Comparative Study On Beagle's LAD Branches Angle Of Subepicardial Intra-myocardium Coronary Artery Between Ultrasonograph And Histopathology

Objective:To investigated the physiological characteristics of blood perfusion of myocardium in coronary circulation by evaluating branches angles between epicardial coronary artery (EPCA)and subepicardial intra-myocardium coronary artery ( SIMCA )( EP-SIMCA-BA ) using transepicardial high-frequency 2D grey scale echocardiography, color Doppler flow imaging(CDFI), myocardial contrast echocardiography(MCE), contrasted with Histopathology. Analyzing the relationship between characteristic of EP-SIMCA–BA and coronary circulation by principle of hydromechanics and characteristic of heart'special motion, and demonstrating its hemodynamics related anatomical characteristics with echocardiographic and patho-histological findings.Methods:⑴Research object:Eight open-chest healthy female beagle models were employed for the research.⑵Images collecting:The connection, orientation and distribution of subepicardial coronary branches from LAD and its distal oblique branch(OB)into sub-epicardium or intra-myocardium were observed with high-frequency transepicardial echocardiography , 2D gray scale imaging, CDFI and MCE. Then, longitudinal and short-axis dynamic image of left ventricular anterior wall with epicardial and subepicardial coronary arteries were acquired⑶Patho-histological slice making and tissue staining: Beagles were executed for euthanasia after echocardiographic image collection by 10% KCL solution or 32.5% BaCL2 solution, which can keep myocardium at end diastole or end systole; The heart were isolated and fixed in 6% formaldehyde solution for further patho-histological slice, stained by Masson.⑷Observating under optical microsope and taking photos: The connection, orientation and distribution of subepicardial coronary branches from LAD and its distal OB into sub-epicardium or intra-myocardium were observed, and photos in region of interesting (ROI) have been taken.⑸Images analysis:In dynamic images of 2D gray-scale ultrasound, CDFI , MCE and photos of patho-histological slice , longitudinal axis views of LAD, superior segments and inferior segments of oblique branch (SSOB, ISOB) were analyzed, as well as two selected SIMCA were displayed, and EP-SIMCA-BA were measured with tne Photoshop software at the end systole and end diastole (i.e.,θs,θd) ,respectively.⑹Statistics analysis:All statistical analysis were performed with Excel 2007 and SPSS13.0 software, all measurement data were expressed as mean±standard deviation (?x±s), test of normality and homogeneity of variance were executed before analysis. When compared means of EP-SIMCA-BA among LAD, SSOB, and ISOB, One-Way ANOVA test and LSD were adopted, and paired samples T Test was used for difference identification of EP-SIMCA-BA in end systole and end diastole, Correlation analysis with Pearson and Bland-Altman plot were used for difference identification of EP-SIMCA-BA in longitudinal axis views of 2D gray-scale imaging and patho-histological slice'photos. Correlation analysis with Pearson and Bland-Altman plot were used for repeatability. or datas abnormal distribution,nonparametric statistics as Wilcoxon(rank sum test)or Spearman(rank correlation) was adopted. The criteria was defined asα=0.05, a value of P <0.05 was considered significant.Results:⑴Echocardiographic and Patho-histological findings:The branches of LAD penetrated vortically into subepicardium from epicardial main trunk with an acute eruption angle either in longitudinal or short-axis section. The angles were reduced from the proximal trunk of LAD to distal part of its branch in the longitudinal sections. The prolonged lines of extension arteries in longitudinal direction was perpendicular obliquely to the longitudinal lines of its superior vessels in the middle myocardial layer of left ventricular anterior wall. EP-SIMCA-BA at left ventricular apex were almost 90°for less myocardial movement. The angles of some coronary artery branches from the vessels of inferior part at apex were obtuse with the reversed originated angle of these branches. At the bifurcation point, a proliferated endarterium cushion was noticed and projected into the chamber of vessels.⑵Eruption angle measurement:In longitudinal sections of 2D gray scale imaging: The angle differences were statistically significant between LAD and ISOB, SSOB (P<0.01), and angles decreased along LAD(i.e., LAD > SSOB > ISOB). Otherwise, there was no the significant difference ofθs andθd between LAD and SSOB except theθs andθd at ISOB( P<0.01).In longitudinal sections of pathological slices: There were significant eruption angle differences between LAD and ISOB, SSOB and ISOB (P<0.01) except between LAD and SSOB(P>0.05). Otherwise, there was no the significant difference ofθs andθd in the same group(P>0.05) .There were uncorrelation of angle between 2D grey scale imaging and patho-histological slice by Spearman(rank correlation)(P>0.05). LAD, ISOB at the end diastole and LAD at the end systole were expressed just consistency changing trends in Bland-Altman plots.There was high degree corrlation of angle measurements between two observers (r =0.76~0.92, P<0.001 or P<0.05). consistency changing trends of two observers'measuring angles of EP-SIMCA-BA were almost noticed in Bland-Altman plots at the end diastle in images of 2D gray-scale imaging and patho-histological slice'photos.Conclusions:⑴The branches of LAD vortically erupted into subepicardium from epicardium in an acute angle with gradual decrease from proximal trunk of LAD to the distal part, apparently changing with cardiac cycle at the distal part. Connecting by acute angle between trunk of superior vessel and interior branch was in line with principle of hydromechanics and characteristic of heart'specially systolic and diastolic motion.⑵The characteristic of coronary artery'wall architecture and branches erupting into subepicardium from epicardial part of LAD vortically and intra-myocardium coronary artery (IMCA) revolving witn myocardium'rotation, which were accorded with the bio-mechanic characteristic of heart'twist motion.⑶The combinated application of high-frequency transepicardial echocardiography, CDFI and MCE was helpful to contiguously observe the characteristics of connection, orientation and distribution of subepicardial coronary branches from trunk into sub-epicardium or intra-myocardium,and the space-time reltationship between diversity of EP-SIMCA-BA and heart motion.⑷The morphological findings from this study will foster the better understanding of the physiological characteristics of blood perfusion of myocardium in coronary circulation and provide animal experimental evidence and methods for the further research of human coronary circulation, as well as the anatomic fundament of coronary artery circulation function for coronary artery interventional therapy.