| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 1996;61:949-955
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, Barnes Hospital, St. Louis, Missouri
 |
Abstract |
|---|
 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Methods. Donor lungs were flushed with modified Euro-Collins solution (4°C) after prostaglandin E1 administration (250 µg), inflated with 550 mL (inspired oxygen fraction = 1.0), and stored for 24 hours at 1°C. Left lung allotransplantation was performed in 13 mongrel dogs. Immediately after reperfusion the recipient right pulmonary artery and bronchus were ligated to permit assessment of allograft function during a 6-hour postreperfusion period. Allograft gas exchange (every 15 minutes) and hemodynamics (every 60 minutes) were assessed. Peripheral blood PMN CD11b expression was determined by flow cytometry. After sacrifice allograft bronchoalveolar lavage fluid PMN count and allograft tissue myeloperoxidase activity were measured. Two groups were studied: In group I (n = 8) lidocaine hydrochloride was added to the donor flush (20 mg/L) solution. In addition lidocaine was given to the recipient at the time of thoracotomy (intravenous bolus of 4 mg/kg), followed by a continuous infusion of 4 mg•kg-1•h-1 during implantation and the assessment period. Three dogs that did not reach effective lidocaine blood levels at the time of reperfusion (3 to 4 µg/mL) were excluded from analysis. Group II animals (n = 5) received no lidocaine.
Results. Gas exchange in group I was superior throughout the assessment period (p < 0.05). Bronchoalveolar lavage fluid PMN count in group I was reduced (0.36 x 106 PMN/mL versus 6.2 x 106 PMN/mL; p < 0.03). Group I allograft myeloperoxidase activity was 0.17 U • mg-1 • min-1 compared with 0.28 U•mg-1•min-1 in group II (p < 0.01). In lidocaine-treated animals PMN CD11b expression was maintained at basal levels 2 hours after reperfusion, compared with group II, in which upregulation of CD11b was observed. Lower lobe wet/dry ratio was not different in the two groups.
Conclusions. Our observations indicate that lidocaine reduces reperfusion injury and inhibits PMN adhesion and subsequent migration to the lung allograft.
|
Introduction |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Although lung transplantation has been established in the last 10 years as therapy for end-stage lung disease, early graft dysfunction continues to represent a major and unpredictable clinical problem. Lung allograft dysfunction increases morbidity and mortality. The pathophysiology of reperfusion injury is not completely understood. A number of factors have been shown to play a role in its pathogenesis. Allograft ischemic time and conditions of preservation such as temperature [1], inspired oxygen fraction [2], and state of inflation [3] are important. The reperfusion injury may be mediated by neutrophil (PMN) granulocytes [4, 5] via oxygen free radicals [6, 7]. Superior flushing and preservation techniques have reduced the frequency of early graft dysfunction. Our laboratory recently demonstrated that posttransplantation lung reperfusion injury can be modulated by a number of strategies, for example, nitric oxide inhalation [8] and continuous prostaglandin E1 administration [9]. As in myocardial ischemia-reperfusion studies [10–12], postischemic injury in lung allografts could be reduced by leukocyte depletion [4, 5] and administration of monoclonal antibodies against adhesion molecules [13]. In a rat model we demonstrated that combined administration of monoclonal antibodies against intracellular adhesion molecule-1, CD11a, and CD18 resulted in reduced accumulation of neutrophils in the lung tissue and superior gas exchange [14]. However, at present PMN depletion and monoclonal antibodies are of limited use as therapeutic agents in the clinical setting.
Lidocaine reduces reperfusion injury. In a canine model of lung autotransplantation after 2 hours of warm ischemia gas exchange was superior in animals receiving lidocaine during reperfusion (Y. Tanaka, personal communication, 1995). In a number of myocardial [15–17] and spinal cord [18] ischemia studies lidocaine decreased reperfusion injury. Cationic anesthetic agents such as lidocaine inhibit membrane-dependent responses of human granulocytes on stimulation [19]. Lidocaine is a known inhibitor of neutrophil adherence, as demonstrated in a variety of experiments in vitro [19, 20] and in vivo [20–23]. A recent study demonstrated that lidocaine inhibits upregulation of PMN CD11b/CD18 in vitro [24]. Expression of CD11b/CD18 induces firm attachment of PMNs to endothelial cells, activates adhesion-dependent oxygen radical production [25], and initiates subsequent migration into the tissue [26].
We evaluated the effect of lidocaine on reperfusion injury in a canine model of left lung allotransoplantation.
|
Material and Methods |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Donor Procedure
Donor animals were anesthetized with thiopental sodium intravenously (10 mg/kg) followed by atropine (0.5 mg), and intubated with an endotracheal tube (9 mm). Mechanical ventilation was established (Bennet MA1; Puritan Bennet, Inc, Overland Park, KS) with 100% oxygen at a tidal volume of 550 mL at a rate of 12 breaths/min and 5 cm H2O of positive end-expiratory pressure. After a median sternotomy, the superior and inferior venae cavae, the ascending aorta, the main pulmonary artery (PA), and the trachea were isolated. Animals were heparinized (400 U/kg) before insertion of a curved, metal-tipped cannula (Sarns, Inc, Ann Arbor, MI) through a pursestring suture in the main PA just distal to the valve. Before administration of the flush solution, 250 µg of prostaglandin E1 (Prostin VR Pediatric; The Upjohn Company, Kalamazoo, MI) was injected directly into the main PA. Cardiac inflow was occluded by ligation of the superior and inferior venae cavae 20 seconds after the infusion of prostaglandin E1. The inferior vena cava was cut proximal to the ligation and the tip of the left atrium was excised for decompression of the PA flush. The lungs were perfused immediately, at a pressure of 40 cm H2O, with 50 mL/kg of cold (4°C) Euro-Collins solution, modified by the addition of 4 mmol/L MgSO4 and 32.7 g/L glucose. During the flush the lungs were cooled topically by flooding the chest with cold (4°C) saline solution (0.9%). The flushing pressure was monitored through a transducer between the flushing tube and the PA cannula. When the flushing was completed, the trachea was clamped at end-inspiration (tidal volume, 550 mL) and the heart-lung block was excised. The harvested organs were stored in modified Euro-Collins solution (1°C) for 24 hours before implantation.
Recipient Procedure
Anesthesia of recipient animals was achieved in the same way as in the donors, and ventilation was accomplished with an adjustable-rate Harvard pump respirator (model 613; Harvard Apparatus, South Natick, MA) with 98.5% oxygen and 1.5% halothane. A femoral arterial line and a Swan-Ganz catheter were placed and continuously transduced (Hewlett-Packard 1290A, Andover, MA). Left pneumonectomy was performed. The contralateral main PA and upper and lower right bronchus were mobilized and encircled separately with umbilical tapes. The donor left lung was separated from the heart-lung block, and left single-lung allotransplantation was performed using standard techniques [27]. The allograft was topically cooled with ice slush during implantation. The left atrial anastomosis was performed first, using a continuous everting mattress suture. The PA and the bronchus were anastomosed by a continuous over-and-over suture. After reperfusion of the allograft a Millar pressure transducer was placed in the left atrium and two chest tubes were inserted and evacuated by underwater seal drainage. The contralateral upper and lower lobe bronchi and main PA were ligated. At this point ventilation was adjusted to 15 breaths/min at a tidal volume of 550 mL and 5 cm H2O positive end-expiratory pressure (Bennet MA1; Puritan Bennet, Inc). This ventilatory change was made so as to precisely maintain inspired oxygen fraction and positive end-expiratory pressure levels during the assessment period. The chest was closed with three umbilical tapes and continuous muscular and skin sutures. Animals were then placed supine for the subsequent 6-hour assessment period.
Study Groups
In group I (n = 8) lidocaine hydrochloride (Spectrum Chemical Mfg Corp, Gardena, CA) was added to the modified Euro-Collins flushing solution at a concentration of 20 µg/mL. In addition an intravenous bolus of lidocaine (4 mg/kg) was given to the recipient at the time of thoracotomy, followed by a continuous infusion of lidocaine (4 mg•kg-1•h-1 in 0.9% NaCl) throughout the implantation procedure and the 6-hour assessment period. With this method of administration we avoided lidocaine blood levels greater than 5 µg/mL at the time of reperfusion, as ligation of the contralateral PA with high levels was poorly tolerated. Lidocaine blood levels could not be obtained during the experiment and therefore no adjustment of the dosage could be made. Preliminary experiments demonstrated that serum levels less than 3 µg/mL were not effective. Three dogs did not reach effective blood levels at the time of reperfusion and were excluded from analysis. Group II (n = 5) served as control; no lidocaine was administrated. Lidocaine was stored as powder, and solutions were prepared before use.
All animals received humane care in compliance with the ``Principles of Laboratory Animal Care'' formulated by the National Society for Medical Research and the ``Guide for the Care and Use of Laboratory Animals'' prepared by the National Academy of Science and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Assessment
During the assessment period anesthesia was maintained with intravenous administration of thiopental sodium. Systemic arterial, PA, central venous, and left atrial pressure were recorded continuously. Cardiac output was measured hourly (model COM-1; American Edwards Laboratories, Santa Ana, CA). Arterial and mixed venous blood were collected for gas analysis every 15 minutes.
Lidocaine levels in the peripheral blood were measured by fluorescence polarization immunoassay. Samples were drawn just before reperfusion, 30 minutes and 2 hours after reperfusion, and at the end of the assessment period. Six hours after reperfusion the animals were sacrificed. Upper lobe allograft samples were submitted to histologic examination and tissue myeloperoxidase (MPO) assay. The allograft lower lobe was used for wet/dry ratio measurement.
Myeloperoxidase Assay
Donor and recipient lung samples were frozen immediately and stored at -70°C until assay. Quantitative MPO activity was determined as previously described [27]. Frozen lung tissue (100 mg) was homogenized in 1 mL of 0.5% hexadecyltrimethylammonium bromide (to release MPO from the primary granules of the PMNs), 5 mmol/L EDTA, and 50 mmol/L potassium phosphate buffer (pH 6.2) with a Broeck tissue grinder (Kontes Glass Co, Vineland, NJ). The homogenate was centrifuged at 10,000 g for 15 minutes at 4°C. The supernatant was assayed for total soluble protein by the method of Pierce Laboratories [28] and for MPO activity. Enzyme activity was measured spectrophotometrically: 10 µg of fivefold supernatant was combined with 0.6 mL Hanks' bovine serum albumin, 0.5 mL of 100 mmol/L potassium phosphate buffer (pH 6.2), 0.1 mL 0.05% H2O2, and 0.1 mL of 1.25 mg/mL o-dianisidine. Color development was stopped by addition of 1% NaN3 after 5 and after 20 minutes at room temperature. The optical density was measured at 460 nm with a spectrophotometer (PMQ II; Carl Zeiss, Germany). The color development from 5 to 20 minutes was linear. Enzyme activity is expressed as change in optical density units per minute per milligram of tissue protein (
OD•min-1•mg-1).
Immunofluorescence Analysis by Flow Cytometry
Neutrophil CD11b expression was detected by indirect immunofluorescence and flow cytometry. Whole blood analysis [24] was performed, and the samples were kept on ice to minimize PMN activation by handling and warming of the samples. The primary antibody was an anti-human CD11b antibody (monoclonal antibody 904), which is cross-reactive in the dog [29] and has been used for characterization of canine CD11b [30]. It was generously provided by Dr James Griffin (Dana Farber Cancer Institute, Boston, MA).
Peripheral arterial blood was collected into tubes containing EDTA. The labeling was performed 2 hours after reperfusion. Five hundred milliliters of peripheral blood was diluted with 500 µL labeling buffer (0.1% bovine serum albumin in Hanks' buffered saline solution) and incubated at 4°C with 5 µg monoclonal antibody 904 for 30 minutes. After two washes with phosphate-buffered saline solution the pellet was resuspended in 500 µL labeling buffer and further incubated for 30 minutes at 4°C with 5 µg fluorescein isothiocyanate–conjugated F(AB`)2 fragment goat anti-mouse immunoglobulin G (Organon Teknika Corp, West Chester, PA). Incubation was finished by two times washing with phosphate-buffered saline solution. Red blood cells were removed by hypotonic lysis. Positive controls were stimulated with 5 µg/mL phorbol 12-myristate 13-acetate, diluted in dimethyl sulfoxide (both Sigma Chemical Co, St. Louis, MO), for 20 minutes at 37°C. For the negative control an anti-human monoclonal antibody was used. The cells were fixed in 4% paraformaldehyde (Sigma Chemical Co) and stored at 4°C in the dark. Flow cytometry was performed on a FACscan (Becton Dickinson, San Jose, CA). A granulocyte window was set in comparison with isolated canine granulocytes, and only this fraction was analyzed for surface fluorescence.
Statistical Analysis
All values are given as the mean ± standard error of the mean. One-way analysis of variance with repeated measures was used to compare an overall difference between groups. Factorial one-way analysis of variance was additionally performed for comparison of each assessment point. Differences were considered significant at the p less than 0.05 level.
|
Results |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
).
|
Mortality
Two animals in group II died due to cardiopulmonary failure during the 6-hour assessment period. One animal in group I, which was excluded from analysis because of an insufficient lidocaine level, died 3 hours after reperfusion due to cardiopulmonary failure. The other animals in group I survived the 6-hour assessment period.
Gas Exchange
Throughout the 6-hour assessment period gas exchange in lidocaine-treated animals was superior to that of the control group. For arterial oxygen tension this difference was statistically significant from 15 minutes to the end of the assessment (p < 0.05). Differences in arterial carbon dioxide tension reached statistical significance from 120 minutes to the end of the assessment (Fig 1).
|
).
|
Bronchoalveolar Lavage Fluid Neutrophil Count
The neutrophil count in the bronchoalveolar lavage fluid in lidocaine-treated animals was reduced from 7.16 x 106 ± 1.93 x 106 PMN/mL in group II to 0.37 x 106 ± 0.21 x 106 PMN/mL in group I (p < 0.03).
Myeloperoxidase Assay
Allograft MPO activity in group I was significantly reduced compared with group II (1.73 ± 0.021 OD• mg-1•min-1 versus 2.82 ± 0.016 OD•mg-1•min-1; p < 0.01). The MPO activity in normal unflushed lung tissue was 0.11 ± 0.001 OD•mg-1•min-1. This equals a 65% reduction of PMN migration to the lung allograft (Fig 3).
|
). On bronchoalveolar lavage fluid PMNs collected at the end of the 6-hour assessment period high expression of CD11b was detected (data not shown).
|
|
Comment |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
The suppressive effect of local anesthetics (xylocaine) on PMN adherence to venular endothelium was demonstrated by the use of intravital microscopy [21]. Invasion of white cells from canine jugular and femoral veins into adjacent damaged tissue was blocked by lidocaine [22]. The same authors investigated migration of indium-111–labeled leukocytes from canine jugular veins during administration of tocainide, a primary amine analogue of lidocaine. Migration of leukocytes across interendothelial junctions, accumulation between endothelial cells and basement membrane, and subsequent damage to the endothelium were reduced by tocainide. The authors of that study suggested that leukocyte-induced damage occurring in sterile inflammation might be reduced by the use of local anesthetic drugs [23].
In a rabbit model of sterile peritonitis lidocaine prevented delivery of PMNs to the site of inflammation [21]. Neutrophil count in the peritoneal exudate after 6 hours was 2% of controls. Methylprednisolone-treated animals had exudate counts of 40% in comparison with controls. In studies of acute myocardial ischemia and reperfusion, lidocaine reduced infarct size [15, 16]. In a pig model the reduction in infarct size was associated with decreased adherence of granulocytes to vascular endothelium in the ischemic myocardium and inhibition of other PMN functions such as superoxide anion release [17].
The mechanism by which lidocaine inhibits PMN adherence remains unclear. A recent publication demonstrates that lidocaine in vitro inhibits upregulation of CD11b/CD18 and superoxide (O2-) release from human PMN granulocytes on stimulation by the chemotactic peptide FMLP (N-formyl-methionyl-leucyl-phenylalanine) and rhG-CSF (recombinant human granulocyte colony-stimulating factor) [24]. The authors of that study suggested that lidocaine may interfere with the postreceptor signal transduction pathway. Lidocaine may alter the expression of membrane effector molecules because local anesthetic-treated PMNs undergo dramatic morphologic changes: pseudopod formation ceases, ruffling of the membrane is reduced, and the cells take on a round, smooth configuration [21].
Lidocaine inhibits fast sodium as well as slow calcium channels [31]. Early activation events are similar in endothelial cells and leukocytes. An increase in free cytosolic Ca2+ in both leukocyte and endothelial cells is an important early signaling event in leukocyte-endothelial adhesion [32]. In activated endothelial cells a free cytosolic Ca2+ increase has a regulatory function on vascular permeability [33], PMN adhesion [34], and PMN migration [35]. In monocytes the increase in cytosolic Ca2+ regulates the high-affinity function of CD11b/CD18 [36]. Lidocaine might be effective in endothelial cells as well as in PMNs by inhibiting signals of activation.
To measure the effect of lidocaine on PMN activation and upregulation of adhesion molecules we detected the expression of CD11b on PMNs. In the control animals CD11b expression increased 2 hours after reperfusion. The increase corresponds with findings in human patients recovering from sepsis [37]. In only 1 lidocaine-treated animal a slight upregulation of CD11b was detected 2 hours after reperfusion. Bronchoalveolar lavage fluid PMN count and PMN migration to the lung allograft, assessed by allograft tissue MPO assay, were significantly reduced in lidocaine-treated animals. In group II bronchoalveolar lavage fluid PMNs exhibited an increased expression of CD11b, indicating a CD11b-dependent mechanism of PMN migration to the lung allograft. These observations suggest that lidocaine inhibits PMN adhesion, thereby diminishing accumulation of PMNs in the lung allograft. Lidocaine might have additional effects on preservation and reperfusion due to its blocking effect on ion channels, by inhibiting calcium and sodium inflow into the ischemic cell. An increased membrane stability after lidocaine administration is exemplified by an increased resistance of erythrocytes against hypotonic lysis [38]. Further, cationic anesthetic agents inhibit membrane-dependent responses of PMNs on stimulation, including release of lysosomal enzyme and superoxide anion production [19, 39].
Unfortunately lidocaine has a small therapeutic index. Our preliminary results demonstrated that lidocaine levels less than 3 µg/mL at the time of reperfusion were not effective. In the range between 3 and 7 µg/mL no significant hemodynamic changes were observed, but with high lidocaine blood levels ligation of the contralateral PA after reperfusion resulted in severe hypotension and cardiac failure. Gas exchange was superior in lidocaine-treated animals throughout the assessment period. We speculate that the variation of the outcome 6 hours after reperfusion is due to PMN-independent mechanisms and small differences in flushing and preservation. This experimental model creates a very severe reperfusion injury by using a long preservation time and direction of the entire blood flow to the transplanted lung.
In summary, our observations indicate that lidocaine reduces lung allograft reperfusion injury and inhibits PMN adhesion and subsequent migration to the lung allograft. We speculate that the main effects of lidocaine are inhibition of PMN-mediated injury and possibly prevention of calcium and sodium overload of the ischemic cell after reperfusion. The results in this severe but clinically relevant large animal model should lead to further research with more specific inhibitors of PMN adherence to evaluate the contribution of PMNs to lung allograft reperfusion injury.
|
Acknowledgments |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
Footnotes |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Address reprint requests to Dr Patterson, Department of Surgery, Washington University School of Medicine, Suite 3108, Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110.
|
References |
|---|
|
TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Related Article
This article has been cited by other articles:
|
|
 |
|
  Y. Toyoda, J. Thacker, R. Santos, D. Nguyen, J. Bhama, C. Bermudez, R. Kormos, B. Johnson, M. Crespo, J. Pilewski, et al. Long-Term Outcome of Lung and Heart-Lung Transplantation for Idiopathic Pulmonary Arterial Hypertension Ann. Thorac. Surg., October 1, 2008; 86(4): 1116 - 1122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Vinten-Johansen Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 481 - 497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Reed General thoracic surgery and the Southern Thoracic Surgical Association: the second 25 years Ann. Thorac. Surg., November 1, 2003; 76(90050): S14 - 16. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Salvatierra, R. Guerrero, M. Rodriguez, A. Alvarez, F. Soriano, R. Lopez-Pedrera, R. Ramirez, J. Carracedo, F. Lopez-Rubio, J. Lopez-Pujol, et al. Antithrombin III Prevents Early Pulmonary Dysfunction After Lung Transplantation in the Dog Circulation, December 11, 2001; 104(24): 2975 - 2980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Stammberger, J. Hamacher, S. Hillinger, and R. A. Schmid sCR1sLeX ameliorates ischemia/reperfusion injury in experimental lung transplantation J. Thorac. Cardiovasc. Surg., December 1, 2000; 120(6): 1078 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Stammberger, R. A. Schmid, S. Hillinger, T. Singer, O. M. Schob, A. Zollinger, and W. Weder Effect of a short period of warm ischemia after cold preservation on reperfusion injury in lung allotransplantation Eur. J. Cardiothorac. Surg., April 1, 1999; 13(4): 442 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Schmid, A. Zollinger, T. Singer, S. Hillinger, J. R. Leon-Wyss, O. M. Schob, K. Hogasen, G. Zund, G. A. Patterson, and W. Weder Effect of soluble complement receptor type 1 on reperfusion edema and neutrophil migration after lung allotransplantation in swine J. Thorac. Cardiovasc. Surg., July 1, 1998; 116(1): 90 - 94. [Abstract] [Full Text]
|
|
|
|
|
|
A. Halldorsson, M. Kronon, B. S. Allen, K. S. Bolling, T. Wang, S. Rahman, H. Feinberg, and R. S. Hartz Controlled Reperfusion After Lung Ischemia: Implications For Improved Function After Lung Transplantation J. Thorac. Cardiovasc. Surg., February 1, 1998; 115(2): 415 - 425. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
