Ischemia-reperfusion injury (IRI) is one of the most critical phenomena in organ transplantation [
We hypothesize that the activation of cellular and molecular mediators in lung IRI could manifest in very early stages of reperfusion through histological and immunohistochemical changes. Furthermore, possible beneficial effects of intravenous lidocaine could be evidenced in the modification of these changes. The aim of this study is to evaluate the histological and histopathological immunohistochemical changes in the early stages of reperfusion and the possible differences that can occur when administering intravenous lidocaine during a surgical procedure.
The study was carried out at the Animal Facility and Experimental Surgery Unit of Gregorio Marañón University Hospital in Madrid (Spain). Ethical approval for this study (Ethical Committee N° 64/10) was provided by the Animal Research and Experimentation Committee of the institution (Chairperson Doctor Fernando Asensio) on 12 January 2010. A total of 18 Large-White pigs, either sex, were equally divided into three groups. In the control group (CON), an orthotopic pulmonary autotransplantation was performed by means of left pneumonectomy, lobectomy ex situ, reimplantation of the caudal lobe, and reperfusion for 60 minutes. The same protocol was performed on the lidocaine group (LIDO), but an intravenous bolus of lidocaine (Braun Medical SA, Barcelona, Spain) was also administered at 1.5 mg/kg after induction, followed by continuous infusion at 1.5 mg/kg/h.
The third group was the simulated surgery or the SHAM group, in which thoracotomy was performed with bipulmonary ventilation at all times. During the procedure, different hemodynamic parameters and arterial blood studies were analyzed. After 60 minutes of reperfusion of the transplanted lobe, pulmonary biopsies were performed for histological and immunohistochemical studies.
The animals were subjected to 18 hours of fasting for solids but were able to drink water until up to 20 minutes before the intervention. At 20 minutes before the induction, they were premedicated with 20 mg/kg of intramuscular ketamine (Ketalar, Parker Davis, Hameln, Germany) and 0.04 mg/kg of intramuscular atropine (Braun Medical, Barcelona, Spain). Subsequently, a vein in the ear was canalized (Introcan Safety, Braun, Germany, number 22), and the animal was monitored with continuous electrocardiogram, pulse oximetry, and an Ohmeda capnography 5250 RGM (General Electric Health Care, United States). A Servo Ventilator 900 C (Siemens), a blood gas analyzer GEM Premiere 5000, and a Pulse Index Continuous Cardiac Output (PICCO) device (Edwards, Irving, California, United States) were used. Anesthesia was induced with 2.5
Each animal was connected to controlled mechanical ventilation through a ventilator. An inspired fraction of oxygen of 60% and tidal volume of 8-10 ml/kg were administered to maintain normocapnia (end-tidal CO2 of 35-40 mmHg). Anesthesia was maintained with fentanyl at a dose of 2.5
After these procedures, the animals were placed in right lateral decubitus. A left thoracotomy was performed through the fourth intercostal space with a lower costoctomy, and left pneumonectomy was carried out. When sectioning the left main bronchus, unipulmonary ventilation was initiated with the orotracheal tube progressing to the right main bronchus under control by fiberoptic bronchoscopy. Pulmonary protection strategies were initiated, and the tidal volume was decreased to 6 ml/kg.
Once the left bronchus was sectioned, the pulmonary artery and the caudal pulmonary vein were occluded with forceps, and the cranial pulmonary vein was ligated. The left pulmonary artery was occluded with a protected clamp near the bifurcation of the main pulmonary artery and sectioned distally, leaving a margin of 5 to 10 mm to allow for the arterial anastomosis to be performed in the reimplantation. The cranial pulmonary vein was ligated near the atrium and sectioned. To complete the pneumonectomy, the pulmonary vein of the caudal lobe was clamped near the mouth of the mediastinal lobe vein, sectioned one or two millimeters from the clamp, and sutured with a 6/0 polypropylene continuous stitch. Systemic heparinization with 300 IU/kg (Mayne Pharma Spain) was performed using a bolus prior to the occlusion of the pulmonary artery.
Next, an ex situ cranial lobectomy was performed. The graft was antegradely and retrogradely perfused with a solution from the University of Wisconsin at 10-15°C while ventilating simultaneously with an Ambu bag, at 12 breaths per minute, with ambient air.
Subsequently, the caudal lobe was reimplanted by bronchial-bronchial, arterio-arterial, and veno-auricular anastomoses. The tube was withdrawn into the trachea to allow ventilation of the implant, and reperfusion was started. The reperfusion was maintained for 60 minutes, after which the animal was euthanized by deepening anesthesia and cardioplegia with intravenous potassium chloride.
Hemodynamic and arterial blood gas studies were carried out at three points: baseline (30 minutes after the anesthetic induction, just before the thoracotomy and with the animal already hemodynamically stabilized and in bipulmonary ventilation), prereperfusion (before reperfusion and ventilation of the left caudal lobe after it has already been reimplanted), and postreperfusion (60 minutes after reperfusion of the left caudal lobe). After 60 minutes of reperfusion, histological samples were collected by lung biopsy of the implanted lobe. Biopsies were preserved in formaldehyde.
For arterial blood gas studies, partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2), and blood pH values were measured from blood samples obtained from the femoral artery. For hemodynamic studies, the mean arterial pressure, heart rate, stroke volume variation, and cardiac output were evaluated using the PICCO system.
To perform histological studies, the samples were fixed in a 10% buffered formaldehyde solution for 24 hours. They were then included in paraffin blocks for subsequent sectioning in 4
In samples indicating the presence of inflammatory cells, the presence or absence of cells of the monocyte-macrophage system, neutrophils, or lymphocytes was described. Congestion was defined as the presence of alveolar capillaries or small vessels that were dilated and filled with red blood cells. Edema was defined as thickening of the alveolar or interlobar septa. The three parameters were classified with a quasi-quantitative scale of 0 to 3 (0 absence, 1 scarce presence, 2 moderate presence, and 3 abundant presence) [
On the other hand, the expression of markers of inflammation and apoptosis in the histological preparations was determined through immunohistochemical detection. Monoclonal and polyclonal antibodies (Ab) were used against CD68 (1 : 100, MBS370038, MyBioSource), which is an antigen expressed mainly in cells of the monocyte-macrophage system; monocyte chemoattractant protein-1 (MCP-1) (1 : 100, MBS2027425, MyBioSource), which is involved in the inflammatory response; Bcl-2 (1 : 100, 8C8, Novus Biologicals), an antiapoptotic protein; and caspase-9 (1 : 100, orb1677, Biorbyt), a proapoptotic protein. Tissue sections were incubated with hydrogen peroxide after rehydration to water. Then, they were incubated with the serum of the animal in which the secondary antibody was raised in and triton-buffer (Tritón-X100, Sigma-Aldrich). Afterwards, biopsies were incubated with the primary antibody for 24 hours, at 4°C, in the dark. Finally, they were washed in phosphate-buffered saline (0.1 M, pH 7.4) and incubated with biotin conjugated secondary antibody (1 : 200, 1 hour, room temperature, in the dark). All samples were processed, fixed, stained, and imaged in parallel, under the same conditions.
A total of 10 microphotographs were taken at random at 40 magnifications from these immunohistological preparations. All photographs were taken with a Leica DMRB photomicroscope and a Nikon DS-Fi1 camera and cataloged by two observers who were blinded to the treatment group to which the samples belonged.
The 10 images were obtained by starting from the central zone of the histological preparation and moving in a clockwise direction. Each image was used to carry out a descriptive study to detail the type of cell that was positive for any of the antibodies tested in the immunohistochemistry study, the location of the staining (cytoplasmic, nuclear, or mixed), and its intensity in a quasi-quantitative study. In the case of CD68, MCP-1, and Bcl-2 expression studies, two independent observers who were unaware of the origin of the sample counted the numbers of cells marked. If there was a discrepancy, the photo was reviewed, and an agreement was reached. Then, the average of the 10 values was assigned to the sample. Given the broad expression of caspase-9 in some stains, it was measured with a semiquantitative scale (0 absence of staining,
A database was created using the program IBM SPSS Statistics 24 for Mac. The qualitative and quasi-quantitative variables are expressed as a frequency (percentage). The quantitative ones are presented as mean (typical error) when they follow a normal distribution. The quasi-quantitative variables were treated as qualitative variables and analyzed using a chi-squared test to detect differences between the study groups. The results are expressed in a contingency table. The Kruskal-Wallis test was used to identify significant differences in the quantitative variables between groups. Subsequently, the Mann–Whitney test was used to analyze the pairs of specific samples and to find significant differences. Statistical significance was established with
In the analysis of the general variables, no statistically significant differences were observed between any of the groups with respect to the weight of the animals (34 (1) CON vs. 39 (2) LIDO vs. 37 (1) kg SHAM,
Hemodynamic and blood test results.
Group | BAS | PRER | POSR60 | |
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MAP (mmHg) | CON |
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HR (bpm) | CON |
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SVV (%) | CON |
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PaO2 (mmHg) | CON |
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PaCO2 (mmHg) | CON |
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pH | CON |
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Results are presented as
No statistically significant differences were found between the three groups in the comparison of hemodynamic values and the arterial blood gas analysis.
Histopathological assessment of inflammation in ischemia-reperfusion lung injury by hematoxylin-eosin staining. Samples were collected 60 minutes after reperfusion.
Group | Degree | Inflammation ( |
Infiltration | ||||||
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MM ( |
Lymphocytes | Neutrophils | |||||||
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CON | Absence | 1 | 16.7% | 1 | 16.7% | 2 | 33.3% | 6 | 100% |
Scarce | 4 | 66.7% | 5 | 83.3% | 3 | 50% | 0 | 0% | |
Moderate | 1 | 16.7% | 0 | 0% | 1 | 16.7% | 0 | 0% | |
Abundant | 0 | 0% | 0 | 0% | 0 | 0% | 0 | 0% | |
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LIDO | Absence | 5 | 83.3% | 5 | 83.3% | 5 | 83.3% | 6 | 100% |
Scarce | 1 | 16.7% | 1 | 16.7% | 1 | 16.7% | 0 | 0% | |
Moderate | 0 | 0% | 0 | 0% | 0 | 0% | 0 | 0% | |
Abundant | 0 | 0% | 0 | 0% | 0 | 0% | 0 | 0% | |
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SHAM | Absence | 5 | 83.3% | 6 | 100% | 5 | 83.3% | 6 | 100% |
Scarce | 1 | 16.7% | 0 | 0% | 1 | 16.7% | 0 | 0% | |
Moderate | 0 | 0% | 0 | 0% | 0 | 0% | 0 | 0% | |
Abundant | 0 | 0% | 0 | 0% | 0 | 0% | 0 | 0% |
MM: monocyte-macrophage system;
Histopathological assessment of edema and congestion in ischemia-reperfusion lung injury by hematoxylin-eosin staining. Samples were collected 60 minutes after reperfusion.
Group | Degree | Edema | Congestion | ||
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CON | Absence | 0 | 0% | 1 | 16.7% |
Scarce | 5 | 83.3% | 5 | 83.3% | |
Moderate | 1 | 16.7% | 0 | 0% | |
Abundant | 0 | 0% | 0 | 0% | |
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LIDO | Absence | 3 | 50% | 4 | 66.7% |
Scarce | 3 | 50% | 1 | 16.7% | |
Moderate | 0 | 0% | 1 | 16.7% | |
Abundant | 0 | 0% | 0 | 0% | |
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SHAM | Absence | 3 | 50% | 6 | 100% |
Scarce | 3 | 50% | 0 | 0% | |
Moderate | 0 | 0% | 0 | 0% | |
Abundant | 0 | 0% | 0 | 0% |
Immunohistochemical detection of CD68, MCP-1, Bcl-2, and caspase-9 in lung biopsies. Samples were collected 60 minutes after reperfusion. Area of peripheral lung parenchyma consisting mostly of well-preserved alveolar wall. Strong nuclear or cytoplasmic staining macrophages or pneumocytes are identified. Images captured with 40x magnification objective (the mark scale corresponds to 30
Quantitative analysis of CD68, MCP-1, and Bcl-2 positive cells. Samples were collected 60 minutes after reperfusion. The results are expressed as the average of the number of positive cells found in 10 images of 0.35 mm2. CON: control group; LIDO: group treated with lidocaine; SHAM: simulated surgery group. (a)
Quasi-quantitative analysis of caspase-9 positive cells. Samples were collected 60 minutes after reperfusion. The results are expressed as the frequency of cases in each group. No significant differences were observed in the caspase-9 expression among groups (
In our experimental model of pulmonary autotransplantation, lidocaine attenuated the degree of inflammation of the transplanted lung 60 minutes after reperfusion. Furthermore, it decreased the degree of infiltration by monocyte-macrophage cells, which was observed microscopically in hematoxylin-eosin staining. Although the antiarrhythmic or anesthetic effects of lidocaine are the best known, different experimental and clinical studies have shown its ability to scavenge oxygen radicals and superoxides and inhibit the function of granulocytes [
Different biomarkers have been used to study induced lung damage inflammation [
In a model of one-lung IRI in mice, Altemeier et al. [
Similarly, Sasagawa et al. [
The present results complement previously published results, in which intravenous lidocaine in an animal lobectomy model showed a decreased expression of inflammatory and proapoptotic mediators [
Microvascular dysfunction mediates many of the local and systemic phenomena of IRI damage and especially affects arterioles, capillaries, and venules. In postcapillary venules, the recruitment and transmigration of leukocytes also compromise the integrity of the endothelial barrier and increase oxidative phenomena, thus producing tissue edema [
Pulmonary edema is considered the greatest exponent of tissue damage produced by IRI and is not evident in the early phases of ischemia-reperfusion, which were analyzed in the present study. For this reason, we consider this phenomenon to be mainly due to the early time at which the samples were obtained, despite not seeing differences between the degrees of congestion and edema of the lungs treated with lidocaine compared to those that were not treated.
The final result of IRI in the lung is a form of combined cell death, which is characterized by the simultaneous occurrence of necrosis and apoptosis [
In general, apoptosis induced in cells in culture usually shows signs of apoptosis much earlier (between 5 and 10 hours) than cells in tissues (between 11 and 14 days) [
Lidocaine plays a controversial role in apoptosis. Some suggest that the drug inhibits it, while others report that it induces it. Other studies report lidocaine effectively promotes apoptosis but only in specific cell lines, such as tumor cells rather than healthy tissues [
It can be assumed that the plasma concentrations used in our experiment would be around 5
In our experimental model of lung transplantation, intravenous lidocaine was associated with an attenuation of the histological markers of lung damage in the early stages of reperfusion. Our current findings suggest that intravenous lidocaine can decrease the inflammatory and apoptotic response in lung IRI, although the biological pathway remains unclear. Further clinical trials are needed in order to acknowledge its effects on ischemia-reperfusion injury in humans. Although challenging, additional studies, with longer reperfusion times and deeper histological assessment, are also warranted.
Data supporting the findings of this study are available from the authors upon request.
These data were presented as an abstract at the American Society of Anesthesiologists’ (ASA) Annual Conference in 2018.
The authors declare that they have no conflicts of interest.
We would like to thank Encarnación Muñoz, laboratory technician, for her constructive suggestions, her awareness of immunohistochemical techniques, and her commitment in assisting this research. This work was funded through the grant ISCIII (13/00700 and 13/00002), cofunded by the ERDF/ESF, “Investing in your future,” the Fundación Mutua Madrileña (FMM 14/08), and the UCM-BS (GR/14, 920210) program.