Amelioration of postischemic reperfusion injury by antiarrhythmic drugs in isolated perfused rat lung.

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


Introduction
Lung transplant is rapidly becoming a clinical alternative for patients with end-stage lung disease. Preservation of the cadaver lung during transplant has been a major impediment to the wide clinical use of transplant procedures. Reperfusion of transplanted lung previously subjected to brief period of ischemia causes irreversible tissue injury (1). Ischemia-reperfusion injury has been extensively studied in many organs, including brain, heart, intestine, and kidney (2)(3)(4)(5)(6)(7). Little work has been done to elucidate the mechanism of reperfusion injury in the lung. Reactive oxygen species has been implicated in the etiology of ischemia-reperfusion injury (3,(8)(9)(10). Antiarrhythmic drugs have been used as membrane stabilizers and were found to prevent microvascular permeability resulting from acute lung injury (11). The mechanism by which these antiarrhythmic agents diminish lung edema, however, is unclear and their ability to reduce postischemic reperfusion injury of the lung has not been tested. Recently we reported that antiarrhythmic agents such as lidocaine, quinidine, and procainamide are potent hydroxyl radical scavengers and were found to inhibit lipid peroxidation (12). Since free radicals of oxygen are important in initiating lipid peroxidation (13,14), and lipid peroxides are known to be produced during postischemic reperfusion of lung (15), we developed the hypothesis that antiarrhythmic drugs may protect lungs from reperfusion injury. Here we present evidence that antiarrhythmic drugs such as lidocaine, quinidine, and procainamide attenuate postischemic reperfusion injury in isolated perfused rat lung and these drugs were effective in preventing the accumulation of cyclooxygenase products during reperfusion of ischemic lung.

Ea Vivo Lung Preparation
Male Sprague-Dawley rats (Harlan's Sprague-Dawley, Dublin, VA) weighing 300 to 500 g were anesthetized with 64.8 mg/kg, ip, pentobarbital sodium (Anthony Products Co., Arcadia, CA). A tracheostomy was performed that permitted ventilation with a Harvard rodent ventilator (model 683) at 62 strokes per min, a tidal volume of 2.3 to 3 ml, and positive end expiratory pressure of 2.5 cm H20. The inspired gas mixture was air mixed with 5% CO2 (Analyzed, Industrial Gas and Supply Co., Radford, VA). Subsequently a median sternotomy was performed, heparin (200 IU) was injected into the right ventricle, and cannulas were placed in the pulmonary artery and left ventricle. The heart, lungs, and mediastinal structures were removed en bloc and suspended from a Fort-250 rigid linear force transducer (World Precision Instruments, New Haven, CT) to monitor any weight change and placed in a humidified chamber. The lungs were perfused by a masterflex pump (Cole Parmer Instruments, Chicago, IL) with Krebs-Hanseilet buffer at a constant flow of 0.05ml/min/g bw. The Krebs-Hanseilet buffer contained 118 mM NaCl, 4.7 mM KCl, 1.17mM MgSO4, 25 mM NaHCO3, 1.18 mM KH2PO4, 1.90 mM CaCl2, 11.1 mM glucose and 4% bovine serum albumin (66,000 mw, Sigma Chemical Co., St. Louis, MO). The pH of the perfusate was maintained between 7.35 and 7.45 by periodic addition of sodium bicarbonate.
The first 50 ml of lung effluents were discarded to eliminate circulating blood elements from the vascular space of the lung. Subsequently a recirculating mode was established with 50 ml of perfusate. Pulmonary artery pressure (Pa) was constantly monitored with a transducer blood pressure BPLR-0111 (WPI). Peak airway Environmental Health Perspectives pressure (Paw) was constantly monitored by a PNEU-01 (WPI) pressure transducer. The pressure transducers were calibrated with a blood pressure measurement instrument (The Lumiscope Co.,[S-Nelkin Home Care, APCC] Edison, NJ).
Mean pulmonary Pa, change in lung weight, and mean peak Paw were constantly monitored through the pressure and linear force transducers connected to an amplifier bridge (WPI). The bridge was connected to a MacLab-4 (WPI) that in turn was connected to a Macintosh SE computer. The data were recorded by Scope version 3.1 software for the MacLab system (WPI).

Inducion ofLung Ischemia
Isolated lungs were perfused for 10 min to ensure a stable preparation and were then subjected to ischemic injury for 60 min by stopping ventilation and perfusion. The lungs were inflated by instillating 2 ml of gas mixture into tracheal cannula before occlusion at the start of the ischemic period. Lung inflation was done to facilitate reperfusion after ischemia. Throughout the 60min ischemia the lungs and perfusate were kept at 37°C.

Lung Reperfision
Reperfusion after ischemic interval was started slowly and the flow rate was increased such that a mean pulmonary Pa of 14 mm Hg was never exceeded. Within 5 min of the onset of the reperfusion the perfusate flow was increased to the original flow rate present before the ischemic period (0.05 ml/min/g, bw). During reperfusion the perfusate reservoir and the lungs were maintained at 37 to 38°C. Lungs were reperfused for 30 min while they were ventilated with the same gas mixture.

Experimental Groups
Five experimental groups were studied. The first group of six lungs underwent 60 min of ischemia followed by 30 min of reperfusion. The same protocol was maintained for groups 2, 3, and 4 (n = 6) with the exception that lidocaine, quinidine, and procainamide at 5, 10, and 20 mg/kg bw, respectively, were added to the perfusate. This dose was calculated taking the blood volume at 8% of the body weight. Group 5 served as control and underwent no ischemia. The drugs were added to the lung perfusate at the onset of lung perfusion prior to ischemia.

Measurement ofLung Injury
Wet-to-Dry Lung Weight Ratio. At the end of each experiment the left main stem bronchus was transected and the left lung was isolated for the determination of the wet-to-dry lung weight ratio. Lungs were weighed and placed in a convection oven (Model 605, Precision Scientific Inc., Chicago, IL) at 1200C and weighed daily for 3 days. Lung weight at 72 hr was reported as dry weight because no further weight loss occurred after that time.
Pulmonary Artery Pressure. Mean pulmonary Pa was measured for 10 min during the preischemic period and entire postischemic period after the full flow was resumed. Percentage change was calculated taking the difference of the mean pre-and postischemic pulmonary artery pressures.
Peak Airway Pressure. Mean peak Paw was monitored for 10 min of preischemic period and 30 min during postischemic period. Percentage change was calculated taking the difference of pre-and postischemic pressures.

Measurement ofCydooxygenase Metabolites
TxB2 and 6-keto-PGFia, the stable metabolites of TxA2 and prostacyclin, respectively, were measured as indicators of cyclooxygenase metabolite production. Samples (1.8 ml) of pulmonary venous effluent were collected immediately before the onset of ischemia, and at 5, 10, and 20 min of reperfusion. Time-matched samples were also obtained in the uninjured controls. Measurements of TxB2 and 6-keto-

Statistical Analysis
Values were expressed as mean ± SEM. Groups were compared using one-way analysis of variance and the Tukey's multiple comparison test using SAS statistical software (SAS Institute, Cary, NC). Data for PGFIa and TXB2 were analyzed by two-way analysis of variance taking preischemia and ischemia as factors. A p value of less than 0.05 was considered significant.

Effect ofAntiarrhythmic Drugs on
Wet-to-Dry LungWeight Ratio Lung weight remained stable in uninjured control lungs during the 100 min of perfusion. Lungs subjected to ischemia reperfusion had increased lung weight at the end Effects of antiarrhythmic drugs on wet-to-dry lung weight ratio of ischemic-reperfused rat lung. Wetto-dry lung weight ratios obtained after 100 min of lung perfusion in experiments exposed to ischemia reperfusion (I/R) and I/R with lidocaine, quinidine, and procainamide 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 treatment group, n = 6. of 30 min of reperfusion as recorded by a MacLab-4 connected to a linear force transducer (data not shown). Wet-to-dry lung weight ratio, a measure of edema formation, was significantly higher (p< 0.000 1) in ischemia-reperfused lungs compared to uninjured controls ( Figure 1). The antiarrhythmic drugs lidocaine, quinidine, and procainamide at single doses of 5, 10, and 20 mg/kg bw, respectively, significantly (p < 0.000 1) reduced the wetto-dry weight ratios compared to ischemiareperfused lungs (Figure 1).

Effect on Pulmonary Pressure
Pulmonary Pa remained stable over the 100 min of perfusion in the control lungs not subjected to ischemia. Lungs subjected to 60 min of ischemia were allowed to reach a stable Pa 10 min after the onset of reperfusion. The Pa was found to be significantly higher (p< 0.008) 10 Figure 2. Effects of antiarrhythmic drugs on pulmonary arterial pressure of ischemic-reperfused rat lung. Uninjured control lungs were perfused for 100 min. Lungs subjected to ischemia reperfusion underwent 10 min of preischemia followed by 60 min of ischemia and 30 min of reperfusion. Lidocaine, quinidine, and procainamide at doses of 5, 10 and 20 mg/kg bw, respectively, were added to the perfusion buffer at the beginning of perfusion. Results were expressed as percent change in mean pulmonary arterial pressure between preischemia and postischemic reperfusion. *p<0.0001 and **p<0.008 compared to the control. For each treatment group, n=6.  Figure 3. Effects of antiarrhythmic drugs o way pressure of ischemic-reperfused Uninjured control lungs were perfused fo Lungs subjected to ischemia reperfusion un( min of preischemia followed by 60 min of is( 30 min of reperfusion. Lidocaine, quinidinE cainamide at doses of 5, 10 and 20 mg/kg t tively, were added to the perfusion bul beginning of perfusion. Results were expres cent change in mean peak airway pressur preischemia and postischemic reperfusion. compared to control. **p<0.0001 cor ischemia-reperfused lung. For each treatr n= 6. aFirst 10 min of perfusion. bSamples taken 5 min after reperfusion. CControl lungs not subjected to is perfused for 100 min. d60-min ischemia followed by 30 min of reperfusion. Lidocaine, quinidine and pr at 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 the ischemia-induced lungs, 5 min of reperfusion resulted in 7-and 2-fold increases in the production of the cyclooxygenase metabolites, 6-keto-PGFia and TxB2, respectively (Table 1). These increases were ide significantly (p < 0.05) higher than either control preischemic lung or preischemic lung with addition of drugs in the perfusate ( Table 1). The time course of accumulation of these metabolites in lung effluents was studied at 5, 10, and 20 min postischemia. Both the 6-keto-PGFIa and TxB2 levels were found to remain elevated (p<0.0001) in the lung effluent collected up to 20 min after reperfusion compared to effluent collected from the same lung prior to ischemia and when compared to time-matched lung effluent samples from control 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 lungs derwent 10 subjected to ischemia reperfusion and chemia and treated with lidocaine, quinidine, and proe, and pro-cainamide at 5, 10, and 20 mg/kg bw, bw, respec-respectively, had significantly less ffer at the (p<0.0001) 6-keto-PGFta and TxB2 com-;sed as per-pared to untreated lungs that were sube between jected to similar ischemia reperfusion mp<0.aOdl conditions (Figures 4, 5). When the levels mpared to of PGF1I and TxB2 were compared in dine, and procainamide at 5, 10 and 20 mg/kg bw, respectively, significantly reduced the Pa respectively, of ischemiareperfused lungs compared to untreated reperfused lungs (Figure 2).
Effect on Peak Airway Pressure Peak Paw remained stable over the 100 min of perfusion in the uninjured control lungs. Lungs subjected to ischemia and reperfusion had a significantly higher (p< 0.000 1) Paw after 30 min of reperfusion. Lungs subjected to ischemia reperfu-sion but treated with lidocaine (5 mg/kg bw), quinidine (10 mg/kg bw), and procainamide (20 mg/kg bw) had significantly (p< 0.000 1) reduced Paw compared to the untreated ischemia-reperfused lungs. These data are presented in Figure 3. Effect ofAntiarrhythmic Drugs on Cydooxygenase Product Formation Measurements of 6-keto-PGFia and TxB2 in lung effluents collected prior to ischemia and in the time-matched lung effluents from the uninjured control lungs showed Discussion A major source of damage in postischemic reperfusion injury is believed to be the generation of oxygen-free radicals and other toxic oxygen metabolites (9,17,18). These radicals and metabolites have been implicated in postischemic reperfusion injury in the heart, kidney, intestine, brain, and other organs (14,(18)(19)(20). Studies in the postischemic reperfusion injury of the lungs also implicate toxic oxygen metabolite as a source of damage (8,17,21,22). In our present study, antiarrhythmic drugs, Volume 102, Supplement 10, December 1994  Effects of antiarrhythmic drugs on 6-keto-PGFia on ischemic-reperfused rat lung. Samples for 6-keto-PGFa, measurements were obtained prior to ischemia as well as 5, 10, and 20 min after reperfusion or at time-matched points in control lungs not subjected to ischemia. Lidocaine, quinidine, and procainamide at 5, 10, and 20 mg/kg bw, respectively, were added to the perfusate at the beginning of the perfusion period. 6-keto-PGF1a was measured by the scintillation proximity assay method on extracted samples in duplicate. *p<0.0001 compared to control and drug-treated postischemic-reperfused lungs. **p<0.05 when compared with control lungs or lungs treated with quinidine or procainamide but significantly lower than the ischemia-reperfused lungs. 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, and 20 min after reperfusion or at time-matched points in control lungs not subjected to ischemia. Lidocaine, quinidine, and procainamide at 5, 10, and 20 mg/kg bw, respectively, were added to the perfusate at the beginning of the perfusion period. TxB2 was measured by the scintillation proximity assay method on extracted samples in duplicate. *p<0.0001 compared to control and drug-treated ischemia-reperfused lungs. For each treatment group, n= 6.
such as lidocaine, quinidine, and procainamide, significantly reduced pulmonary edema, pulmonary Pa and Paw. These drugs also inhibited the formation of cyclooxygenase metabolites known to be produced during reperfusion of ischemic lungs (23). Although these agents have numerous systemic and local effects on various biological tissues, both in vivo and in vitro (24,25), their ability to ameliorate postischemic reperfusion injury in the lungs has not previously been recognized.
The mechanism by which the antiarrhythmic agents are effective against reperfusion injury may be explained, in part, by their antioxidant properties. In a recent study, Stelzner et al. (11) found that these drugs protect lungs against thioureainduced injury in rats. As oxygen radicals are likely to be produced during metabolism of thiourea (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 101 Msec-1 for quinidine, lidocaine and procainmide, respectively) and inhibitors of lipid peroxidation (12). Therefore, it is likely that these drugs protect pulmonary tissue against postischemic reperfusion injury by preventing lipid peroxide formation and/or scavenging -OH.
The concentration of three antiarrhythmic drugs in the perfusat were 40, 120, and 250 pg/ml for lidocaine, quinidine, and procainamide, respectively. However these drugs are also known to bind to plasma albumin, thereby reducing the availability of free drug for therapeutic action (26). Since our perfusate contained 4% bovine serum albumin, the actual concentrations of free drugs may be much less than indicated. The LD50 and pharmacokinetics of these drugs have not yet been elu-cidated in rats. Taking into account the doses of 4, 20, and 30 mg/kg bw for lidocaine, quinidine, and procainamide in the study by Stelzner et al. (11), our doses of 40, 120, and 250 pg/ml for rats are significantly smaller. Detailed pharmacokinetic studies of these drugs in rats are needed to determine the typical nontoxic plasma levels of these drugs in rats.
Because toxic oxygen metabolites can directly cause cell injury or lead to the production of other mediators, such as arachidonate metabolites, we measured the effect of antiarrhythmic agents on the cyclooxygenase metabolites thromboxane and prostacyclin during the reperfusion of ischemic lungs. Postischemic reperfusion caused a significant elevation of both the thromboxane and prostacyclin levels as measured by their stable metabolites TxB2 and 6-keto-PGFia. Similar increases of these metabo- Preischemia metabolites, produced secondary to the production of oxygen radicals, may in turn, cause tissue damage (16). However, these authors have concluded that cyclooxygenase metabolites may not be the sole source of injury because protection by inhibitors of cyclooxygenase product formation was not complete. Burghuber et al. (27) have also reported similar findings and concluded that cyclooxygenase products are not primarily responsible for lung damage. It has been shown that lidocaine is not an inhibitor of prostaglandin biosynthesis in vitro (28), but rather increases the production of prostacyclin (29). Our data demonstrate that all these antiarrhythmic drugs inhibit cyclooxygenase product formation. Since -OH are known to be responsible for the release of arachidonic acid from membrane phospholipids (30), it is possible that these drugs may not inhibit prostaglandin formation per se but might inhibit the liberation of arachidonic acid from phospholipids by scavenging OH radicals.
Nevertheless, the lung damage imposed by ischemia reperfusion could be the concommitant actions of both toxic oxygen products (21) and cyclooxygenase metabolites (23). We conclude that the inhibition of cyclooxygenase product formation and the attenuation of postischemic lung injury by these drugs may, in part, be due to the removal of toxic oxygen metabolites generated during the reperfusion of the lung.