Secondary Logo

Journal Logo

Original Articles – Cardiovascular

Tramadol reduces myocardial infarct size and expression and activation of nuclear factor kappa B in acute myocardial infarction in rats

Zhang, Lin-Zhonga,b; Guo, Zhenga,b,c

Author Information
European Journal of Anaesthesiology: December 2009 - Volume 26 - Issue 12 - p 1048-1055
doi: 10.1097/EJA.0b013e32832c785d

Abstract

Introduction

Cardioprotection for ischaemia–reperfusion injury has been a focus for studies in the past [1–3]. However, fewer studies have been reported on cardioprotective measures in cardiac ischaemic injury, which is often seen in clinical practice, such as myocardial infarction (MI) prior to reestablishment of coronary circulation, and in some clinical manoeuvres, including cardiac surgery and preservation of donor hearts for transplantation. Therapeutic measures initiated during acute myocardial ischaemia may be crucially affecting the pathology in ischaemia–reperfusion. Prevention of injury could be of clinical value. Some anaesthetics show a cardioprotective effect [1,4,5], but the underlying mechanism remains elusive.

Tramadol, a centrally acting analgesic, currently used for the treatment of moderate-to-severe pain in clinical practice, has shown a cardioprotective effect against myocardial ischaemia–reperfusion injury in isolated rat heart. However, the mechanism of this effect is still unclear [6]. Whether tramadol produces a protective effect in cardiac ischaemia injury is unknown.

Nuclear factor kappa B (NF-κB) is a redox-sensitive transcription factor regulating a battery of genes of inflammatory mediators and has important implications for the initiation and development of pathogenesis in a variety of inflammatory diseases. NF-κB was also demonstrated to be involved in cardiac ischaemia–reperfusion damage [7–13], the inflammatory response in various cardiac diseases [12–14], including hypertensive heart disease [15,16], and myocarditis [17]. The NF-κB system represents the most important transcription-regulatory element in the cell adhesion molecule promoters and plays a key role in regulating cytokine-induced leucocyte adhesion [18–21]. Intercellular adhesion molecule-1 (ICAM-1) was shown to be associated with leucocyte-mediated injury in the ischaemia–reperfusion pathological process [22–26].

In this study, we examined the hypothesis that tramadol produces cardioprotective effects on acute MI via modulation of the NF-κB pathways. A rodent model of acute MI induced by permanent coronary artery occlusion (CAO) was used. Changes in the expression and activation of NF-κB and expression of ICAM-1 during acute MI in the animals with and without pretreatment with tramadol were compared and analysed.

Methods

The experiments conformed to the guidelines for the care and use of laboratory animals [National Institutes of Health (NIH) guide for the care and use of laboratory animals, NIH publication no. 80-23, revised 1996 (website: http://grants.nih.gov/grants/olaw/olaw.htm)] and were approved by the Institutional Animal Care and Use Committee of Shanxi Medical University. Sixty-one adult male Sprague–Dawley rats, weighing 260 ± 20 g were used for this study. They were given free access to food and water except before the study.

Protocol

The infarct size of the myocardium insult caused by acute ischaemia induced by permanent CAO in rats was measured by computer morphometry of tetrazolium-stained sections. The expression of NF-κB p65 and activation of NF-κB were examined using immunohistochemical assay and flow cytometry (FCM), respectively. Real-time quantitative reverse transcription–PCR (qRT–PCR) was carried out to investigate the expression of NF-κB p65 mRNA and ICAM-1 mRNA. Experiments were carried out in the animals, randomly divided into three groups: sham surgery group, CAO group and tramadol group (T + CAO group), in which animals were treated with tramadol (402H, Grunenthal, Aachen, Germany; 12.5 mg kg−1, intravenously, in 0.35 ml 10 s−1) 15 min before CAO. The dose of tramadol had been proved to be effective in the attenuation of nociceptive neuronal activity of thalamic neurons induced by the CAO in our previous preliminary study (data not shown).

Preparations of the acute myocardial infarction model

The procedure of CAO and sham surgery was performed as previously reported [27,28]. Briefly, the rat's pericardium was opened through an incision in the fourth left intercostal space for the animals in the CAO and sham surgery groups under anaesthesia with sodium pentobarbital (induction with 65 mg kg−1, intraperitoneally, and maintained with 15 mg kg−1 h−1, intravenously). The anterior part of the heart was exposed. At this stage, the chest was closed in the animals in the sham surgery group. A suture was made around the left anterior descending branch of the coronary artery, and ligation of the artery was performed in the animals in the CAO groups with or without pretreatment with tramadol. Then the chest was closed under sustained negative pressure. Spontaneous respiration was restored. The length of the time taken for the open-chest procedure in the animals in the CAO and sham surgery groups was not significantly different (5 ± 1 min).

Animals in the CAO and T + CAO groups were subjected to 6 h of ligation. Coronary artery ligation was confirmed by changes in the ECG during the experiment and by autopsy at the end of each test. Anaesthesia was maintained, and ECG and pulse oxygen saturation (Spo2) were monitored until the end of the observation period. The femoral artery and the tail vein were cannulated for measurements of arterial pressure and administration of testing agents and supplemental anaesthetics and the continuous administration of physiological saline (1 ml h−1) during the experiment. The depth of anaesthesia was monitored by observation of the changes in the size of the pupils and the depth and the pattern of respiration.

Measurement of myocardial infarct size

As scheduled, at 6 h of CAO, rats were given a lethal dose of sodium pentobarbital and Evans blue (10%, 1.5 ml, Sigma-Aldrich Co., Saint Louis, Missouri, USA) through the tail vein to delineate the ischaemic area at risk (AAR; unstained by Evans blue) from the nonischaemic area (dyed blue). The heart was excised, and the left ventricle (LV) was sectioned into five or six transverse slices (2 mm) in a plane parallel to the atrioventricular groove, perpendicular to the long axis from the apex to the base. The slices were then incubated in a 1% triphenyltetrazolium chloride (TTC; Sigma-Aldrich Co.) solution in an isotonic PBS (pH 7.4) for 30 min at 37°C. After fixation in 4% paraformaldehyde (pH 7.4), both sides of each TTC-stained tissue slice were photographed with a digital camera. In the AAR, the infarct area was pale, and the other area was red. Each slice was analysed with NIH free software, NIH Image J 1.40. The AAR was expressed as a percentage of the LV and the infarct area as a percentage of the AAR, that is, AAR/LV and infarct area/AAR, respectively.

Immunohistochemistry

The heart was excised at 6 h of CAO and fixed with 4% paraformaldehyde (pH 7.4), embedded in paraffin and sectioned at 6 μm. The sections were incubated with NF-κB p65 mouse mAb in PBS (1: 250; Santa Cruz Biotechnology, Santa Cruz, California, USA) at 4°C, overnight, and followed by incubation with a biotinylated secondary antibody (1: 200; Zhongshan Goldenbridge, Beijing, China) for 1 h at room temperature. After washing with PBS, sections were treated with peroxidase-ligated streptavidin for 1 h at room temperature and developed for 1 min in diaminobenzidine (Zhongshan Goldenbridge).

The expression of NF-κB in the ischaemic area (defined according to the results of the TTC assay) or the position-matched area of the LV for the CAO, T + CAO and sham groups was examined using an Olympus BX51TF microscope (Olympus Optical Co. Ltd, Tokyo, Japan), and a semi-quantitative image analysis for the immunoreactive products was performed with microimage analysing software (IDA2000, Konghai Science and Technology Inc., Beijing, China). The results were presented as mean optical density and immunoreactive area. Comparisons were made for the sham surgery groups with the animals in the CAO and T + CAO groups.

Quantitative reverse transcription–PCR

Fresh samples of myocardium from the ischaemic area and the position-matched area of the LVs were collected from the CAO (6 h), T + CAO (6 h) and the sham (6 h) groups, respectively. The first-strand cDNA was synthesized according to the standard protocol of the Prime Script RT–PCR Kit (Takara Biotechnology Co. Ltd, Dalian, China). We used 0.4 μg of total RNA from each sample for reverse transcription in a reaction volume of 10 μl. cDNA levels of specific genes were measured by quantitative real-time PCR, which was performed using SYBR Premix EX Taq (Takara Biotechnology Co. Ltd) and a MX3005P Real-Time PCR System (Stratagene, La Jolla, California, USA) with gene-specific primers: NF-κB p65 (NM_199267): 5′-TGAAGAAGCGAGACCTGGAG-3′(forward), 5′-AGGGGTTATTGTTGGTCTGGAT-3′ (reverse), the amplified product was 145 bp; ribosomal protein L32 (Rpl32, NM_013226): 5′-CACAGCTGGCCATCAGAGTCA-3′ (forward), 5′-AAACAGGCACACAAGCCATCTATTC-3′ (reverse), the amplified fragment was 83 bp; ICAM-1 (NM_012967): 5′-GCTTCTGCCACCATCACTGTGTA-3′ (forward), 5′-AGCGCAGGATGAGGTTCTT-3′ (reverse), the amplified fragment was 97 bp. The cDNA (2 μl of 1: 10 diluted) was used for each reaction in a total volume of 25 μl. The program was set for 10 min at 95°C (one cycle); 5 s at 95°C, 20 s at 60°C (40 cycles); 1 min at 95°C followed by 30 s at 55°C (one cycle) and 30 s at 95°C for the melting curves. Dissociation curves were examined to verify the presence of a single amplification product in the absence of DNA contamination, after amplification. Changes in gene expression, presented in folds of the controls (sham), were determined using the delta–delta–Ct method. All PCR reactions were performed in triplicate.

Flow cytometry

Single nucleus suspension was prepared as reported previously [29,30]. In brief, homogenation of the ischaemic/infarct myocardium induced by 6 h of CAO was made by digestion with 0.5% collagenase II for 2 h at 37°C, washed twice with PBS (pH 7.4) and filtered with 200 μm nylon mesh, discarding the undigested tissue. The filtrate was collected and incubated with 1% Triton X-100 (Sigma-Aldrich Co., Saint Louis, Missouri, USA) for 90 min at 4°C. After centrifugation at 1200 rpm for 5 min, the precipitation was collected. The nuclei were confirmed by Wright–Giemsa staining, as shown in Fig. 1d, and adjusted to a density of 1 × 106 ml−1. A volume of 100 μl of the single nucleus suspension was suspended in 0.5 ml of fluorescence-activated cell sorter buffer (0.1% BSA and 0.1% sodium azide in PBS) and incubated at 4°C for 20 min. The nuclei were then washed twice with PBS and incubated with 1 μg NF-κB p65 mouse mAb (1: 100; Santa Cruz Biotechnology) in fluorescence-activated cell sorter buffer for 40 min at room temperature. Then the nuclei were incubated with goat antimouse fluorescein isothiocyanate–immunoglobulin G (1: 100; Invitrogen, Carlsbad, California, USA) and propidium iodide (PI; 1: 500; Beckman Coulter, Inc., Fullerton, California, USA) for 20 min at room temperature, after washing. Analysis was performed using FCM (Elite-Esp, Beckman Coulter, Inc.) according to previous reports [30,31]. The fluorescence intensity was expressed on a logarithmic scale. The activation of NF-κB was detected in nuclei positively expressing both NF-κB p65 and PI.

Fig. 1
Fig. 1

Statistical analysis

Values are presented as mean ± SD. Haemodynamic data were analysed using two-way analysis of variance (ANOVA) and repeated measures analysis. One-way ANOVA followed by a post-hoc Bonferroni's test was performed to analyse the changes in the parameters shown in Figs 1e, 2b, 3, 4 and 5. Statistical significance was defined as a P value of less than 0.05.

Fig. 2
Fig. 2
Fig. 3
Fig. 3
Fig. 4
Fig. 4
Fig. 5
Fig. 5

Results

Seven rats died and were excluded because of intractable ventricular fibrillation (n = 7) occurring after CAO. Fifty-four animals completed the study. There was an obvious elevation in the ST segment after the onset of CAO and throughout the observation, as reported previously [28]. There was no significant change in blood pressure (BP), heart rate (less than 10% of baseline) and Spo2 following the administration of 12.5 mg kg−1 (intravenously, in 0.35 ml 10 s−1) of tramadol (Table 1). Spo2 indicated that there was no hypoxia during the experiment.

Table 1
Table 1:
Changes in haemodynamic and pulse oxygen saturation

Infarct size

MI could be detected in the myocardium insulted by 6 h of CAO (Fig. 2a). In the CAO and T + CAO groups, the infarct size (percentage of risk area) was 44.9 ± 6.8 and 31.6 ± 7.6%, respectively (Fig. 2c). The reduction in infarct size in the T + CAO animals was statistically significant (P < 0.05), compared with that of CAO animals. The AAR (percentage of LV) was 46.4 ± 6.2 and 48.2 ± 5.9% in the CAO and T + CAO groups, respectively (Fig. 2b), presenting no statistical difference in AAR/LV between the two groups (P > 0.05).

Nuclear factor kappa B mRNA and intercellular adhesion molecule-1 mRNA

Acute ischaemic injury induced by 6 h of CAO caused a significant increase in NF-κB mRNA, by 3.08 ± 0.42-fold, whereas only a 1.79 ± 0.11-fold increase in mRNA was observed in the T + CAO animals. Tramadol produced a significant reduction, by 40%, in the number of original copies of NF-κB mRNA (P < 0.05, Fig. 3). ICAM-1 mRNA was markedly elevated in infarct myocardium by 5.28 ± 0.65-fold, whereas less upregulation of mRNA (3.52 ± 0.44-fold) was detected in the T + CAO animals (P < 0.05, Fig. 4).

Immunohistochemistry

In the CAO (Fig. 5b) and T + CAO animals (Fig. 5c) animals, positive immunoreactive products of NF-κB were mainly located in the cytoplasm (Fig. 5d) and nuclei (Fig. 5e), whereas they were rarely observed in the sham group (Fig. 5a). The semi-quantitative analysis showed that, in the ischaemic myocardium of CAO animals, the mean optical density and immunoreactive area regarding the NF-κB immunoreactivity were significantly increased, compared with the sham group (P < 0.05, Fig. 5f and g). Administration of tramadol before the CAO caused a significant decrease in the expression of NF-κB, compared with the CAO group (P < 0.05, Fig. 5f and g). However, the expression of NF-κB in the T + CAO animals was still more than that in the sham group (P < 0.05). Change in the expression of NF-κB protein was identical to that of NF-κB mRNA.

Activation of nuclear factor kappa B

The results showed that there was less activation of NF-κB (5.1 ± 1.2%) in the cardiomyocytes in the sham group. After 6 h of CAO, the ratio of positive nucleus to both NF-κB p65 and PI was significantly increased up to 36.2 ± 4.1% (P < 0.05, compared with sham) in the CAO animals. Immunoreactivity positive nuclei were significantly decreased down to 22.8 ± 3.5% in the T + CAO animals (P < 0.05, compared with the CAO group); however, they were still significantly more than that in the sham group (P < 0.05, Fig. 1).

Discussion

Studies on cardioprotection in myocardial ischaemia may be of value in the preservation of functions of the heart, which occur under temporary disturbances of coronary circulation, for instance in preservation of donor hearts for transplantation. In this study, we observed that tramadol pretreatment reduced the infarct size of the myocardium insulted by acute ischaemia induced by permanent CAO in rats, which is similar to a previous report by Bilir et al. [6]. In this study, we also observed that tramadol pretreatment reduced the infarct size parallel with downregulation of the expression and activation of NF-κB in the myocardium insulted by the CAO, which may suggest an association of cardioprotective effects of tramadol with the activity of NF-κB.

Previous studies [32,33] have revealed that nitric oxide plays an important role in the analgesic effect of tramadol. Kaya et al. [34] observed that tramadol could cause vasodilation of the aorta by increasing nitric oxide production. It was reported that nitric oxide could regulate the activity of NF-κB through stabilization of IκBα, an NF-κB inhibitor, by preventing its degradation by NF-κB, increasing the expression of IκBα, or by both [35,36]. In addition, nitric oxide could also directly cause nitrosylation of the cysteine of NF-κB and decrease the NF-κB DNA-binding activity [37]. It might indicate that nitric oxide interferes with the signal pathway and reduces the inflammatory reaction. Taken together, the studies might indicate that tramadol plays a cardioprotective role through modulation of inflammatory mediators.

The inflammatory reaction of myocardium in acute MI is considered to be a pathophysiological response to injury [38], although NF-κB is a key factor in the inflammatory reaction. The NF-κB family comprises p50, p52, p65 (RelA), c-Rel and RelB, which form various homodimers and heterodimers. The NF-κB dimers in resting cells reside in the cytoplasm in an inactive form and translocate to the nucleus and bind to promoter or enhancer regions of specific genes, when activated, initiating transcription [39,40], leading to alterations in the synthesis and release of proinflammatory cytokines, and thereby initiate a receptor-dependent apoptotic process.

Activation of NF-κB has been reported in an experimental model of myocardial ischaemia, reperfusion or both [14]. Blockade of NF-κB transcriptional activity in vivo, by inhibiting proinflammatory gene expression, resulted in a reduction in the extent of MI [14,41,42]. The present study shows that attenuation of the activation and expression of NF-κB and its mRNA was in line with the reduction of infarct size of the myocardium following pretreatment with tramadol, which might suggest an association between the cardioprotective effect of tramadol and the downregulation of synthesis and activity of NF-κB in acute MI and may indicate a role of the inflammatory reaction in the pathology of acute MI. The simultaneous reduction of ICAM-1 mRNA after pretreatment with tramadol, observed in this study, may serve as supporting evidence indicating the modulation (downregulation) of the activity of NF-κB. The observations may also suggest that the modulation of NF-κB activation may be a novel and future strategy for myocardial protection [42], especially in the early stage of acute MI and donor heart preservation, and tramadol could be a candidate for the application.

Kaya et al. [34] demonstrated a tramadol-induced vasodilatation using an experimental setting of isolated rabbit thoracic aortic rings at a concentration of 10−4 and 3 × 10−4 mol l−1 of tramadol. The vasodilatation effect of tramadol could be a potential factor affecting the physiological stage of the animals, which may further potentially alter the expression and activation of NF-κB. However, in the current study, we detected only a slight change (<10% of baseline level) in BP after administration of tramadol at a dose of 12.5 mg kg−1 (intravenously, in 0.35 ml 10 s−1), which did not suggest a haemodynamic effect on the changes in NF-κB. The difference in the vasodilatation of tramadol, between previous and current studies, could be caused by the difference in experimental setting and drug concentration at the sites of action.

However, the beneficial effects of NF-κB on the preservation of myocardium and cardiac function have been reported previously [11,43,44]. We must keep in mind that the dual effects of NF-κB on the myocardium and cardiac function may be related to, or determined by, the levels of noxious stimuli, including acute ischaemia, the types of cells involved and injury produced as well as the pattern and time course of the transcription process during short or longer ischaemia, about which knowledge is still very limited.

In conclusion, we propose that tramadol pretreatment could reduce the infarct size of the myocardium, induced by 6 h of CAO in rats, and this effect was accompanied by downregulation of the expression and activation of NF-κB, presenting an association between the cardioprotective property of tramadol and the changes in NF-κB in the early stage of acute MI, which may suggest a novel pharmacological approach for clinical applications.

References

1 Minguet G, Joris J, Lamy M. Preconditioning and protection against ischaemia–reperfusion in noncardiac organs: a place for volatile anaesthetics? Eur J Anaesthesiol 2007; 24:733–745.
2 Kobayashi I, Kokita N, Namiki A. Propofol attenuates ischaemia-reperfusion injury in the rat heart in vivo. Eur J Anaesthesiol 2008; 25:144–151.
3 Pagel PS, Krolikowski JG. Transient metabolic alkalosis during early reperfusion abolishes helium preconditioning against myocardial infarction: restoration of cardioprotection by cyclosporin A in rabbits. Anesth Analg 2009; 108:1076–1082.
4 Xu J-J, Wang Y-L. Propofol attenuation of hydrogen peroxide-mediated oxidative stress and apoptosis in cultured cardiomyocytes involves haem oxygenase-1. Eur J Anaesthesiol 2008; 25:395–402.
5 Hieber S, Huhn R, Hollmann MW, et al. Hypoxia-inducible factor 1 and related gene products in anaesthetic-induced preconditioning. Eur J Anaesthesiol 2009; 26:201–206.
6 Bilir A, Erkasap N, Koken T, et al. Effects of tramadol on myocardial ischemia-reperfusion injury. Scand Cardiovasc J 2007; 41:242–247.
7 Kukreja RC. NFkappaB activation during ischemia/reperfusion in heart: friend or foe? J Mol Cell Cardiol 2002; 34:1301–1304.
8 Kin H, Wang NP, Halkos ME, et al. Neutrophil depletion reduces myocardial apoptosis and attenuates NF-kB activation/TNF-a release after ischemia and reperfusion. J Surg Res 2006; 135:170–178.
9 Brown M, McGuinness M, Wright T, et al. Cardiac-specific blockade of NF-kappaB in cardiac pathophysiology: differences between acute and chronic stimuli in vivo. Am J Physiol Heart Circ Physiol 2005; 289:H466–H476.
10 Moss NC, Stansfield WE, Tang R, Selzman CH. Delayed NF-kappaB inhibition still provides cardioprotection from myocardial ischemia and reperfusion. J Am Coll Surg 2007; 205:S95–S96.
11 Valen G, Yan ZQ, Hansson GK. Nuclear factor kappa-B and the heart. J Am Coll Cardiol 2001; 38:307–314.
12 Hall G, Hasday JD, Rogers TB. Regulating the regulator: NF-kappaB signaling in heart. J Mol Cell Cardiol 2006; 41:580–591.
13 Chandrasekar B, Freeman GL. Induction of nuclear factor kappaB and activation protein 1 in postischemic myocardium. FEBS Lett 1997; 401:30–34.
14 Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovasc Res 2002; 53:31–47.
15 Sun Y, Zhang J, Lu L, et al. Aldosterone-induced inflammation in the rat heart: role of oxidative stress. Am J Pathol 2002; 161:1773–1781.
16 Ogata T, Miyauchi T, Sakai S, et al. Myocardial fibrosis and diastolic dysfunction in deoxycorticosterone acetate-salt hypertensive rats is ameliorated by the peroxisome proliferator-activated receptor-alpha activator fenofibrate, partly by suppressing inflammatory responses associated with the nuclear factorkappa-B pathway. J Am Coll Cardiol 2004; 43:1481–1488.
17 Huang H, Petkova SB, Cohen AW, et al. Activation of transcription factors AP-1 and NF-kappa B in murine Chagasic myocarditis. Infect Immun 2003; 71:2859–2867.
18 Baeuerle PA, Henkel T. Function and activation of NF-kB in the immune system. Annu Rev Immunol 1994; 12:141–179.
19 Collins T, Read MA, Neish AS, et al. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J 1995; 9:899–909.
20 Iademarco MF, McQuillan JJ, Rosen GD, Dean DC. Characterization of the promoter for vascular cell adhesion molecule-1. J Biol Chem 1992; 267:16323–16329.
21 Ledbur HC, Park TP. Transcription regulation of intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. J Biol Chem 1995; 270:933–943.
22 Ma X, Lefer DJ, Lefer AM, Rothline R. Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion. Circulation 1992; 86:937–946.
23 Weyrich AS, Ma X, Lefer DJ, et al. In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest 1993; 91:2620–2629.
24 Yamazaki T, Seko Y, Tamatani T, et al. Expression of intercellular adhesion molecule-1 in rat heart with ischemia/reperfusion and limitation of infarct size by treatment with antibodies against cell adhesion molecules. Am J Pathol 1993; 143:410–418.
25 Hartman JC, Anderson DC, Wiltse AL, et al. Protection of ischemic/reperfused canine myocardium by CL18/6, a monoclonal antibody to adhesion molecule ICAM-1. Cardiovasc Res 1995; 30:47–54.
26 Lefer DJ, Flynn DM, Anderson DC, Buda AJ. Combined inhibition of P-selectin and ICAM-1 reduces myocardial injury following ischemia and reperfusion. Am J Physiol 1996; 271:H2421–H2429.
27 Zhang JW, Guo Z. Changes in substance P in myocardial and dorsal root ganglion following coronary artery occlusion in rats. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 2006; 18:201–205.
28 Guo Z, Niu YL, Zhang JW, Yao TP. Coronary artery occlusion alters expression of substance P and its mRNA in spinal dorsal horn in rats. Neuroscience 2007; 145:669–675.
29 Cohen C, Tickman RJ, Derose PB, Whitaker BP. DNA ploidy studies of benign and malignant tumors. Comparison of flow cytometry and image analysis techniques using two types of cytological specimen. Cytopathology 1991; 2:241–259.
30 Foulds S. Flow cytometric method for quantifying nuclear binding of the transcription factor nuclear factor kappa B in unseparated human monocytes and polymorphonuclear cells. Cytometry 1997; 29:182–186.
31 Deptala A, Bedner E, Gorczyca W, Darzynkiewicz Z. Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC). Cytometry 1998; 33:376–382.
32 Okuducu H, Onal SA. Is nitric oxide involved in the antinociceptive activity of tramadol? Findings in a rat model of neuropathic pain. Agri Dergisi 2005; 17:31–40.
33 Dal D, Salman MA, Salman AE, et al. The involvement of nitric oxide on the analgesic effect of tramadol. Eur J Anaesthesiol 2006; 23:175–177.
34 Kaya T, Gursoy S, Karadas B, et al. High-concentration tramadol-induced vasodilation in rabbit aorta is mediated by both endothelium-dependent and -independent mechanisms. Acta Pharmacol Sin 2003; 24:385–389.
35 Spiecker M, Peng HB, Liao JK. Inhibition of endothelial vascular cell adhesion molecule-1 expression by nitric oxide involves the induction and nuclear translocation of IkBa. J Biol Chem 1997; 272:30969–30974.
36 Peng HB, Libby P, Liao JK. Induction and stabilization of IkBa by nitric oxide mediates inhibition of NF-kB. J Biol Chem 1995; 270:14214–14219.
37 Matthews JR, Botting CH, Panico M, et al. Inhibition of NF-kB DNA binding by nitric oxide. Nucleic Acids Res 1996; 24:2236–2242.
38 Mehta JL, Li DY. Inflammation in ischemic heart disease: response to tissue injury or a pathogenetic villain? Cardiovasc Res 1999; 43:291–299.
39 Chen F, Castranova V, Shi X, Demers LM. New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem 1999; 45:7–17.
40 Li C, Kao RL, Ha T, et al. Early activation of IKKb during in vivo myocardial ischemia. Am J Physiol Heart Circ Physiol 2001; 280:H1264–H1271.
41 Morishita R, Sugimoto T, Aoki M, et al. In vivo transfection of cis element ‘decoy’ against nuclear factor-kappaB binding site prevents myocardial infarction. Nat Med 1997; 3:894–899.
42 Sakaguchi T, Sawa Y, Fukushima N, et al. A novel strategy of decoy transfection against nuclear factor-kappaB in myocardial preservation. Ann Thorac Surg 2001; 71:624–629, discussion 629–630.
43 Dawn B, Guo Y, Rezazadeh A, et al. Tumor necrosis factor-a does not modulate ischemia/reperfusion injury in naïve myocardium but is essential for the development of late preconditioning. J Mol Cell Cardiol 2004; 37:51–61.
44 Maulik N, Sato M, Price BD, Das DK. An essential role of NFkB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 1998; 429:365–369.
Keywords:

infarct size; myocardial infarction; nuclear factor kappa B; tramadol

© 2009 European Society of Anaesthesiology