Extremity surgery performed using a limb tourniquet results in skeletal muscle ischaemia and reperfusion (I/R) injury , with local and systemic proinflammatory responses, which can adversely affect patient outcome . In this setting, there is neutrophil activation with increased adhesion molecule expression (CD11b/CD18) , decreased antioxidant status  and increased transendothelial migration .
I/R organ injury is not entirely neutrophil dependent . Paterson and colleagues have shown that frozen stored plasma obtained during tourniquet I/R contains a stable humoral element capable of initiating neutrophil activation, chemotaxis and de novo endothelial cell activation . Endothelial cell damage, in remote organs such as the kidney and myocardium, is in part neutrophil independent and directly mediated by plasma factors . Barry and colleagues  demonstrated that plasma factors augment neutrophil and endothelial cell activation during aortic surgery. Such activation has not been demonstrated after more minor perioperative I/R injuries.
Local anaesthetic agents have demonstrated beneficial effects in animal models of I/R [8,9]. Potential therapeutic mechanisms include anti-inflammatory  and antioxidant actions .
The aim of this study was to examine the effects of lidocaine (in vitro) on adhesion molecule expression associated with tourniquet-induced neutrophil-independent I/R in elective upper limb surgery.
With institutional ethics approval, and written informed consent, seven adult American Society of Anesthesiologists (ASA) Grade I-II patients, undergoing elective upper limb surgery requiring tourniquet application, were studied (Table 1). Anaesthesia was induced with intravenous (i.v.) propofol 2.5 mg kg−1 and fentanyl 1.5 μg kg−1, and maintained with nitrous oxide/oxygen (60% : 40%) and sevoflurane (<2.5%). Venous blood samples (5 mL each, in lithium heparin bottles; Greiner bio-one, Kremsmunster, Austria) were obtained from a dedicated venous cannula (Insyte, Becton Dickinson, UT, USA) in the cubital fossa of the surgical limb, immediately prior to induction of anaesthesia (baseline) and immediately prior to tourniquet application and inflation. A tourniquet (Tourniquet 2500, Medizintechnik, Sulz am Neckar, Germany) was subsequently applied to the upper arm at a pressure of 300 mmHg. At the end of surgery and immediately before release of tourniquet, a third blood sample was obtained. Blood samples were possible at this time despite inflated tourniquet and prior exsanguination. Blood samples were also obtained 15 min, 2 and 24 h following tourniquet release.
Venous blood samples were collected from healthy adult volunteers (n = 7) (age: 30-45, male/female ratio: 4 : 3) to represent non-I/R plasma and to act as a source of isolated neutrophils.
Blood samples (5 mL each, from patients or volunteers) were drawn into lithium heparin bottles (Greiner bio-one, Kremsmunster, Austria), immediately placed on ice and processed within 2 h of collection. After centrifugation at 2500 rpm for 15 min, plasma samples of patients and volunteers were aliquoted and stored at −80°C until use.
Assessment of neutrophil activation
Human neutrophils from healthy adult volunteers (n = 7) were isolated by sequential sedimentation in 6% dextran (molecular weight: 520 000, Sigma, UK) in 0.9% sodium chloride for 40 min at 22°C, centrifugation in Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) at 300 g 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 was then harvested, washed, resuspended in Roswell Park Memorial Institute 1640 culture medium and counted. This method yielded the purity of neutrophils >97%, as determined by Rapi-DiffII (DiaCheM, Lancanshire, England) staining of cytocentrifuged samples, and viability of cells by trypan blue exclusion was >98%.
Incubation of neutrophils with lidocaine and plasma. After isolation, volunteers' neutrophils were pretreated with lidocaine at final concentration of 0.005, 0.05 or 0.5 mg mL−1 or an equal volume of culture medium for 60 min. Lidocaine 0.005 mg mL−1 is a concentration that has been used in in vitro studies to represent a therapeutic concentration . After pretreatment, 300 μL aliquots of isolated neutrophils at a final concentration of 1 × 106 cells mL−1 were incubated for 2 h with 100 μL of I/R plasma obtained from patients (n = 7). Plasma (100 μL) obtained from healthy volunteers (n = 7) served as controls for this experiment (control plasma). N-formylmethionylleuyl-phenylalanine (FMLP) (10−3 M) was used as a positive control for these experiments.
Neutrophil CD11/CD18 expression. Following incubation with control plasma, I/R plasma or FMLP, isolated neutrophils were incubated with 10 μL of R-phycoerythrin-conjugated anti-leu 15 (anti-CR3/CD11b) mouse anti-human mAb (Becton Dickinson, CA, USA) for 20 min, and 10 μL of fluorescence isothiocyanate (FITC)-conjugated anti-CR3/CD18 mouse anti-human mAb (Becton Dickinson, CA, USA) for 20 min at 4°C. Following centrifugation at 250 g for 5 min, the cell pellets were washed. CD11b and CD18 receptors 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 software Lysis II.
Assessment of endothelial cell activation
Endothelial cell isolates and cultures. 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), amphotericin (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 plates (Costar, Cambridge, MA). Confluent endothelial monolayers with tight cell conjunctions were formed after 30 h at 37°C in humidified 5% CO2 in culture.
Incubation of endothelial cells with lidocaine and plasma. Sub-confluent monolayers of HUVEC were incubated with lidocaine at final concentrations of 0.005, 0.05 or 0.5 mg mL−1 or an equal volume of culture medium for 60 min. After pre-treatment, HUVECs were incubated for 4 h in a humidified 5% CO2 incubator with 200 μL of I/R plasma obtained from patients, control plasma from healthy volunteers or 200 μL of recombinant human tumour necrosis factor (TNF)-α (2.5 ng mL−1) as a positive control for these experiments.
ICAM-1 expression. After exposure to plasma, 100 μL of stimulated endothelial cell suspension (1 × 106 cells mL−1) was stained with 10 μL of FITC-conjugated anti-CD54 (anti-intercellular adhesion molecule-1 (anti-ICAM-1)) mouse anti-human mAb (Becton Dickinson, CA, USA) or 10 μL of FITC-conjugated isotype IgG1 control mAb (Becton Dickinson, CA, USA) and incubated for 30 min at 4°C. ICAM-1 expression on endothelial cells was analysed on an FACScan cytofluorometer (Becton Dickinson, CA, USA). The MCF intensity of stained cells was detected on the basis of a minimum number of 5000 cells collected, analysed using the software Lysis II.
Statistical analysis. The Sigma Stat 2.0 for windows (SPSS, Inc., IL, USA) software package was used for all statistical analysis. Data are expressed as percentile intensity of fluorescence compared with time 0 (mean ± SD). Response of isolated neutrophils to patient (baseline) or normal volunteer plasma in terms of adhesion molecule expression is expressed as actual values. Data was normally distributed. Differences in adhesion molecule expression between volunteers and patients were analysed with unpaired, twotailed, t-tests. Differences between lidocaine and control patient groups were analysed by ANOVA and post hoc Student-Newman-Keuls test as appropriate. P < 0.05 was considered significant.
Incubation of isolated neutrophils with FMLP resulted in increased CD11b (183.9 ± 18.8 MCF) and CD18 (34.2 ± 5.2 MCF) expression compared to isolated neutrophils alone (CD11b: 93.3 ± 24.3 MCF, P < 0.01; CD18: 17.9 ± 6.8 MCF, P < 0.01). Neutrophil CD11b and CD18 expression in response to plasma from healthy volunteers (n = 7) was similar to patients (baseline) [(90.7 ± 24.2 vs. 99.5 ± 18.8 MCF, P = 0.8) and (19.7 ± 6.8 vs. 18.5 ± 8.9 MCF, P = 0.9), respectively].
Effect of I/R plasma ± lidocaine on polymorphonuclear neutrophil (PMN) CD11b/CD18 expression. Incubation with I/R plasma 15 min following tourniquet release increased neutrophil CD11b (154.1 ± 34.2%, P = 0.03) and CD18 (129.5 ± 28.8%, P = 0.01) expression compared to baseline (Figs 1 and 2). Treatment of neutrophils with lidocaine (0.05 mg mL−1 and 0.5 mg mL−1 but not 0.005 mg mL−1, 1h) diminished increased CD11b and CD18 expression on exposure to I/R plasma 15 min following tourniquet release compared to control (Figs 1 and 2).
Incubation of HUVEC with recombinant human TNF-α for 4 h resulted in increased ICAM-1 expression compared to HUVEC alone (281.3 ± 75.8 MCF vs. 98.3 ± 32.3 MCF, P < 0.01). Endothelial ICAM-1 expression in response to plasma from healthy volunteers (n = 7) was similar to patients (baseline) [(100.3 ± 62.3 MCF vs. 75.2 ± 28.9 MCF, P = 0.8)].
Effect of I/R plasma ± lidocaine on endothelial cell ICAM-1 expression. Incubation with I/R plasma (15 min and 2 h following tourniquet release) increased ICAM-1 expression compared to baseline [(205.9 ± 24.2%, P < 0.001) and (225.6 ± 57.0%, P < 0.001), respectively] (Fig. 3). Treatment of HUVECs with lidocaine (0.05 mg mL−1 and 0.5 mg mL−1 but not 0.005 mg mL−1) diminished increased ICAM-1 expression on exposure to plasma 15 min and 2 h following tourniquet release compared to control (Fig. 3).
This study demonstrates increased in vitro neutrophil and endothelial cell adhesion molecule expression by plasma obtained during the early reperfusion phase following tourniquet release associated with upper limb surgery, and inhibition of these effects by lidocaine.
The time points chosen in this study were based on previous animal and human studies, which indicate that maximal neutrophil and endothelial activation occur during the first hour following reperfusion [1,14,15]. Sutter and colleagues have reported that upper limb tourniquet-induced I/R is associated with increased neutrophil adhesion molecule expression in the early reperfusion period . In a similarly designed experiment to ours, Welbourn and colleagues  failed to demonstrate the increased CD11b/CD18 expression on neutrophils after incubation with plasma obtained 10 min after lower limb reperfusion. This contrasted with the ability of the same plasma to promote neutrophil diapedesis, a process dependent upon the CD18 receptor . This finding may be due to the fact that these ex vivo studies were confined to plasma samples taken early in the reperfusion period (10 min) and later samples were not assessed for their ability to induce neutrophil activation. Barry and colleagues  demonstrated that plasma factors augment neutrophil and endothelial cell activation during aortic surgery. Incubation with I/R plasma resulted in a significant increase in neutrophil CD11b and endothelial cell ICAM-1 expression 40 min after aortic clamp release. In our study, there was a significant increase in neutrophil CD11b, CD18 and endothelial ICAM-1 expression induced by plasma obtained 15 min following tourniquet release. This was a transient event that had resolved within 2 h for neutrophil CD11b and CD18 expression similar to Barry and colleagues . Increased endothelial ICAM-1 expression did not resolve, however, until >12 h after reperfusion. These effects have not been demonstrated previously in minor perioperative I/R.
Lidocaine has demonstrated protective effects in animal models of I/R injury [8,9]. Lidocaine i.v. infusion started 90 min prior to ischaemia (70 μg kg−1 min−1) decreased myocardial infarct size by 25-30% 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 amide local anaesthetic, ropivacaine inhibits TNF-α-induced upregulation of CD11b/CD18 and L-selectin shedding by human neutrophils in vitro. Ropivacaine has also been shown to inhibit the TNF-α-induced neutrophil-endothelial interaction and neutrophil tissue accumulation . Lidocaine has recently been demonstrated to attenuate cytokine-induced injury in endothelial and vascular smooth muscle cells .
I/R is an important event in the perioperative period, with such injury consequent to surgeries such as aortic , cardiac , orthopaedic and plastic surgery . In addition to potential beneficial mechanisms of action, local anaesthetics are commonly administered in the perioperative period, and rarely cause adverse effects . In this study, the effects of lidocaine on changes in neutrophil and endothelial adhesion molecule expression associated with I/R were concentration dependent. This is similar to its anti-inflammatory  and antioxidant effects . The absence of a therapeutic effect of lidocaine at clinically relevant concentrations (0.005 mg mL−1) limits the clinical applicability of these results. However, a therapeutic benefit may be possible at sites where higher therapeutic plasma concentrations are found such as in tissues at or near the site of injection of local anaesthetic agents .
In the current experiments, incubation of isolated neutrophils with I/R plasma can lead to artefactual upregulation due to neutrophil isolation procedures . However, addition of I/R plasma results in a significant and stepwise increase in markers of neutrophil activation in both isolated neurophils and whole blood as reported by Barry and colleagues using a similar experimental design . Several potential mediators of neutrophil-independent I/R injury including thromboxane, TNF-α, interleukin-1β (IL-1β) and IL-6 have been implicated . IL-1β, TNF-α, thromboxane and the complement activation product complement protein 5a (C5a) have all been shown to appear in plasma after rat hind limb ischaemia [23-25]. The plasma concentrations of these mediators, neutrophil adhesion or transendothelial neutrophil migration were not measured and represent a study limitation. HUVECs are the most often used endothelial cell culture model, although there are few studies comparing their response with other human endothelial cell types from the adult organism. Klein and colleagues  have reported similar in vitro cytokine effects on the expression of adhesion molecules by human umbilical vein, saphenous vein and femoral artery endothelial cells. Study results must also be interpreted against a background of a small sample size and large standard deviations.
In conclusion, we have demonstrated that increased in vitro neutrophil and endothelial cell adhesion molecule expression occurs on exposure to plasma obtained during the early reperfusion phase following tourniquet release after upper limb surgery. This effect is diminished by lidocaine at greater than clinically relevant plasma concentrations.
1. Wakai A, Wang JH, Winter DC, Street JT, O'Sullivan RG, Redmond HP. Tourniquet-induced systemic inflammatory response in extremity surgery. J Trauma
2. Wakai A, Winter DC, Street JT, O'Sullivan RG, Wang JH, Redmond HP. Inosine attenuates tourniquer-induced skeletal muscle reperfusion injury. J Surg Res
3. Sutter PM, Spagnoli GC, Heberer M, et al.
Increased surface expression of CD18 and CD11b in leukocytes after tourniquet ischaemia during elective hand surgery. World J Surg
4. Mathru M, Dries DJ, Barnes L, Tonino P, Sukhani R, Rooney MW. Tourniquet-induced exsanguinations in patients requiring lower limb surgery. An ischemia-reperfusion model of oxidant and antioxidant metabolism. Anesthesiology
5. Simpson R, Alon R, Kobzik L, Valeri CR, Shepro D, Hechtman HB. Neutrophil and non-neutrophil-mediated injury in intestinal ischaemia-reperfusion. Ann Surg
6. Paterson IS, Smith FC, Tsang GM, Hamer JD, Shearman CP. Reperfusion plasma contains a neutrophil activator. Ann Vasc Surg
7. Barry MC, Wang JH, Kelly CJ, Sheehan SJ, Redmond HP, Bouchier-Hayes DJ. Plasma factors augment neutrophil and endothelial cell activation during aortic surgery. Eur J Vasc Endovasc Surg
8. Lesnefsky EJ, VanBenthuysen KM, McMurtry IF, Shikes RH, Johnston Jr RB, Horwitz LD. Lidocaine reduces canine infarct size and decreases release of a lipid peroxidation product. J Cardiovasc Pharmacol
9. Lantos J, Roth E, Temes G. Effects of lidocaine on cerebral lipid peroxidation and neutrophil activation following complete compression ischemia. Arch Int Pharmacodyn Ther
10. Hollmann MW, Durieux ME. Local anesthetics and the inflammatory response: a new theraputic indication. Anesthesiology
11. Kang MY, Tsuchiya M, Packer L, Manabe M. In vitro
study on antioxidant potential of various drugs used in the perioperative period. Acta Anaesthesiol Scand
12. Mikawa K, Akamatsu H, Nishina K, et al.
Inhibitory effect of local anaesthetics on reactive oxygen species production by human neutrophils. Acta Anaesthesiol Scand
13. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest
14. Freischlag JA, Hanna D. Superoxide anion release (O−2
) after ischemia and reperfusion. J Surg Res
15. Welbourn R, Goldman G, Kobzik L, et al.
Neutrophil adhesion receptors (CD18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4. J Immunol
16. Martinsson T, Oda T, Fernvik E, Roempke K, Dalsgaard CJ, Svensjo E. Ropivacaine inhibits leukocyte rolling, adhesion and CD11b/CD18 expression. J Pharmacol Exp Ther
17. Zhang XW, Thorlacius H. Inhibitory actions of ropivacaine on tumor necrosis factor-alpha-induced leukocyte adhesion and tissue accumulation in vivo. Eur J Pharmacol
18. de Klaver MJ, Buckingham MG, Rich GF. Lidocaine attenuates cytokine-induced cell injury in endothelial and vascular smooth muscle cells. Anesth Analg
19. Fransen EJ, Maessen JG, Hermens WT, Glatz JF, Buurman WA. Peri-operative myocardial tissue injury and the release of inflammatory mediators in coronary artery bypass graft patients. Cardiovasc Res
20. Harmon D, Lan W. Effects of systemic local anesthetics on perioperative ischemia reperfusion may be beneficial. Anesth Analg
21. Swanton BJ, Shorten GD. Anti-inflammatory effects of local anesthetic agents. Int Anesthesiol Clin
22. Fearon DT, Collins LA. Increased expression of C3b receptors on polymorphonuclear leukocytes induced by chemotactic factors and by purification procedures. J Immunol
23. Walsh CJ, Leeper-Woodford SK, Carey PD, et al.
CD18 adhesion receptors, tumour necrosis factor, and neutropenia during septic lung injury. J Surg Res
24. Seekamp A, Warren JS, Remick DG, Till GO, Ward PA. Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury. Am J Pathol
25. Barry MC, Kelly CJ, Stokes K, et al.
Glyceryl trinitrate prevents neutrophil activation but not thromboxane release following ischaemia reperfusion injury. Br J Surg
26. Klein CL, Kohler H, Bittinger F, et al.
Comparative studies on vascular endothelium in vitro.
I. Cytokine effects on the expression of adhesion molecules by human umbilical vein, saphenous vein and femoral artery endothelial cells. Pathobiology
Keywords:© 2004 European Academy of Anaesthesiology
ANAESTHETICS, local, lidocaine; ADHESION MOLECULES, cell; INJURY, ischaemia-reperfusion