Induced Remote Ischemic Pre-conditioning on Ischemia Reperfusion Injury in Patients Undergoing Coronary Artery Bypass

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1 ORIGINAL ARTICLE Induced Remote Ischemic Pre-conditioning on Ischemia Reperfusion Injury in Patients Undergoing Coronary Artery Bypass Nasir Ali 1, Farwa Rizwi 2, Afsheen Iqbal 1 and Azhar Rashid 1 ABSTRACT Objective: To determine the role of remote ischemic pre-conditioning (ripc) on myocardium, against ischemia reperfusion injury in patients undergoing coronary artery bypass graft (CABG) surgery by measuring CKMB levels. Study Design: A randomized controlled trial. Place and Duration of Study: The Surgical Department of Armed Forces Institute of Cardiology/National Institute of Heart Diseases, Rawalpindi, from January to June Methodology: One hundred patients with double and triple vessels coronary artery disease were randomized in two groups of 50 each. ripc protocol consisted of 3 x 5 minutes of forearm ischemia, induced by a blood pressure cuff inflated to 200 mmhg, with an intervening 5 minutes of reperfusion, during which the cuff was deflated. Patients in the control group were not subjected to limb ischemia. The protocol of induced ischemia was completed before placing patients on extracorporeal bypass circuit. At the end of surgery serum CKMB levels were measured and compared at 8, 16, 24 and 48 hours from both the groups. Written informed consent was taken from patients. Study was approved by the hospital ethical committee. Results: Remote ischemic pre-conditioning significantly reduced CKMB levels at 8, 16, 24 and 48 hours after surgery with p-values of 0.026, 0.021, and respectively. There was mean reduction of 3 iu/l in CKMB levels, in patients who underwent ripc protocol prior to CABG surgery, compared to control group. Conclusion: This study showed a significant reduction of enzyme marker CKMB in patients subjected to ripc prior to CABG surgery. This suggests lesser degree of myocardial damage compared to control group in CABG patients. Key words: Coronary artery bypass grafting (CABG). Creatine kinase myocardial band (CKMB). Ischemic pre-conditioning (IPC). Remote ischemic pre-conditioning (ripc). INTRODUCTION Ischemic heart disease (IHD) is currently the leading cause of morbidity and mortality in the developed world. 1 According to a report by World Health Organization on ischemic heart diseases in 2005, IHD would become the leading cause of death in the world by the year Patients with severe IHD that require coronary artery bypass graft (CABG) surgery, although protected by techniques such as cross-clamp fibrillation and cardioplegia, still sustain significant myocardial injury as evidenced by perioperative troponin T or I or CK-MB release. 2 Novel treatment strategies are required to limit the myocardial injury sustained by patients undergoing 1 Department of Cardiac Surgery, Armed Forces Institute of Cardiology/National Institute of Heart Diseases, Rawalpindi. 2 Department of Community Medicine, Islamabad Medical and Dental College, Islamabad. Correspondence: Dr. Farwa Rizvi, Community Medicine Department, Islamabad Medical and Dental College, Bara Kahu, Islamabad. farwa.rizvi@hotmail.com Received October 08, 2008; accepted April 28, CABG surgery in order to improve the clinical outcome of this patient group. Ischemic pre-conditioning (IPC) is the most powerful innate mechanism to protect against ischemia reperfusion injury. 3 This involves a brief period of sub-lethal local tissue ischemia that confers protection against a subsequent lethal ischemia. A more intriguing form of ischemic pre-conditioning with potentially greater clinical significance is remote ischemic preconditioning ripc i.e. transient tissue ischemia at a distance may confer subsequent protection of an organ subjected to potentially lethal ischemia. 4 Przyklenk in 1993 and later Birnbaum et al. in 1997 demonstrated the phenomenon of remote ischemic preconditioning in animal models, whereby transient limb ischemia could reduce myocardial injury caused by prolonged coronary artery occlusion. 5 This magnitude of protection in remote pre-conditioning certainly approaches to local ischemic pre-conditioning in the context of myocardial protection in animal models. 6,7 It was observed that remote ischemic pre-conditioning modifies expression of at least 30 proinflamatory genes involved in leukocyte activation. This anti-inflammatory response is more global in remote ischemic preconditioning as compared to local ischemic preconditioning. 8 Journal of the College of Physicians and Surgeons Pakistan 2010, Vol. 20 (7):

2 Nasir Ali, Farwa Rizwi, Afsheen Iqbal and Azhar Rashid In the heart, ripc incurs protection against ischemia reperfusion injury by K + ATP channel dependent mechanism, similar to local pre-conditioning, 9,10 but the details as to how protection is transferred to distant organs is still largely unknown. This may involve humoral factors and autonomic system. 11 Clinical trials of ripc carried out in children undergoing corrective cardiac surgery for congenital heart diseases have shown promising results. 12 Trials of ripc in adults undergoing cardiac surgery are under way at many centers in the world. The rationale for conducting this study in CABG group only was that, since procedure-associated tissue injury is minimum in CABG patients, release of enzyme CKMB will have a better positive predictive value for ischemia rather than surgery associated myocardial injury. This study was designed to determine the protective effects of ripc in reducing myocardial damage against ischemia reperfusion injury by measuring postoperative CKMB levels. METHODOLOGY Between January and June 2008, 100 patients with double and triple vessel disease, who were candidates for coronary artery bypass surgery were prospectively randomized into 2 groups. Group A (n=50) patients undergoing ripc prior to bypass surgery. Group B (n= 0) or controlled group, undergoing bypass surgery without ripc. Random allocation of patients to either ripc group or controlled group was done by lottery method. Ward Incharge was given 100 sealed non-transparent envelops allocating patient to either of the study group. These sealed envelops were sent to operation theatre along with patient s documents. Envelops were opened by the operating surgeon or anaesthetist incharge, and patients were placed in either of the study group, mentioned in the envelop. The ripc protocol comprised of 3 x 5 minutes of forearm ischemia, induced by a blood pressure cuff inflated to 200 mmhg with an intervening 5 minutes of reperfusion during which the cuff is deflated. The control protocol consisted of placing a deflated cuff on upper arm for 30 minutes. This ischimia protocol was adapted from a similar study by Cheng et al. in children undergoing corrective open heart surgery. 12 The ripc protocol was implemented after the patient was anaesthetized and completed prior to placing the patient on extra corporal bypass circuit. All the procedures were performed by a single surgeon to keep the operator dependant variables such as cross clamp time and bypass time within a predictable limit. All patients undergoing elective CABG surgery were included in the study. The exclusion criteria consisted of significant renal or hepatic disease, which could influence the enzyme kinetics in the blood, haemodynamic instability ECG changes and/or raised cardiac enzymes suggesting ongoing ischemia and infarction, acute MI during last 4 weeks, and significant peripheral vascular disease. Written informed consent were taken from all the patients. Project was approved by the ethical committee of the hospital. Study protocol was approved by CPSP review board. Standard non-pulsatile moderate hypothermic cardiopulmonary bypass with a membrane oxygenator was used. Warm blood hyperkalemic cardioplegia delivered through either antegrade or both antegrade and retrograde route in patients with left main stem (LMS) or equivalent disease was used to ensure myocardial protection. All patients were operated under general anaesthesia. An aneroid cuff was placed either on right or left upper arm. In patients undergoing ripc, the cuff was inflated to 200 mmhg for 5 minutes, followed by a 5 minutes interval in which cuff was deflated to allow reperfusion. Three cycles of induced ischemia followed by reperfusion were carried out. Pedical left internal memory artery (LIMA) and saphenous vein grafts were harvested. Patient was heparinized to achieve the activated clotting time (ACT) between 400 to 600 seconds. Aortic and two stage venous cannulation done and cardiopulmonary bypass established. Systemic temperature was lowered to 34 o C. Heart was arrested by giving cardioplegia after clamping aorta. Target vessels anastomosed with conduits. Aortic clamp released after anastomosing LIMA to left anterior descending artery (LAD). Proximal ends of vein grafts anastomosed to aorta. Patient weaned off from cardiopulmonary bypass. Effects of heparin reversed with a measured dose of protamine before decannulation. CKMB in serum was measured on photometric system, Vitalab Selecta. The reagent used was CKMB-FS. CKMB levels 5 times or more, the upper limit of reference range (> 5 ULR) were taken as suggestive of peri operative myocardial infarction. 13 Statistical analyses were performed using statistical software package SPSS version Independent variable was ripc stimulus. Dependent variables were postoperative. CKMB levels measured at 8, 16, 24 and 48 hours after surgery. Confounding variables were cross clamp time, bypass time and body mass index (BMI). Data is presented as mean + SD. Comparison between treatment groups was made with the unpaired student t test. A value of p < 0.05 was regarded as significant. RESULTS No significant difference was noted in terms of age in either group. Mean age in ripc group was years and years in the control group. Patient s characteristics such as co-morbid conditions, signs and symptoms and medication history is given in Table I. 428 Journal of the College of Physicians and Surgeons Pakistan 2010, Vol. 20 (7):

3 Induced remote ischemic pre-conditioning on ischemia reperfusion injury in patients undergoing coronary artery bypass Table I: Patient characteristics. Control (n=50) RIPC (n=50) Age (years) Male/female 42/8 47/3 Diabetes 8 (16%) 11 (22%) Hypercholesterolemia 18 (36%) 21 (42%) Hypertension 22 (44%) 13 (26%) Previous myocardial infarction 8 (16%) 11 (22%) Previous stroke Smoking Current smokers 3 (6%) 02 (4%) Ex-smokers 9 (18%) 07 (14%) Never smoked 38 (76%) 41 (82%) Family history of IHD 04 (8%) 03 (6%) Body-mass index Dyspnoea NYHA class Class I 27 (54%) 24 (48%%) Class II 22 (44%) 22 (44%) Class III 01 (2%) 04 (8%) Angina CCS class Class I 6 (12%) 04 (8%) Class II 31 (62%) 34 (68%) Class III 11 (22%) 12 (24%) Class IV 02 (4%) 00 Ejection fraction > 55% 27 (54%) 23 (46%) 30-55% 19 (38%) 23 (46%) < 30% 04 (8%) 04 (8%) Drug history Aspirin 03 (6%) 05 (10%) β blocker 45 (90%) 43 (86%) ACE-inhibitor/ACE antagonist 22 (44%) 20 (40%) Long-acting nitrates 44 (88%) 42 (84%) Data are given as percentage (%) and SD; CCS=Canadian Cardiovascular Society; IHD= Ischemic heart disease; NYHA=New York Heart Association; RIPC=Remote ischemic pre-conditioning. Pre-operative intra aortic balloon pump (IABP) was placed in 8 individuals, 4 patients of each group. All those individuals with IABP had low left ventricular ejection fraction (LVEF < 30). None of the patients experienced peri-operative myocardial infarction based on the ECG and CKMB levels criteria. There were no major neurological complications in either group. Endarterectomy was carried out in 20 patients, 11 patients from the ripc group and 9 from the control group. Postoperative mild inotropic support was given in 19 patients out of 50 in the ripc group and 22 patients out of 50 in the control group. BMI (body mass index) in patients in ripc group was while in control group BMI was (p=0.363). Number of grafts were slightly more in control group. Mean compared to ripc group (p=0.1552). Bypass time in ripc group was minutes, while in control group it was minutes (p=0.945). Clamp time in ripc group minutes, and in control group was minutes (p=0.883). Postoperative CKMB levels were measured at 8, 16, 24 and 48 hours. Base line CKMB levels in both the groups were similar. Baseline CKMB levels in patient undergoing RIPC protocol were slightly higher than the control group but were statistically insignificant i.e iu/l as opposed to iu/l (p=0.725). Eight hours after surgery CKMB levels were significantly lower in ripc group as compared to control group i.e iu/l in ripc group compared to iu/l in control group with p= Similar trend was noticed at 16, 24 and 48 hours postoperative period. These values are summarized in Table II. Table II: CKMB levels (iu/l) in control and ripc group with p-values at 8, 16, 24 and 48 hours. CKMB n= Mean +SD (iu/l) p-value Control ripc Pre bypass p = hours p = hours p = hours p = hours p = DISCUSSION This study showed that remote ischemic preconditioning mediated by transient limb ischemia can reduce myocardial injury and thus CKMB levels in patients undergoing coronary artery bypass surgery. Cardiopulmonary bypass (CPB) is a highly sophisticated life support system in cardiovascular surgery. The system is used to temporarily perform the functions of the heart (circulation of blood) and lungs (gas exchange) during surgical procedures on the heart and great vessels. In most CPB procedures, a cross clamp is applied to the ascending aorta proximal to the site of aortic cannulation, excluding the coronary arteries from the extracorporeal circuit. This makes it possible to arrest the heart by giving cardioplegia through the aortic root and providing a non-contracting heart and a field without blood in which surgeons can work. However, application of aortic cross clamp itself causes myocardial ischemia. Various types of cardioplegia preparations greatly reduce metabolic O 2 requirement of the heart during X-Clamp application but do not abolish it completely. Reduced oxygen supply to myocardium during ischemic clamp time impairs mitochondrial respiration, depleting cells of ATP with subsequent build-up of anaerobic metabolites, reactive oxygen species and intracellular calcium leading to cell death via necrosis and apoptosis. 14 Although successful reperfusion is mandatory for tissue salvage, re-establishing blood flow institutes a cascade of events similar to an inflammatory response. This involves adhesions of circulatory neutrophils to the vascular wall with subsequent tissue invasion and release of proteases, elastases and reactive oxygen species. While this may be an integral part of healing process, it may also contributes to tissue injury. 15 Journal of the College of Physicians and Surgeons Pakistan 2010, Vol. 20 (7):

4 Nasir Ali, Farwa Rizwi, Afsheen Iqbal and Azhar Rashid Endogenous protective mechanisms against a range of tissue insults have been identified in all mammalian species. The best characterized is ischemic preconditioning, whereby sub-lethal ischemia induces a state of protection against subsequent prolonged ischemia. Although this phenomenon was described almost 15 years ago, it is slow to be translated into clinical practice. Several clinical trials have recently reported that remote ischemic pre-conditioning reduces myocardial injury after major cardiovascular surgery. 16 This phenomenon has been extensively investigated in myocardium of many species, where it can reduce infarct size by up to 75%. 17 Mechanisms involved in ischemic pre-conditioning are very complex. They induce an early phase preconditioning, which occurs in minutes after applying sublethal ischemia and late phase pre-conditioning that occurs 24 hours after the pre-conditioning stimulus and lasts up to 72 hours. 18 In early pre-conditioning, number of stimuli, generated during hypoxia such as adenosine, bradykinin, endogenous opiates and reactive oxygen species, activate complex second messenger system that converge on mitochondria of ischemic tissue to activate K + -ATP channels, which is responsible in maintaining cell homeostasis and protecting it from ischemic insult. 19 A late phase of pre-conditioning also termed as second window of protection occurs 24 hours after the pre-conditioning stimulus. This is more prolonged than early phase and lasts up to 72 hours. The prolonged interval (24hours) between preconditioning event and its renewed protection after 24 hours is consistent with new protein synthesis which triggers events inside the target cells, similar to early pre-conditioning. 20 Although local ischemic preconditioning phenomenon has been widely studied in animal models, it has failed to establish as a regular practice in medicine, This is due to the fact that preconditioning stimulus has to be applied directly to the organ which is at risk, which poses obvious logical difficulties, when the organ is the human heart or brain. The protective effects of remote ischemic preconditioning in animal models have been demonstrated. The magnitude of this protection approaches that of local ischemic pre-conditioning in the context of myocardium. Detailed mechanism remains unclear how protection is transferred from one tissue to another. 21 In ripc there is evidence for a role of autonomic nervous system in pre-conditioning distant organs. Studies have shown that this protection can be abolished by ganglionic blockers like hexamethonium, without affecting local pre-conditioning. 22 However, unidentified humoral mediators have also been suggested in other studies since protection is being transferred from on animal to another by administration of a plasma fraction or whole blood. 23 This clinical study was designed to prove or otherwise the concept of remote ischemic pre-conditioning by measuring postoperative CKMB levels in CABG patients. This study was carried on relatively stable patients undergoing CABG surgery to keep the patient and disease associated variables within predictable limits. However, one might expect that in high-risk patients or in procedures with extended cross clamp and bypass time remote ischemic pre-conditioning might confer an even greater reduction in myocardial injury. This study is based on measuring post procedure CKMB levels. 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