Protective effect of tacrolimus on myocardial ischemia/reperfusion injury in rats

1University of Health Sciences, Ankara City Hospital, Department of Cardiovascular Surgery, Ankara/TURKEY 2Gaziantep University, Vocational School of Health Services, Department of Operating Room Services, Gaziantep/TURKEY 3Tepecik Training and Research Hospital, Department of Pediatric Cardiovascular Surgery, Izmir/TURKEY 4University of Health Sciences, Ankara City Hospital, Department of Pediatric Cardiovascular Surgery, Ankara/TURKEY 5Gaziantep University, Medical Faculty, Department of Medical Pharmacology, Gaziantep/TURKEY 6Dokuz Eylul University, Faculty of Medicine, Department of Cardiovascular Surgery, Izmir/TURKEY Takrolimusun sıçanlarda miyokardiyal iskemi / reperfüzyon hasarı üzerindeki koruyucu etkisi Original Article


Introduction
Myocardial ischemia/reperfusion (I/R) injury leads to severe arrhythmias and associated with high risk of mortality [1].
Accumulation of reactive oxygen species and cardiomyocyte apoptosis play a role in the pathogenesis of myocardial I/R injury [2]. Inflammatory cascade is activated with reperfusion and cardiomyocyte death and apoptosis occur during this period. Although percutaneous coronary intervention (PCI) has a positive effect on survival of patients with acute myocardial infarction (AMI), there is evidence suggesting that PCI increases the heart failure risk in patients with AMI, as it leads to myocardial I/R that result with myocardial injury and cardiomyocyte death [3]. Thus, novel therapeutic aproaches are needed for preventing myocardial I/R injury.
Tacrolimus, also known as FK506, is a calcineurin inhibitor, and acts as an immunsuppresive agent. It is used typically in organ transplantations to reduce the risk of graft rejection and autoimmune diseases [4]. Several studies have revealed that tacrolimus has an antioxidative and antiapoptotic effects [5].
It blocks the catalytic activity of calcineurin and this leads to gene repression that regulates the production of adhesion molecules and cytokines. Due to this repression with tacrolimus, inflammatory cell response, platelet activation, neutrophil adhesion and aggregation decrease in I/R injury [6].
In this study, we aimed to investigate the protective effect of tacrolimus on myocardial I/R injury in a rat model by using hemodynamical, histopathological, and biochemical evaluations.

Animals
Adult male Wistar Albino rats (mean weight, 252±20 g; age, 46-54 days) were included to this study. Rats were housed in a climate-controlled room (temperature 25±2oC; humidity 50-60%) on a 12 h light/dark cycle and ad libitum access to food and water. All the study protocols were approved by the Insti-

Study Groups
Rats were randomly assigned into three groups: Group 1 (sham, n=4), Group 2 (I/R+saline, control, n=7), Group 3 (tacrolimus+I/R, n=7). Tacrolimus (0.1 mg/kg body weight; Prograf R ampul, Fujisawa Pharmaceutical Co., Japan) was administered as an intravenous infusion in the first 15 min of reperfusion after 45 min ischemia period [7]. Rats in the control group were administered with the same volume of saline.

Anesthesia and Monitorization
Ketamine (Ketalar R flacon, 100 mg/ml, Alfasan International, Holland) 35 mg/kg and xylazine (Alfazyne R, 20 mg/ml, Alfasan International, Holland) 5 mg/kg were used for anesthesia. Entubation was performed via tracheostomy. Mechanical animal respiratory vehicle (Ugo Basile SRL, Rodent Ventilator, Italy) was used with the 100% O2 support, 15 ml tidal volume, and respiratory rate of 60/min. Carotid artery was catheterized with 24 G branule for continous pressure monitorization and jugular vein was catheterized with the same way for saline and tacrolimus administration. Heart rate and blood pressure were followed continously by using ECG-pressure monitore during the operation and data were recorded. During the experiment, body temperature was kept at 37°C with the use of a desk lamp.

Surgical Procedure
Left thoracotomy was performed to reach the heart. Following the exploration of the heart, 600 IU/kg heparin sodium (Nevparin R vial, 5000 IU/ml, Mustafa Nevzat Pharmaceutical Company, Turkey) was given intravenously to prevent thrombosis in the coronary artery at the begining of the 15 min hemodynamic stabilization period. After that, 6/0, 10 mm atraumatic prolene suture with snare tape was used for left anterior descending artery (LAD) occlusion. Ischemia was achieved by tightening the snare. Duration of ischemia was 45 min. Reperfusion was initiated by releasing the snare. Successful occlusion was confirmed by a reduction in arterial blood pressure.
Reperfusion continued for 3 hours. After sacrification, the heart was removed and sent to pathology laboratory within icy water immediately. Sham group was underwent a similar surgical procedure wherein coronary artery was not occluded.

Calculation of Area at Risk and Infarct Size
Cardiac area at risk and infarct size determination was performed as described previously [8]. At the end of experiments, the left anterior descending coronary artery was occluded again at the same site as previously, and 3 ml of a 2% solution of Evans blue dye was infused into the jugular vein catheter to distinguish between perfused and non-perfused (area at risk) sections of the heart. The Evans blue solution stains the perfused myocardium, while the occluded vascular bed remains uncoloured. Then the heart was excised. Both atria and the roots of the great vessels were removed. The entire ventricle was cut, from the apex to the base, into slices of 3-4 mm, the right ventricular wall was removed, and the area at risk (pink) was separated from the non-ischemic (blue) area. The area at risk was cut into small pieces and incubated with a 1% solution of 2,3,5-triphenyltetrazoliumchloride (TTC, in 20 mM phosphate buffer, pH 7.4) stain for 30 min at 37oC, to visualize the infarct area. The area at risk of infarction was colored brick red, and the infarcted area within the region at risk remains pale yellow (i.e. necrotic area). To fix the color difference in the sections after the procedure, tissues were kept in 10% formalin solution in 20 min, and the field was analyzed by transferring the images to the computer with the help of video image (Image Pro-plus-Media Cybernetics, Silver Spring, Maryland, USA). The ischemic damage area of the myocardium was measured using a morphometric program for each heart slice, and averages of these measurements were presented.

Biochemical Measurements
Intracardiac blood samples were collected at the end of the experiment. The samples were centrifuged at 2400 g, 4oC,

Histopathological Examination
The sections that were sliced from apex to the bottom into four pieces for the calculation of the infarct area were processed with hematoxylin-eosin while they were kept in 10% formalin solution. Myofibrillar edema, myocytolysis, focal hemorrhage and polymorphonuclear leukocyte (PMNL) infiltration were examined using light microscope after this procedure, and pathological scoring was performed as: 0-none, 1-mild, 2-moderate, 3-severe.

Statistical Analysis
All data are expressed as means±S.E.M. or the percentage incidence. Statistical analyses were performed using SPSS 13.0 for Windows program. Statistical analysis between two experimental groups was performed using a Student's t test. Statistical comparison of more than two groups was performed by a one-way analysis of variance followed by Student-Newman-Keuls multiple comparisons test. The Mann-Whitney Utest was used to detect significant differences between histopathological scores. In all tests, p values less than 0.05 were considered to be statistically significant. 304 COMAKLI Et al.

Results
Tables 1 and 2 summarize mean arterial blood pressure and heart rate in all groups, respectively.
Occlusion of the LAD coronary artery produced a marked decrease in blood pressure. Tacrolimus had no marked effect on mean arterial blood pressure and heart rate. There were no marked differenes in pH, pO2, pCO2, and hematocrit values between the groups as shown in Table 3. Additionally, no significant change in CK-MB levels was observed (Table 3).
Area at risk and infarct size in myocardial sections of control and tacrolimus groups was shown in Figure 1. Although there were no change in area at risk, infarct size was markedly reduced in tacrolimus group when compared to control group.
Histopathological evaluations of all groups were presented in Table 4. Myofibrillar edema, myocytolysis, focal hemorrhage and PMNL infiltration were markedly increased in I/R control group when compared to sham group. Tacrolimus significantly attenuated these parameters (Table 4).

Discussion
AMI is the major cause of mortality and morbidity worldwide.
The purpose of the treatment choices of patients with MI is to reduce acute myocardial ischemic injury and limit MI size by maintaining effective myocardial reperfusion. However, reperfusion can itself cause cardiomyocyte death known as myocardial I/R injury [9]. Myocardial I/R injury activates the inflammatory response that involves production of oxidants, activation of complement and infiltration by polymorphonuclear neutrophils [10]. Myocardial necrosis, apoptosis and stunning may ocur as a result of this cascade [11]. Infarct size is the most important indicator of long-term mortality and chronic heart failure, so limiting the extent of necrosis during MI has a vital importance. Studies concerning the therapies that focus on to reduce reperfusion injury are available in the literature [12].
Tacrolimus is an immunosuppressive drug and typically used for organ transplantation, atopic dermatitis, various autoimmun and inflammatory diseases [13]. It shows its effect by inhibiting calcineurin, thus; cytokines, lymphokines and adhesion molecules that play role in immune and inflammatory response are being repressed [14].  its the PMNL accumulation and protects against myocardial I/R injury in rats [28]. We have found a statistically significant difference in terms of PMNL infiltration between tacrolimus group and control group.
Our findings have shown that there were no statistically significant differences in blood gas parameters including pH, pO2, pCO2, and hematocrit between the groups. Similar results were obtained by Mohebbi and colleagues who showed that there was no change in systemic acid-base status with tacrolimus treatment after 9 days [29].

Conclusion
In conclusion, our data showed that tacrolimus has cardioprotective effects in myocardial I/R injury in rats. Although various studies in different animal models have been published, further clinical studies are required for determining the tacrolimus efficacy on myocardial I/R injury.