Does Early Posttreatment with Lidocaine Attenuate Endotoxin-induced Acute Lung Injury in Rabbits?

This study was conducted according to the guidelines of the animal care review board of Kobe University School of Medicine. Thirty-two male Japanese white rabbits weighing 2.0-2.4 kg were used in this study and randomly divided into four groups (n = 8 for each group) in a blinded manner as follows: rabbits in group S-S received saline alone without lidocaine posttreatment; group S-L received saline with lidocaine posttreatment; group E-S received endotoxin from E. coli (055:B5 from the same lot, Difco, Detroit, MI), without lidocaine posttreatment; and group E-L received endotoxin followed by lidocaine (Figure 1).

After the rabbits were sedated with 4 mg *symbol* kg sup -1 ketamine, tracheostomy was performed aseptically, and a 3.5-mm noncuffed endotracheal tube was inserted and tied in place. Anesthesia was maintained with continuous infusion of ketamine at a rate of 4 mg *symbol* kg sup -1 *symbol* h sup -1. The lungs of the rabbits were ventilated with an infant ventilator (IV100B, Sechrist, Anaheim, CA) at an inspired Oxygen sub 2 concentration of 40%. Tidal volume was set to 10 ml *symbol* kg sup -1 measured by pneumotachograph. Respiratory rate was adjusted to produce an initial arterial CO²tension (Pa^CO2) of 35-42 mmHg; Pa sub CO²was maintained at less than 50 mmHg throughout the study period.

Via femoral cutdown, a catheter was placed in the distal aorta to monitor arterial pressure and to take samples for blood gas analysis. Pulmonary arterial pressure was continuously monitored with a pulmonary artery catheter (3-French, Baxter, Chicago, IL) inserted through the right internal jugular vein. Central venous pressure was also monitored with a catheter inserted through the femoral vein. The animals were placed on a heating pad under a radiant heat lamp so that the body temperature could be kept at 37.7-40.3 degrees Celsius. Lactated Ringer's solution was intravenously administered at a rate of 8 ml *symbol* kg sup -1 *symbol* h sup -1.

Immediately after the baseline measurement of lung mechanics, hemodynamics, peripheral leukocyte and platelet counts, and arterial blood gas analysis, groups S-S and S-L received infusion of saline for 60 min. Rabbits in groups E-L and E-S received endotoxin 100 micro gram *symbol* kg sup -1 over a 60-min period, with or without lidocaine treatment. Groups E-L and S-L received a bolus of lidocaine 2 mg *symbol* kg sup -1 (Fujisawa, Osaka, Japan), 10 min after the end of administration of endotoxin (E-L) or saline (S-L), followed by continuous infusion of lidocaine at a rate of 2 mg *symbol* kg sup -1 *symbol* h sup -1 until the rabbits were killed. This infusion rate of lidocaine was also used in our previous study. [6].

All rabbits were killed 6 h after the start of endotoxin treatment by injection of thiamylal. In groups S-L and E-L, arterial blood samples were obtained at 0, 0.5, 1, 2, 3, and 4 h after the start of administration of lidocaine and at the time of killing to measure plasma concentrations of the drug using fluorescence polarization immunoassay (TDX system, Abbott, North Chicago, IL).

Measurement of Lung Mechanics. During each experimental period, we obtained arterial blood specimens for analyzing arterial Oxygen²tension (Pa^O2), Pa^CO2, and pH using an automatic blood gas and electrolyte analyzer (ABL2, Radiometer, Copenhagen, Denmark) and for counting the number of peripheral leukocytes and platelets (counter, Coulter Electronics, Harkenden, United Kingdom). Immediately after the start of mechanical ventilation (before infusion of saline or endotoxin), at the end of administration of endotoxin, and immediately before the rabbits were killed (after the period of observation), lung mechanics were measured by the passive expiratory flow-volume technique as described by LeSouef et al. [7]The air flow was measured with a Fleish 00 pneumotachograph and a differential pressure transducer (MP-45, Validyne Engineering, Northridge, CA). Airway pressure was measured at the proximal end of the pneumotachometer with a semiconductor pressure transducer (P-300 501G, Copal Electronics, Tokyo, Japan). The volume was measured for each breath by digital integration of air flow using a respiration monitor (Aivision, Tokyo, Japan) and a personal computer (PC9801 VM11, NEC, Tokyo, Japan). The lungs were inflated and the air flow was interrupted at 20 cmH sub 2 O. The occlusion was rapidly released after airway pressure reached a plateau. Compliance and resistance of the total respiratory system were then calculated by means of the personal computer.

At the end of the experiment, after the thorax was opened, blood (15 ml) was drawn into a heparinized syringe (20 U *symbol* ml sup -1) from the pulmonary artery for chemiluminescence assay (see below). The rabbits were killed by administration of thiamylal after sampling of blood. The heart and lungs were then removed en bloc by observers blinded to the nature of experiment.

Ratio of Lung Wet Weight to Dry Weight. The left upper lobe was weighed and then dried to constant weight at 60 degrees Celsius for 24 h in an oven. The ratio of wet weight to dry weight (W/D weight ratio) was calculated to assess tissue edema.

Preparation of Bronchoalveolar Lavage Fluid and Measurements. Through the right mainstem bronchus 40 ml saline with ethylenediamine tetraacetic acid-2Na at 4 degrees Celsius was slowly infused and withdrawn. This procedure was repeated five times. Indomethacin was added to the bronchoalveolar lavage fluid (BALF) to inhibit further metabolism of arachidonic acid to prostaglandins during analysis. The BALF was analyzed for cell count and cell differentiation. A cytocentrifuged preparation (Cytospin 2, Shandon Southern Products, United Kingdom) of the BALF was stained with Wright-Giemsa for cell differentiation. The cells present in the fluid were counted with the Coulter counter and the Burker-Turk method. [8].

The fluid was centrifuged at 250g at 4 degrees Celsius for 10 min to remove the cells. The cell-free supernatant was divided into several aliquots and stored at -70 degrees Celsius until assayed. The following substances, metabolites, and mediators in the BALF were then measured. (1) The activated complement components C3a and C5a were quantified by radioimmunoassay (Amersham, Bucks, UK). (2) Albumin concentrations were measured by nephelometry with immunoglobulin G fraction of goat antirabbit albumin (Cappel, PA). (3) Concentrations of tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1 beta, IL-6, and IL-8 were measured by enzyme immunoassay (Amersham, Bucks, United Kingdom). (4) Concentrations of thromboxane A²(TxA²) and prostacyclin were quantified by radioimmunoassay (Amersham) as thromboxane B²(TxB²) and 6-keto-prostaglandin F¹alpha, the stable metabolites of TxA²and prostacyclin, respectively.

Chemiluminescence Assay. Reagents. Cypridina luciferin analogue (CLA) (2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-a]-pyrazine-3-one), dimethyl sulfoxide, Hank's balanced salt solution (HBSS), Histopaque-1119, Histopaque-1077, N-formyl-L-methionyl-L-luecyl-L-phenylalanine (FMLP), and zymosan A were obtained from Sigma Chemical (St. Louis, MO).

The CLA was dissolved to 56 micro gram *symbol* ml sup -1 in distilled water. The solution was stored in 1-ml aliquots at -80 degrees Celsius. The CLA concentrations were based on E410 nm = 8900 M sup -1 *symbol* cm sup -1. [9]FMLP 5 mg was dissolved in 1.14 ml DSMO. The solution was stored at -80 degrees Celsius until the time of the assay. Just before use, the stored solution was diluted with 50% dimethyl sulfoxide-50% HBSS to 100 micro Meter. Zymosan A was opsonized by the method of Nishida et al. [10]with modification. Zymosan A was suspended in HBSS at a concentration of 2 mg *symbol* ml sup -1 and heated in a boiling water bath for 100 min, washed twice with HBSS, and opsonized with pooled serum in a shaking water bath for 30 min at 37 degrees Celsius. The opsonized zymosan (OZ) was then washed twice, resuspended in HBSS to a concentration of 20 mg *symbol* ml sup -1, and stored at -80 degrees Celsius until use.

Isolation of Neutrophils. Histopaque-1119, Histopaque-1077, and whole blood were layered in a test tube and centrifuged at 700g for 30 min at room temperature. The layer containing granulocytes (at the interphase between Histopaque-1077 and Histopaque-1119) was transferred to another tube. The cells were washed in HBSS and centrifuged twice at 200g for 10 min. The resultant leukocytes were suspended to 1 x 10⁷cells *symbol* ml sup -1 in HBSS and were kept at 0 degree Celsius for no longer than 3 h before use. The cell analysis showed that more than 96% of the cells were neutrophils, and the trypan blue dye exclusion test confirmed that more than 95% of the cells were viable.

Measurement of Chemiluminescence. Measurement of chemiluminescence was made by the method of Sugioka et al. [11]The incubation mixture contained 4 x 10⁵white blood cells (WBC), 20 micro liter FMLP or 80 micro liter OZ, 50 micro liter 40 micro Meter CLA, and HBSS to a total volume of 2 ml. Cells and HBSS were preincubated for 3 min and the reaction initiated by the simultaneous addition of the other two components. CLA-dependent luminescence, which is thought to reflect primarily Oxygen²sup - production, was monitored with a luminescence reader (Lumicounter-1000, Nichion, Chiba, Japan). During luminescence measurement, the incubation mixture was agitated at 37 degrees Celsius in the luminescence reader. Ketamine used as an anesthetic in the current study has been shown to have no effect on Oxygen²sup - production by neutrophils at doses used in the clinical setting. [12].

Shortly after the rabbits were killed (< 5 min), the left lower lobe was fixed by instillation of 10% formaldehyde solution through the left lower bronchus at 20 cmH²O. The specimens were embedded in paraffin wax, and stained with hematoxylin and eosin and examined under a light microscope. Lung injury was scored as 0 (minimal damage) to 4+ (maximal damage) according to combined assessments of alveolar congestion; hemorrhage and edema; infiltration or aggregation of neutrophils in air space or vessel wall; thickness of alveolar wall, and hyaline membrane formation by two observers unaware of the group assignment of the animal.

Data except lung injury score are expressed as means plus/minus SEM; data on lung injury score are given as medians and range. The degree of attenuation of lung injury by lidocaine was calculated from the following formula: percentage attenuation = 100 x (b - c)/(b - a), where a = value in S-S group; b = value in E-S group; and c = value in E-L group. This value indicates the degree of efficacy of lidocaine treatment in each subject: 0% indicates that the mean scores for groups E-S and E-L were equal (no attenuation of lung injury by lidocaine), and 100% indicates that the mean scores for group E-L and S-S were equal (maximum attenuation). Statistical analysis was performed by repeated-measures analysis of variance for continuous variables, except for lung injury score, for which the Kruskall-Wallis rank test was used. P < 0.05 was deemed significant. When analysis of variance indicated a significant difference, Bonferroni's multiple-comparison test was used to determine which groups were significantly different from each other.

No rabbits died of endotoxemia. In groups S-L and E-L, plasma lidocaine concentrations were maintained between 1.2-2.3 micro gram *symbol* ml sup -1. As shown in Figure 2, Pa^CO2in groups S-S and S-L remained at a level exceeding 150 mmHg, whereas Pa^O2in group E-S gradually decreased to 89 mmHg during the experiment. In group E-L, however, the decrease in this parameter was attenuated maximally by 29% (P < 0.05). The values of Pa^CO2in four groups were similar (38 to 40 mmHg) and gradually increased to 50 plus/minus 1.2 and 52 plus/minus 1.3 in groups E-L and E-S, respectively. The alveolar -- arterial difference in Oxygen²tension was increased in groups E-L and E-S as Pa^O2decreased. Lidocaine attenuated the increase in the alveolar -- arterial difference in Oxygen²tension maximally by 31% (P < 0.05). The heart rate, arterial blood pressure, or central venous pressure did not differ among the four groups at any point (data not shown). Infusion of endotoxin rapidly increased pulmonary arterial pressure, with a peak reached at the end of endotoxin infusion. Lidocaine posttreatment failed to attenuate the pulmonary hypertension (Table 1). Peripheral blood leukocyte counts decreased with infusion of endotoxin, reached their nadir 1-2 h after the end of endotoxin infusion, and remained low during the experiment. Lidocaine posttreatment also failed to attenuate the decrease in leukocyte. Peripheral blood platelet counts decreased gradually in the endotoxin-treated group (E-S and E-L). No effect of lidocaine on platelet counts was observed.

Neither compliance nor resistance immediately after the start of mechanical ventilation and at the end of endotoxin treatment was different among the four groups (Table 2). Compliance 6 h after the start of treatment with endotoxin was greater in group E-L than in group E-S (attenuation 29%; P < 0.05). In contrast, resistance in group E-L 6 h after endotoxin was similar to that in group E-S (attenuation 27%).

The lung W/D weight ratio was calculated as a parameter of lung edema. The ratio increased in rabbits receiving endotoxin (E-S and E-L) compared with those receiving saline (S-S and S-L) (Table 3). Lidocaine posttreatment attenuated the increase in W/D weight ratio (attenuation 43%; P < 0.05).

Recovery in BALF in the three groups was 83-89%, indicating no difference between the four groups. Table 3shows that the total number of leukocytes recovered in BALF was significantly higher in groups E-S and E-L compared with that in group S-S. Leukocyte counts in BALF were not significantly different in groups E-L and E-S (attenuation 15%). Differential counts revealed that BALF leukocytes in group S-S were mostly macrophages. Polymorphonuclear cells accounted for 1% of the WBC in BALF obtained from group S-S. In contrast, the ratio of polymorphonuclear cells to total WBC increased to 12% in group E-S and 8% in group E-L (P > 0.05).

Albumin concentrations in the supernatant of BALF were higher in endotoxin-treated rabbits (E-S and E-L) than in the saline-treated rabbits (S-S and S-L). Lidocaine posttreatment significantly decreased the albumin concentrations in endotoxin-treated rabbits (E-L) (attenuation 37%). Table 3indicates that the concentrations in BALF of C3a and C5a, which are known to be chemotactic factors, were similar in groups E-L and E-S. The BALF concentrations of cytokines significantly increased in endotoxin-treated rabbits (E-S and E-L). The TxB²concentration in BALF increased in group E-S compared with that in group S-S. Posttreatment with lidocaine failed to attenuate the increase. In contrast, there were no differences in BALF concentrations of 6-keto-prostaglandin F¹alpha among the four groups.

The CLA-dependent chemiluminescence (representing Oxygen²sup - production) by neutrophils isolated from the pulmonary artery blood in group E-S was significantly higher compared with that in group S-S when stimulated by OZ or FMLP (Table 4). Posttreatment with lidocaine attenuated the increase in chemiluminescence (attenuation 75% for OZ and 64% for FMLP).

The results of the grading of lung damage are summarized in Table 5. The score for E-L group was statistically less than that for group E-S. Light-microscopic findings in group E-S included hemorrhage and edema, thickened alveolar septa, and the presence of inflammatory cells in alveolar spaces; these changes were slightly less pronounced in group E-L.

In the current study, we have shown that early posttreatment with lidocaine slightly attenuated deterioration of oxygenation in endotoxin-treated rabbits (E-S and E-L). Lidocaine posttreatment also attenuated endotoxin-induced pulmonary edema as assessed by W/D weight ratio. The drug was effective morphologically (assessed by lung injury score) and functionally (assessed by compliance) for the acute lung injury. In contrast, lidocaine posttreatment did not decrease the release of chemotaxins (C3a, C5a, TNF alpha, IL-1 beta, and IL-8 in BALF) in rabbits receiving endotoxin (E-L). There were no differences between the groups E-L and E-S in the percentage of alveolar neutrophils recovered in BALF and leukocyte counts in peripheral blood. These observations suggest that lidocaine posttreatment could not inhibit the release of the chemotaxins, resulting in failure to reduce accumulation of leukocytes in the lung.

The pulmonary arterial pressure increased in rabbits receiving endotoxin (E-S and E-L), peaking at the end of endotoxin treatment. The pulmonary hypertension continued until 3 h after the end of endotoxin treatment. Lidocaine had no effect on this increased pulmonary arterial pressure. No difference in TxB²concentrations in BALF was observed in endotoxin-treated groups with or without lidocaine (E-S or E-L). This failure of lidocaine to decrease TxB²concentrations may be responsible for failure of lidocaine to decrease of pulmonary artery pressure. Lidocaine posttreatment lessened the endotoxin-induced increase in BALF concentrations of albumin, which is an index of endothelial hyperpermeability leading to pulmonary edema. The beneficial effect of lidocaine on pulmonary edema, which is assessed by W/D weight ratio and pathologic changes, may be due to attenuation of vascular hyperpermeability. Successful use of lidocaine to reduce lung extravascular protein accumulation, as an index of endothelial hyperpermeability, in thiourea-induced lung injury has been reported. [13].

We began the lidocaine treatment 10 min after the end of endotoxin infusion. We chose this timing because significant pathophysiologic events (pulmonary hypertension and profound leukopenia) have already occurred at this time. [6]It is well known that pulmonary hypertension and neutropenia are initial events in endotoxin-induced lung injury. [14-16].

We have hypothesized that posttreatment with lidocaine attenuates lung injury by the suppression of activation of neutrophils, as does pretreatment. Thus we evaluated the effect of lidocaine on acute lung injury until approximately 5 h after starting treatment with lidocaine, focusing our attention on the period of activating neutrophils and macrophages. As in the control group of the current study, endotoxin causes increases in production and release of cytokines, Oxygen²sup -, thromboxanes, and complement components, resulting in accumulation of neutrophils in lung, deterioration of oxygenation, and increase in extravascular lung water within 6 h. Lung vascular permeability to proteins increased between 2 and 6 h after endotoxin injection. [17,18]Chemiluminescence response reveals that neutrophils in blood are activated early after endotoxin, followed by metabolic exhaustion with a minimal chemiluminescence response after 2 h. The response is augmented thereafter by new granulocytes liberated from the bone marrow.* Accumulation of neutrophils in affected tissue probably became peaked by 6 h after endotoxin administration, because the WBC counts in peripheral blood have started increasing.

We previously reported that lidocaine administered before endotoxin reduced endotoxin-induced lung injury. [6]Lidocaine pretreatment attenuates the deterioration of pulmonary hypertension, peripheral leukopenia, oxygenation, lung compliance, W/D ratio, and WBC counts, percentage of polymorphonuclear cells in total WBC, albumin in BALF, and Oxygen²sup - production by neutrophils with stimulation. However, lidocaine posttreatment was effective only in oxygenation, lung edema (assessed by albumin concentration in BALF, W/D ratio, and morphologic examination), and lung compliance. Lidocaine attenuated chemiluminescence of neutrophils to the same degree regardless of the timing of administration (attenuation 65% and 83% [pretreatment] and 64% and 75% [posttreatment], FMLP- and OZ-stimulated chemiluminescence, respectively). This attenuation of Oxygen²sup - production, which is known to be a major factor involved in lung injury as a final step, is a putative mechanism for reducing lung edema by lidocaine in this study.

Lidocaine after endotoxin failed to suppress chemical mediators despite improved pulmonary hyperpermeability. Some mediators that concern lung injury induced by endotoxin were not measured in the current study (e.g., protease derived from neutrophils, phospholipase A², platelet-activating factor [PAF], and endothelin). Protease from activated neutrophils and macrophages directly attacks lung matrix, causing destruction of lung structure. Lidocaine stabilizes the cell membrane to decrease the release of proteases from neutrophils or macrophages. [19]Phospholipase A²activated by endotoxin causes degradation of phospholipid to arachidonic acids, as substrates of eicosanoids. TxA², produced by endothelium, platelets, and pulmonary macrophages, causes early phase pulmonary hypertension lasting about 90 min after starting endotoxin infusion. [20,21]It was not affected by lidocaine posttreatment probably due to its prompt release. As the results of this study showed that the concentration of TxA²and 6-keto-prostaglandin F¹alpha concentration in BALF were similar in groups E and E-L, lidocaine posttreatment would not prevent activation of phospholipase A sub 2. PAF directly and indirectly increases pulmonary vascular resistance and permeability. In the rat model, within 20 min of intraperitoneal injection of endotoxin, blood and lung PAF concentrations increased for more than 120 min. [22]TNF alpha stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release PAF 1-4 h after TNF alpha administration in vivo. [23]The PAF and proteases remain as yet to be determined to clarify the mechanisms through which lidocaine posttreatment attenuated endotoxin-induced lung injury.

Cytokines, such as IL-1, TNF-alpha, and IL-6, might have been induced and released at the early phase of post endotoxin administration, namely before starting lidocaine treatment. These cytokines injure lung not directly but through activating many cell types, including neutrophils and macrophages. Lidocaine might be effective in the control of the next phase related to cytokines, because even pretreatment with lidocaine possessed partial effect on attenuation of cytokine secretion. Fletcher and Ramwell reported that posttreatment with the drug only for 3 h successfully decreased mortality (as assessed 72 h after endotoxin) due to endotoxin shock in baboons and dogs. [24]Our observations were inconsistent with their study. Lidocaine suppressed Oxygen²sup - production from neutrophils but did not cut off the cytokine cascade that may be responsible for the late phase of lung injury.

In conclusion, lidocaine administered after endotoxin slightly attenuated deterioration of oxygenation probably due to reduction of edema. A comparison of these findings with our previous findings [6]suggests that posttreatment with lidocaine may have only limited clinical application in patients with adult respiratory distress syndrome.

* Dwenger A, Regel G, Ellendorff B, Schweitzer G, Funck M, Limbrock H, Sturm JA, Tscherne H. Alveolar cell pattern and chemiluminescence response of blood neutrophils and alveolar macrophages in sheep after endotoxin infection. J Clin Chem Clin Biochem 28:163-168, 1990.