Thirty domestic pigs of either sex were used in this study. The present study has been carried out in accordance with the Guide for the Care and Use of Laboratory Animal as adopted and promulgated by the U.S. National Institutes of Health and has been approved by the Institutional Animal Care and Use Committees of the Faculty of Medicine, Chiang Mai University. The pigs were anesthetized by a combination of atropine (0.04 mg/kg), telazol (4.4 mg/kg), and xylazine (2.2 mg/kg), and maintained by 1.5-3.0% isoflurane delivered in 100% oxygen. The vital signs including femoral arterial blood pressure (BP), heart rates (HR), lead II electrocardiogram, respiratory rate and core temperature were continuously monitored for the entire study. The PaO2, end-tidal CO2 and blood gases were measured and maintained under physiologic condition. Under artificial respiration, a 34-mm platinum coated titanium coil electrode catheter (Guidant Corp.) was inserted into the right ventricular apex and a 68-mm electrode catheter at the junction between right atrium and superior vena cava (Chattipakorn et al., 2006d,Kanlop et al., 2008f,Shinlapawittayatorn et al., 2006c,Sungnoon et al., 2008a). These 2 electrodes were used to deliver strong stimulus during VFT and DFT determination. Median sternotomy was done and the heart was suspended in a pericardial cradle. The left anterior descending coronary artery (LAD) was dissected from its surrounding tissues.
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Two protocols were performed in this study; one in pigs with normal hearts without ischemia (n=14) and another in pigs with ischemic/reperfusion hearts (n=16). In each experimental model, pigs were divided equally into 2 groups; G-CSF and vehicle. In each pig, cardiac electrophysiological parameters, including diastolic pacing threshold (DPT), effective refractory period (ERP), VFT and DFT were determined at the beginning of the study. G-CSF (0.33ïg/kg/min) (Takahama et al., 2006e) and vehicle (normal saline solution of similar amount) were administered intravenously for 30 minutes in G-CSF and vehicle groups, respectively. In the intact heart model, the cardiac electrophysiological parameters were determined before and after the drug or vehicle administration. In the ischemic/reperfusion model, the LAD was ligated at 5 centimeters above a distal branch of LAD to perform a complete regional occlusion. The ischemic period was sustained for 45 minutes and then the LAD was reperfused (Krug et al., 1966). During the first 20 minutes of occlusion, defibrillation shock would be delivered to terminate VF if VF spontaneously occurred. The time interval for the first VF to occur after LAD occlusion was determined in each pig. Once the spontaneous VF disappeared or did not occur within 20 minutes, VF would be electrically induced for the VFT determination (Qin et al., 2002a). The DFT was also determined simultaneously with VFT determination. At the time of reperfusion, G-CSF or vehicle was infused again and all parameters were determined at 30 minutes after the reperfusion (Chattipakorn et al., 2006c,Kanlop et al., 2008e). Illustration of the study in the ischemic-reperfusion protocol is shown in Figure 1.
A train of 10 S1 stimuli (square, 5-ms pulse width, 500-ms interval) was delivered from the tip of RV pacing electrode to determine the DPT. The pacing current was initially at 0.1 mA and increased in 0.1-mA steps until all stimuli exerted their ventricular responses. This pacing current was defined as the DPT. The ERP was determined by delivering an S2 stimulus after the last S1. The S1-S2 interval was initially set at 350 ms and was decremented in 10-ms steps until an S2 stimulus could not elicit a ventricular response. The last S1-S2 interval that elicited a ventricular response was defined as an ERP (Kanlop et al., 2008d).
VFT and DFT determination protocol: VF was induced by delivering a stimulus during the vulnerable period (T-wave of lead II ECG). The coupling interval for VF induction was determined by delivering a train of 10 S1s for 3 times and the interval between the last S1 and the peak-T wave from each train was determined. The average value of these intervals was used as a coupling interval to deliver an S2 stimulus for VF induction (Kanlop et al., 2008c,Chattipakorn et al., 2000a,Chattipakorn et al., 2000b,Chattipakorn et al., 2000c). A biphasic S2 shock (Ventak ECD, Guidant Corp.) was delivered through shocking electrodes for VFT and DFT determination. For VFT determination, the S2 shock strength was initially at 100 V. This strength was reduced or increased in 10-V steps depending on whether VF induction was successful or failed, respectively (Kanlop et al., 2008b). The lowest shock strength required for inducing VF was defined as the VFT (Kanlop et al., 2008a). There was a minimum interval of 4 minutes between each VF episode to allow the hemodynamic status to return to physiological conditions (Sungnoon et al., 2008b). The DFT was determined using a three-reversal up/down protocol (Chattipakorn et al., 2006b,Shinlapawittayatorn et al., 2006b). In brief, the defibrillation shock was initially at 400 V and delivered after 10 seconds of VF. The shock strength was decreased or increased in 80-V steps depending on whether the shock succeeded or failed to terminate VF, respectively. This process was repeated with an increment or decrement of 40-V and 20-V steps. The lowest shock strength required for successful defibrillation after the third reversal was defined as the DFT (Chattipakorn et al., 2006a,Shinlapawittayatorn et al., 2006a).
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G-CSF preparation: G-CSF (Neupogen®) was used for intravenous infusion. A vial of Neupogen® of 1.0 ml contains 30 MU (=300 µg) of filgrastim. G-CSF 1.0 ml was diluted in 9 ml dextrose saline solution (30µg/ml) and intravenously infused for 30 minutes at the rate of 0.011ml/kg/min (0.33µg/kg/min).
Infarct size and area at risk (AAR) measurement: At the end of each experiment, the heart was removed and irrigated with normal saline to wash out blood from chambers and vessels. The LAD was occluded again at the same site previously done during the ischemic period. The catheters were inserted into the right and left coronary ostia for Evan blue infusion. The area which could not be infused by Evan blue was defined as area of no blood flow during ischemic period. The heart was frozen and cut horizontally into 5-mm thick slices, starting from the apex until 5 mm above the occluding site. Then, each slice was immersed in Triphenyltetrazolium chloride (TTC) at least 25 minutes (Takahama et al., 2006d,Khalil et al., 2006), after which the area with viable tissue was seen in red. The area that was not stained with Evan blue was defined as area at risk (AAR). The area which demonstrated neither blue nor red was defined as the infarct site. The area measurements were performed with Image tool software version 3.0. The infarct site was calculated depending on the weight of each slice according to Reiss et al.'s formula (Riess et al., 2009):
Total areaTotal infarct size = ∑ [( Infarct size of slice n x weight of slice n)]
Total weight of slices
Total areaTotal AAR = ∑ [( AAR of slice n x weight of slice n)]
Total weight of slices
Cardiac mitochondria study protocol
In this protocol, cardiac mitochondria were isolated from the hearts of wistar rats using the technique described previously (Larche et al., 2006), and the protein concentration was determined according to Bicinchoninic Acid (BCA) assay (Walker, 1994). The morphology of the isolated cardiac mitochondria was confirmed using the electron microscope. In this protocol, H2O2 (2 mM) was used to mimic the oxidative stress condition occurring during I/R injury (Yang and Cortopassi, 1998a). Cardiac mitochondria were divided into 3 treatment groups (n=5 in each group): 1) a control group, 2) H2O2 treated group, 3) G-CSF (100 nM) plus H2O2 treated group. In group 2, cardiac mitochondria were incubated with H2O2 for 5 minutes, whereas in group 3 cardiac mitochondria were pretreated with G-CSF (100 nM) for 30 minutes followed by H2O2 application for another 5 minutes. At the end-point, cardiac mitochondria in each group were determined for mitochondrial swelling, mitochondrial ROS production, and mitochondrial membrane potential changes.
Determination of cardiac mitochondrial swelling
The change in the absorbance of the mitochondrial suspension (0.4 mg/ml) at 540 nm (A540) was determined using a microplate reader (Ruiz-Meana et al., 2006). The decrease in the absorbance of the mitochondrial suspension indicated mitochondrial swelling.
Determination of cardiac mitochondrial ROS production
The dye dichlorohydro-fluorescein diacetate (DCFDA) was used to determine the level of ROS production in cardiac mitochondria (Novalija et al., 2003). DCFDA could pass through the mitochondrial membrane, and was oxidized by ROS in the mitochondria into DCF. Fluorescence was determined at λex 485 nm and λem 530 nm using a fluorescence microplate reader. The ROS level was expressed as arbitrary units of fluorescence intensity of DCF.
Determination of cardiac mitochondrial membrane potential changes
The dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to determined the change in the mitochondrial membrane potential (Di Lisa et al., 1995,Tong et al., 2005). JC-1 was characterized as a cation and remained in the mitochondrial matrix as a monomer (green fluorescence) form. However, it could interact with anions in the mitochondrial matrix to form an aggregate (red fluorescence) form. JC-1 monomer fluorescence (green) was excited at 485 nm and the emission was detected at 530 nm. JC-1 aggregate fluorescence (red) was excited at 485 nm and the emission fluorescence was recorded at 590 nm. Mitochondrial depolarization was indicated by a decrease in the red/green fluorescence intensity ratio.
Values were expressed as mean ± SD. Analysis of covariance (ANCOVA) was used to assess whether the inequality of the baseline values affected the results after the treatment. Comparisons of variables before and after the drugs or vehicle administration (comparison within group) were performed individually in each group using the Paired, two-tailed student's t- test. Comparisons among treatments of cardiac mitochondria were made by one-way ANOVA followed by the Fisher post-hoc test. P < 0.05 was considered statistically significant.
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