of arachidonic acid from phospho-lipids by scavenging OH radicals.

Nevertheless, the lung damage imposed byischemia reperfusion could be the concom-mitant actions of both toxic oxygen prod-ucts (21) and cyclooxygenase metabolites(23). We conclude that the inhibition ofcyclooxygenase product formation and theattenuation of postischemic lung injury bythese drugs may, in part, be due to theremoval of toxic oxygen metabolites gener-ated during the reperfusion of the lung.

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