Evaluation Of The Protective Effects Of Nicorandil, An ATP Sensitive K～+ Channel Opener, On Ischemic-reperfused Canine Heart With Myocardial Contrast Echocardiography

Evaluation of the protective effects of Nicorandil, on ATP sensitive K~+ channel opener, on ischemic-reperfused canine heart with myocardial contrast echocardiographyIntroductionRepeated ischemia and reperfusion may render heart more resistant to the subsequent prolonged ischemia. The phenomenon was termed myocardial ischemic preconditioning. It consists of two stages, the early protection and delayed protection. The latter is more clinically relevant for its longer persistence. The typical ischemic preconditioning is less feasible clinically because of complex manipulation under specially required conditions. The pharmacological preconditioning has important significance for clinical practice. It is believed that ATP sensitive K~+ channel play an important role in the process. In the present study, we evaluated the protective effects of nicorandil, an ATP sensitive K~+ channel opener, on canine heart model with myocardial contrast echocardiography technique, which can access myocardial perfusion dynamically.Material and MethodAnimal model preparationMongrel dogs with either sex were anesthetized with intravenous injection of pentobarbital sodium and intubated for mechanical ventilation. Supplements administration was added as needed for stable anesthesia. Femoral vein was exposed for administration of fluids, drug and contrast media. A vinyl catheter was introduced into abdominal aorta for continuous monitoring of arterial pressure with multi-function heart monitor. Standard ECG was monitored for heart rate and ST segment change. Dogs were draped and median sternotomy was performed. The pericardium was incised longitudinally and suspended as a cradle to expose the heart. LAD, just after the first diagonal branch, was isolated. Thin tape was passed underneath the LAD for flow occlusion. Self-made water bath with warm normal saline was placed on heart surface, which served as an acoustic interface. Echo-probe was placed on the heart at left ventricle long axis with slight clockwise rotation. Coronary flow detection program was initiated to detect intra-coronary blood flow signal. LAD was gently snared until flow signal loss within the distal LAD. This indicated the total occlusion of LAD. Meanwhile the thickness change of left ventricular anterior wall was observed with two-dimensional echocardiography.After 3 hours of total LAD occlusion, the affected area was reperfused for 2 hours.1,Part One: Evaluation of the early protective effects of nicorandil26 mongrel dogs of either sex were randomly divided into following groups. Control group (n=6): left anterior descending artery(LAD) was occluded for 3 hours then the heart was reperfused for another 2 hours. Preconditioning group (n=7): 3 cycles of 5min ischemia with 5min of reperfusion were conducted for preconditioning before LAD occlusion. Nicorandil group (n=7): before LAD occlusion, nicorandil was infusion intravenously, first at a dosage of 100μg/kg as bolus injection and followed by 30min continuous infusion at a dosage of 30μg/kg/min. after nicorandil pretreatment, the LAD was occluded with the same protocol described in the control group. Glibencamide group (n=6): 10 min before infusion of nicorandil (same as that in the nicorandil group, glibencamide was injected intravenously with a dosage of 0.3 mg/kg. The LAD was managed in the same manner.2, Part Two: Evaluation of the late protective effects of nicorandil19 mongrel dogs of either sex were randomly divided into the following 3 groups. Control group (n=6): left anterior descending artery (LAD) was blocked for 3 hours then the heart was reperfused for 2 hours. Nicorandil group (n=7): nicorandil was infusion intravenously, first at dosage of 100μg/kg as bolus injection and followed by 30min continuous infusion at dosage of 30μg/ kg/min. 24 hours after nicorandil pretreatment, dog model was prepared and same LAD occlusion protocol was followed. Glibencamide group (n=6):10 min before infusion of nicorandil (same as that in the nicorandil group, glibencamide was injected intravenously with a dosage of 0.3 mg/kg. 24 hours later, the same protocol as the above group was followed.3, Parameters for evaluationA. Left ventricle ejection fraction: before ischemia (baseline), during ischemia and 120min after reperfusion, left ventricle ejection fraction was calculated with Simpson's method. And the ejection fraction recovery rate was calculated with the formula (EF at 120min after reperfusion/baseline EF)B. Myocardial contrast echocardiography (MCE) protocol: intermittent harmonic imaging was performed in the para-sternal short-axis plain at midpapillary muscle level with the HP 5500 Sonos system. Water bath was placed on heart as an acoustic interface. Mechanical index was set to 1.6 at the beginning of the experiment, the gain, the depth and focus were adjusted at the baseline and the myocardium was almost black and only endocardial borders was visible. The adjustments were kept unchanged throughout the procedure. The baseline images were acquired. Self-made contrast media was injected intravenously as bolus infusion. End-systolic images, triggered by ECG R wave were obtained at pulse intervals ranging of 7 cycles. And images of end diastolic phase 7 cardiac cycles at AD Time-Intensity mode were recorded with papillary muscle level at short axis plain. All of the data were stored on magnetic optic disk for later retrieval.MCE data sampling and analysis: acoustic densitometry (AD) program was used for offline analysis. Myocardial video density time-intensity curves at baseline, 30min, 60min and 120min after reperfusion were analyzed with AD technique for calculation of peak intensity (PI), half time of descent (HT), and area under the curve (AUC), the ratios of these parameters of the lowperfused myoeardium to that of the normal myocardium were also calculated (PIr, HTr, AUCr). Areas of contrast media loss during ischemia (indicating risk area) and during reperfusion (indicating infarct area) were calculated also. The ratios of infarction area to risk area were calculated for comparison with pathologic stain results.C. Pathologic stain for infarction evaluation: at the conclusion of the experiment, the heart was arrested by intravenous administration of saturated KCL. The heart was removed. Evans blue dye was injected into left main, with LAD re-occluded. Then the heart was cut into slices with 3～5mm thickness. The slices were immersed in a solution of 2% triphenyhetrazolium chloride at 37℃for 20min. The infarct myocardium was identified as the region of white color, while the risk area was labeled as brick red. Both of the infarction and risk area were calculated and ratio of infarction to risk area was calculated also.Results1, Part oneIn the nicorandil group, the infarct size was significantly smaller than the control group, it is shghfly higher than the preconditioning group, but the difference didnt reach significant level. The infarct area measured by MCE was well correlated with the result from pathological stain analysis. MCE and AD analysis showed that myocardial perfusion at micro vascular level was better ameliorated in nicorandil group, reflected by higher PI and AUC and shorter HT in the group (p＜0.05). And the left ventricular systolic function was well preserved in nicorandil group indicted by higher ejection fraction. The protection afforded by nicorandil was similar to that in the ischemic preconditioning group and these effects were totally blocked by pretreatment with K~+ channel blocker, glibencamide.2, Part Two24 hours after pretreatment with nicorandil, the infarct area in nicorandil group was still significantly smaller than the control group. After reperfusion, the micro vascular perfusion in the risk area was better than the control group, showed by higher PI, AUC indicating higher blood flow and volume in the area, and shorter HT, only at 120min after reperfusion, representing rapid clearance of contrast media. Also, the left ventricular systolic function was well preserved also. Pretreatment with Glibencamide before nicorandil administration can totally eradicated these protective effects from nicorandil, indicating that K~+ channel plays an important role in the process of late preconditioning process.ConclusionPretreatment with nicorandil can reduce the infarct size, ameliorate micro vascular perfusion and preserve left ventricular systolic function in canine heart model of 3 hours ischemia followed by 2 hours reperfusion. And 24 hours after pretreatment with nicorandil, it can provide significant cardio protective effects on canine myocardium. All these effects can be blocked with glibencamide. Myocardial contrast echocardiography can not only observe the risk and infarct area dynamically but also can evaluate the myocardial perfusion at micro vascular level, indicating the MCE may be used as an effective technique in the research of myocardial ischemic and reperfusion.