Ischaemia-reperfusion injury is encountered during a variety of medical and surgical procedures . Neutrophils are the effectors of tissue alterations such as vascular damage and increased vascular permeability associated with ischaemia-reperfusion injury [1-3]. Increased adherence of circulating neutrophils to the vascular endothelium is an essential early event in the initiation of an inflammatory response after a period of ischaemia-reperfusion . Hypoxia-reoxygenation promotes neutrophil-endothelial interactions by upregulating expression of neutrophil and endothelial adhesion molecules including CD11b, CD18  and ICAM-1 . IL-1β is a pro-inflammatory cytokine that plays a central role in the endothelial response to hypoxia-reperfusion .
Local anaesthetic agents are widely used in the perioperative period. Lidocaine has demonstrated beneficial effects in animal models of ischaemia-reperfusion injury [8,9]. Potential mechanisms include anti-inflammatory  and antioxidant  actions. The effect of lidocaine on neutrophil and endothelial adhesion molecule expression, and on endothelial IL-1β induced by hypoxia-reoxygenation are not known.
With institutional ethical approval, and having obtained written informed consent, 40 mL of venous blood was withdrawn from five healthy adult volunteers. Exclusion criteria were major surgery within the past 6 months, concurrent medication and recent (1 month) infection. Freshly discarded human umbilical cords were obtained from the delivery suite of the Erinville Hospital, Cork.
Assessment of neutrophil activation
Neutrophils from healthy volunteers (n = 5) were isolated by sequential sedimentation in 6% Dextran (mol. wt. 520 000 Da, Sigma, UK) in 0.9% sodium chloride for 45 min at 22°C, centrifugation in Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) at 300g for 30 min to pellet granulocytes and remaining erythrocytes, and centrifugation of the resuspended pellet over an 81% isotonic Percoll (Sigma, UK) gradient at 350 g for 15 min to pellet erythrocytes. The diffuse layer at the interface containing neutrophils then was harvested, washed, resuspended in medium and counted, and the cells were kept at 0°C for at least 30 min before use. Viability of cells by trypan blue exclusion was >98%.
Preparations of isolated neutrophils were maintained in RPMI 1640 supplemented with 10% fetal calf serum, and 2 mmol L-glutamine, 100 μg mL−1 streptomycin, and 2.5 μg mL−1 amphotericin B at a concentration of 1 × 106 cells mL−1 in ultra low attachment bottle (Falcon, Becton Dickinson, CA, USA) at 37°C. After isolation, volunteers' neutrophils were pretreated with lidocaine at a final concentration of 0.005, 0.05 or 0.5 mg mL−1, or an equal volume of RPMI 1640 culture medium for 60 min. Lidocaine 0.005 mg mL−1 is a concentration that has previously been used in in vitro studies to represent a therapeutic concentration .
Group assignments were as follows:
• Group A: No lidocaine, no hypoxia-reoxygenation (normal control).
• Group B: Lidocaine, no hypoxia-reoxygenation (lidocaine control).
• Group C: No lidocaine, hypoxia-reoxygenation (experimental control).
• Group D: Lidocaine, hypoxia-reoxygenation (experimental group).
Human neutrophils were subjected to hypoxic conditions as described previously  by suspending the cells in Hanks Balanced Salt Solution, which was purged with N2 gas for 30 min and the partial pressure of O2 (PO2) then measured to assess the O2 depletion. PO2 and pH were measured in the cells suspended in Hanks Balanced Salt Solution before (25.8-34.9 kPa) and after hypoxia (9.88-10.9 kPa) and reoxygenation using a blood-gas analyser (Eschweiler System C 2000, Keil, Germany). Neutrophils (Groups C and D) were incubated in the O2-depleted solution for 90 min at 37°C in a modular incubator continuously purged with an anoxic gas mixture (93% N2: 5% CO2: 2% O2) and PO2 was measured at the end of the incubation time. Neutrophils were reoxygenated by suspending the cells in normoxic solution after centrifugation, and PO2 was again measured. Groups A and B were exposed to room air at 37°C for the study period. Viability of the cells was measured at the end of the experiment by a trypan blue exclusion test, and was unchanged after hypoxia and hypoxia-reoxygenation.
Following hypoxia-reoxygenation, neutrophils were incubated with 10 μL of PE-conjugated anti-leu 15 (anti-CR3/CD11b) mouse anti-human antibody (Becton Dickinson, CA, USA) for 20 min, and 10 μL of fluorescein-isothiocyanate-conjugated anti-CR3/CD18 mouse anti-human antibody (Becton Dickinson, CA, USA) for 20 min. Following centrifugation at 250 g for 5 min, the cell pellets were washed.
Due to considerable variation of CD11b/CD18 expression depending on the neutrophil donor, the data were normalized to the respective basal value (before incubation with conditioned medium), and expressed as percent increase. CD11b and CD18 expression were analysed on a Fluorescence-Activated Cell-sorter Scanner (FACScan cytofluorometer, Becton Dickinson, CA, USA). The mean channel fluorescence (MCF) intensity of stained cells was detected on the basis of a minimum number of 5000 cells collected analysed using the FACScan Research Software version B.
Assessment of endothelial cell activation
Human umbilical vein endothelial cells (HUVECs) from fresh placental cords were isolated by previously described methods  and grown until confluence at 37°C in humidified 5% CO2. The growth medium consisted of complete Medium 199 supplemented with 20% fetal calf serum, penicillin (100 U mL−1), streptomycin sulphate (100 μg mL−1), fungizone (0.25 μg mL−1), heparin (16 U mL−1), endothelial cell growth supplement (75 μg mL−1) and glutamine (2 mmol L−1). In all experiments reported herein, HUVECs were used as individual isolates between passages 3 and 5. At confluence, HUVECs were detached from the culture flask by trypsinization using 0.05% trypsin/0.02% ethylenediamine tetra-acetic acid and seed out on 24-well culture plate (Costar, Cambridge, MA, USA). Confluent endothelial monolayers with tight cell conjunctions were formed after 30 h at 37°C in humidified 5% CO2 in culture.
Sub-confluent monolayers of HUVEC were pretreated with different concentrations of lidocaine (0.005, 0.05 and 0.5 mg mL−1) or with equal volumes of Medium 199 for 60 min. Endothelial cell monolayers were exposed to hypoxia for 24 h in a modular incubator chamber (Billups-Rothenberg) flushed with a gas mixture (1% O2: 5% CO2: 94% N2) to purge it of atmospheric air. Reoxygenation was induced by exposing cells to ambient air in normoxic culture incubator for another 24h, and the supernatants and cells were collected.
One hundred microlitres of stimulated endothelial cell suspension (1 × 106 cells mL−1) were stained with 10 μL of fluorescein-isothiocyanate-conjugated anti-CD54 (anti-ICAM-1) mouse anti-human antibody or 10 μL of fluorescein-isothiocyanate-conjugated isotype IgG1 control mouse anti-human antibody and incubated for 30 min at 4°C. ICAM-1 expression on endothelial cells was analysed on a FACScan flow cytometer. The MCF intensity of stained cells was detected on the basis of a minimum number of 5000 cells collected and analysed using the software Lysis II as well. Endothelial supernatant IL-1β concentrations were determined in cell media using enzyme-linked immunosorbent assays (Quantikine R&D Systems Europe Ltd., Abingdon, Oxon, UK) according to manufacturer instructions. Concentrations were estimated at the end of hypoxia-reoxygenation. The sensitivity for IL-1β was 1 pg mL−1. The inter- and intra-assay precisions for IL-1β for the range of values obtained in this study were 4.1-8.4% and 2.8-5.4%.
Statistical analysis. The Sigma Stat 2.0 for Windows (SPSS, Inc., Chicago, IL, USA) software package was used for all statistical analyses. The data obtained were analysed using repeated measures analysis of variance (ANOVA, and post hoc Student-Newman-Keuls or unpaired t-test as appropriate. A P value of <0.05 was considered significant. Data are presented as mean ± SD.
Exposure to 90 min hypoxia followed by 15 min of reoxygenation produced upregulation of neutrophil CD11b (94.33 ± 40.65 MCF vs. 34.32 ± 6.83 MCF, P = 0.02) (Fig. 1) and CD18 (109.84 ± 35.44 MCF vs. 59.05 ± 6.71 MCF, P = 0.03) (Fig. 2) expression compared to normoxia. Neutrophil CD11b and CD18 expression was similar in Group A (normal control) and Group B (lidocaine control) at 15 min reoxygenation (lidocaine 0.5 mg mL−1, CD11b [34.32 ± 6.83 MCF vs. 28.12 ± 6.29 MCF, P = 0.9] and CD18 [59.05 ± 6.71 MCF vs. 53.36 ± 7.51 MCF, P = 0.9], respectively). This result rules out a direct drug effect of lidocaine. Treatment of neutrophils with lidocaine (0.5 mg mL−1 but not 0.005 and 0.05 mg mL−1) diminished increase in CD11b (lidocaine 0.5 mg mL−1, 40.52 ± 13.19 MCF; lidocaine 0.05 mg mL−1, 57.53 ± 10.65 MCF; lidocaine 0.005 mg mL−1, 65.60 ± 14.65 MCF) compared to Group C (experimental control) (94.32 ± 40.65 MCF, lidocaine 0.5 mg mL−1, P = 0.04; lidocaine 0.05 mg mL−1, P = 0.07; lidocaine 0.5 mg mL−1, P = 0.1) (Fig. 1). Treatment of neutrophils with lidocaine (0.005, 0.05 and 0.5 mg mL−1) decreased CD18 expression (lidocaine 0.005 mg mL−1, 71.07 ± 10.14 MCF; lidocaine 0.05 mg mL−1, 65.79 ± 16.03 MCF; lidocaine 0.5 mg mL−1, 59.16 ± 20.98 MCF) compared to control (109.84 ± 35.44 MCF, lidocaine 0.005 mg mL−1, P = 0.03; lidocaine 0.05 mg mL−1, P = 0.01; lidocaine 0.5 mg mL−1, P = 0.01) (Fig. 2). ICAM-1 expression. Hypoxia and reoxygenation resulted in increased HUVEC ICAM-1 expression (146.62 ± 16.78 MCF vs. 47.29 ± 9.85 MCF, P < 0.001) (Fig. 3) compared to normoxia. Endothelial cell ICAM-1 expression was similar in Group A (normoxia) and Group B (lidocaine control) at 4 h (lidocaine 0.5 mg mL−1, 47.29 ± 9.85 MCF vs. 34.76 ± 5.87 MCF, P = 0.7). This result rules out a direct drug effect of lidocaine. Treatment of HUVECs with lidocaine (0.005, 0.05 and 0.5 mg mL−1) decreased ICAM-1 expression (lidocaine 0.005 mg mL−1, 133.25 ± 16.05 MCF; lidocaine 0.05 mg mL−1, 106.33 ± 14.92 MCF; lidocaine 0.5 mg mL−1, 73.23 ± 12.57) compared to control (146.62 ± 16.78 MCF, lidocaine 0.005 mg mL−1, P = 0.03; lidocaine 0.05 mg mL−1, P < 0.001; lidocaine 0.5 mg mL−1, P < 0.001) (Fig. 3).
IL-1β concentrations. Hypoxia-reoxygenation increased HUVEC supernatant IL-1β concentrations (3.41 ± 0.36 vs. 2.65 ± 0.21 pg mL−1, P = 0.02) compared to normoxia (Fig. 4). Endothelial supernatant IL-1β concentrations in lidocaine (0.005 and 0.05 mg mL−1) treated HUVECs were similar (lidocaine 0.005 mg mL−1, 2.96 ± 0.57 pg mL−1; lidocaine 0.05 mg mL−1, 2.85 ± 0.58 pg mL−1) to control (3.40 ± 0.36, lidocaine 0.005 mg mL−1, P = 0.4; lidocaine 0.05 mg mL−1, P = 0.1) (Fig. 4). Treatment of HUVECs with lidocaine (0.5 mg mL−1) decreased supernatant IL-1β concentration (3.41 ± 0.36 vs. 2.25 ± 0.21 pg mL−1, P = 0.04) compared to control (Fig. 4).
Lidocaine diminished increased neutrophil CD11b/CD18, endothelial ICAM-1 expression and endothelial supernatant IL-1β concentrations associated with hypoxia-reoxygenation. The effect of lidocaine treatment was concentration dependent. Lidocaine at clinically relevant concentrations (0.005 mg mL−1) decreased neutrophil CD18 and endothelial ICAM-1 expression but not endothelial IL-1β concentrations.
Neutrophils and endothelial cells have important roles in ischaemia and reperfusion injury [1-3]. Neutrophils release free radicals and enzymes that damage the endothelial membrane . The endothelium in turn when activated releases cytokines that attract and facilitate neutrophil migration [2,5]. Therapeutic strategies that inhibit neutrophil and endothelium responses to ischaemia-reperfusion ameliorate tissue injury . The complexity of the clinical situation is difficult to replicate in vitro. Models of ischaemia-reperfusion have been described that replicate neutrophil  and endothelial responses [6,7] to ischaemia-reperfusion.
Hypoxia-reoxygenation promotes neutrophil-endothelial interactions by upregulating expression of neutrophil and endothelial adhesion molecules including CD11b, CD18  and ICAM-1 . IL-1β is a pro-inflammatory cytokine that plays a central role in the endothelial response to hypoxia-reperfusion . Tumour necrosis factor-α concentrations are also increased during ischaemia-reperfusion injury . It results in increased endothelial cell production of IL-6, IL-8 and IL-1β  and induces the expression of several adhesion molecules , which participate in neutrophil-endothelial cell interactions that occur at sites of inflammation and ischaemia-reperfusion . Our in vitro models of hypoxia-reoxygenation resulted in increased expression of neutrophil CD11b/CD18 and endothelial cell ICAM-1 adhesion molecules, and endothelial supernatant IL-1β concentrations. This is consistent with the findings of Scannell and colleagues (neutrophil CD11b)  and Mataki and colleagues (endothelial cell ICAM-1) .
Hypoxia results in increased polymorphonuclear (PMN)  and endothelial  adhesion molecule expression. The mechanism of activation cannot be postulated from this study but sustained increased intracellular calcium concentrations [18,19], and inhibition of mitochondrial ATP production by hypoxia has been reported .
Ischaemia and reperfusion is an important event in the perioperative period . A therapeutic role for local anaesthetics to limit ischaemia-reperfusion injury in the perioperative period has been suggested . Lidocaine has demonstrated protective effects in animal models of ischaemia-reperfusion injury [8,9]. Lidocaine intravenous (i.v.) infusion (70 μg kg−1 min−1) commenced 90 min prior to ischaemia decreased myocardial infarct size by 30% after a 90-min ischaemic insult in a canine model . Lidocaine (10 mg kg−1 i.v.) administered 10 min before ischaemia, not only prevented the increase of malondialdehyde concentrations during ischaemia, but also resulted in a significant transient decrease 10 min after the start of reperfusion in a canine cerebral ischaemic model .
The mechanism by which lidocaine inhibits neutrophil adherence remains unclear. Lidocaine in vitro inhibits upregulation of CD18 and superoxide release from human neutrophil on stimulation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) . The authors suggest that lidocaine may interfere with the post-receptor signal transduction pathway. Lidocaine may alter the expression of membrane effector molecules because local anaesthetic-treated neutrophils undergo dramatic morphologic changes: pseudopod formation ceases, ruffling of the membrane is reduced and the cells take on a round, smooth configuration . The present study confirms inhibitory and concentration-dependent effects of lidocaine, similar to its anti-inflammatory  and antioxidant effects . Lidocaine decreases neutrophil respiratory burst in a dose-dependent manner . Mikawa and colleagues  have shown that lidocaine and mepivacaine concentrations (100-fold higher than clinically relevant ones) decreased reactive O2 species production by human neutrophils. Lidocaine at clinically relevant plasma concentrations (0.005 mg mL−1)  diminished increased neutrophil CD18 and endothelial ICAM-1 expression, but did not diminish increased neutrophil CD11b expression and endothelial supernatant IL-1β concentrations. Lidocaine at higher concentrations (0.5 mg mL−1) diminished increased neutrophil CD11b expression and supernatant IL-1β concentrations. This increased concentration (0.5 mg mL−1) may be achieved in tissues at or near the site of injection of local anaesthetic .
Study limitations include the use of in vitro experiments using maximal stimulation and absence of determination of the mechanism of effect. Extrapolation of in vitro studies to complex clinical scenarios needs to be done with extreme caution. In this study we focused on a limited set of adhesion molecules and cytokines, and demonstrated a selective inhibition of only some of these adhesion molecules and cytokines by lidocaine at therapeutically relevant concentrations. Complement, immune complexes and natural antibodies to a cellular neoantigen play a critical role in ischaemia-reperfusion injury . The effect of lidocaine on neutrophil-independent ischaemia-reperfusion injury was not examined in this study.
In conclusion, we have demonstrated that lidocaine (at clinically relevant concentrations) diminishes increased neutrophil CD18 and endothelial ICAM-1 expression associated with hypoxia-reoxygenation. These findings would suggest further in-depth studies of the protective effects of lidocaine in ischaemia-reperfusion injury are warranted.
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