Experimental And Clinical Studies Of Noninvasive Evaluation Of Anthracycline-induced Myocardial Damage During The Subclinical Stage By New Techniques Of Ultrasound

BackgroundAnthracycline(ANTH) such as adriamycin(ADM) and epirubicin(EPI) is one of the most effective antineoplastic agents for the treatment of a variety of malignancies. However, the therapeutic benefit of these agents has been limited by their myocardial toxicity which is primarily related to the cumulative dose of the drug administered.In such cases,which may result in congestive heart failure. So,early detection of myocardial damage might enable implementation of preventive measures that could reduce the likelihood of congestive heart failure later in life. Various techniques have been advocated for the detection of anthracycline-induced myocardial damage, including serial measurements of cardiac enzymes, electrocardiography,echocardio-graphy and radionuclide angiography. These techniques, however,do not necessarily improve the sensitivity and/or specificity of detecting the early,subclinical stage of anthracycline-induced myocardial damage. Therefor,an feasible method should be applied to provide quantitative information for detecting the early phase of anthracycline-induced myocardial damage.In the current study .we assess the subclinical stage of anthracycline-induced myocardial damage using integrated backscatter (IBS),quantitative tissue velocity imaging(QTVI) and tissue tracking imaging(TTI). Objective1. To evaluate the experimental value of IBS and QTVI measurement in detecting adriamycin-induced myocardial damage during the subclinical stage in rabbits. And to assess adriamycin-induced myocardial damage model in rabbits during the subclinical stage through dynamicly monitoring using IBS and QTVI.And then to assess congestive heart failure induced by adriamycin in rabbits.2. To study the changes of left ventricular systolic function evaluated by TTI and to determine cardiomyocyte apoptosis in adriamycin-induced myocardial damage in rabbits treated with three different body weight of adriamycin by immunohistochemical method and by transmission electron microscope(TEM).And to study the relationship between changes of left ventricular systolic function and cardiomyocyte apoptosis.3. To evaluate the value of IBS and QTVI measurement in detecting epirubicin-induced myocardial damage during the subclinical stage in breast cancer patients after operation. And to assess their left ventricular function by Tei index obtained by QTVI.MethodsPart oneFourty-two New Zealand purebred white rabbits were stochastically divided into four groups(10,10,10 and 12 rabbits in groups A,B,C and D respectively),and rabbits in groups B,C and D were given 2 mg/kg body weight of ADM intravenously once a week for 2 weeks,4 weeks and 8 weeks,and total dose 4 mg/kg,8 mg/kg, 16 mg/kg body weight respectively .Ten control rabbits received 2 ml/kg body weight of normal saline once a week for 8 weeks.After 12 weeks,IBS,QTVI and conventional echocardiographic parameters were obtained. These parameters were as follows:left ventricular end diastolic diameter(LVDd),left ventricular end systolic diameter(LVDs), interventricular septum thickness diastolic(IVSTd),left ventricular pesterior thickness diastolic(PWTd), left ventricular ejection fraction(LVEF),fraction shortening(FS),E velocity(E),cyclic variation of integrated backscatter(CVIB), normalized integrated backscatter(IBS%), systolic peak velocity(Vs), systolic acceleration(a),early diastolic peak velocity(Ve) and E/Ve. The pathological examination was determined after ultrasonography at 12th week.The rabbits in groups A,C and D were detected by conventional echocardiography, IBS and QTVI every week before being given adriamycin to order to observe the changes of the parameters.Part twoThe animal and group were the same as the part one.After 12 weeks.the systolic mitral annular displacement(MAD) at the mitral annulus (posterior interventricular septum and left ventricular lateral wall) were measured by TTI(Ds) and by M-mode echocardiography(Dm),and average values of two points were taken.The pai'ameters of left ventricular ejection fraction(LVEF) were also obtained by 2D echocardiography using Simpson's rule.Apoptotic cardiomyocytes were detected by TEM.And the index of apoptotic cardiomyocytes was examined by using the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling method(TUNEL).The expression of Bcl-2 and Bax proteins was also determined by immunohistochemical method.and Bcl-2/Bax was calculated.Part threeA total of 133 female breast cancer patients after operation were divided into three groups,40 patients in group A without chemotherapy served as the control group. The patients in groups B and C were given the classic chemotherapy(EPI,Cyclophosphane and Fluorouracil).45 patients in group B were given a total dose of EPI 180～240 mg/m~2,48 patients in group C were given a total dose of EPI 360～420mg/m~2. IBS, QTVI and conventional echocardiographic parameters were obtained. These parameters included LVDd, LVDs, IVSTd, PWTd, FS, LVEF, E velocity/A velocity(E/A),CVIB, IBS%,Vs,Ve,atrial contraction(Va) and Ve/Va.And isovolumic contraction time(ICT), isovolumic relaxation time(IRT) and ejection time(ET) were also deteced by QTVI,and then Tei indices were calculated. ResultsPart one1. Conventional echocardiography at 12th week No significant difference of conventional echocardiographic parameters were found between groups A and B(p>0.05). LVDd and LVDs of group C enlarged slightly within normal range(p>0.05),and LVEF, FS also slightly decreased within normal range(p>0.05),but E velocity decreased significantly compared with those in group A(p<0.05).LVDd and LVDs in group D enlarged significantly compared with those in groups A and C(p<0.01),IVSTd and PWTd in group D weakened significantly compared with those in groups A and C(p<0.05), LVEF,FS and E in group D velocity decreased significantly compared with those in groups A and C(p<0.01),and four cases showed pericardium in group D.2. IBS at 12th week No significant difference of IBS parameters were found between groups A and B.IVS-IBS% and PW-IBS% were significantly higher in group C than those in group A(p<0.05),IVS-CVIB and PW-CVIB in groups C were lower than those in group A(p<0.05). IVS-IBS% and PW-IBS% were significantly higher in group D than those in groups A and C(p<0.01),IVS-CVIB and PW-CVIB in group D were significantly lower than those in group A(p<0.01).3. QTVI at 12th week No significant difference of QTVI parameters were found between groups A and B. Average Vs,a and Ve were significantly lower in group C than those in group A(p<0.01).E/Ve was significantly higher in group C than that in group A(p<0.01).Average Vs,a and Ve were significantly lower in group D than those in groups A and C(p<0.01).E/Ve was significantly higher in group D than those in groups A and C(p<0.01).4. Pathology at 12th week The pathological examination demonstrated that myocardial pathology in group A was normal,howevere the damages increased from groups B,C to group D.5. The results of group A and C every week (1)No significant difference of conventional echocardiographic parameters were found between two groups except E velocity.E velocity was significantly lower in group C than that in group A at 7th week.(2) IVS-CVIB and PW-CVIB in group C were considerably lower than those in group A at 3rd week(p<0.05);At 4th week, IVS-IBS% and PW-IBS% were significantly higher in group C than those in group A (p<0.05).(3)Vs,a and Ve were significantly lower in group C than those in group A at 7th week (p<0.05) and at 8th week(p<0.01).6. The results of group A and D every week (1)E velocity was significantly lower in group D than that in group A at 4th week(p<0.05) and at 8th week (p<0.01).LVDd and LVDs in group D enlarged significantly compared with those in group A at 8th week(p<0.05) and at 12th week(p<0.01).(2)CVIB in group D was considerably lower than that in group A at 3th week(p<0.05) and at 12th week(p<0.01). IBS% were significantly higher in group C than that in group A at 4th week(p<0.05) and at 8th week(p<0.01).(3)Vs,a and Ve were significantly lower in group C than those in group A at 4th week(p<0.05) and at 7th week(p<0.01).Part two1. Conventional echocardiography There was no significant difference between groups A and B(p>0.05).Average Dm was significantly lower in group C than that in group A(p<0.01),LVEF was slightly lower in normal range in groupC(p>0.05).Average MAD determined by M-mode echocardiography(Dm) and LVEF were significantly lower in group D than those in groups A and C(p<0.01).2. TTI There was no significant difference between groups B and A(p>0.05). Average Ds was significantly lower in group C than that in group A(p<0.01). Average MAD determined by TTI(Ds) was significantly lower in group D than that in groups A and C (p<0.01).3. Mean Ds were closely correlated with mean Dm(p<0.001, p<0.001,p<0.01, p<0.04) and LVEF(p<0.001,p<0.001,p<0.01,p<0.04)in four groups.4. Apoptotic cardiomyocytes There were no apoptotic cardiomyocytes in groups A and B by TEM,however apoptotic cardiomyocytes were found in groups C and D.5. Apoptotic index There were no apoptotic cardiomyocytes in groups A and B,however the indices of apoptotic cardiomyocytes exmined by TUNEL were (3.03±0.78) in groups C and (16.78±3.51) in group D.The index of apoptotic cardiomyocytes were significantly higher in group D than those in group C(p<0.01).6. The expression of Bcl-2 and Bax protein There were no significant difference between groups A and B(p>0.05).The expression of Bcl-2 protein was significantly lower in groups C and D than that in group A(p<0.01),however the expression of Bax protein was considerably higher in groups C and D than that in group A(p<0.01).Bcl-2/Bax was significantly lower in groups C and D than that in group A(p<0.01).The expression of Bcl-2 protein was considerably lower in group D than that in group C,however the expression of Bax protein was significantly higher in group D than that in group C.Bcl-2/Bax was considerably lower in group D than that in group C.7. Ds in groups C and D were closely correlated with the index of apoptotic cardiomyocytes (r=-0.69, p<0.01),the expression of Bcl-2 protein(r=0.66,p<0.01) and the expression of Bax protein (r=-Q.76, p <0.01).Part three1. Conventional echocardiography The conventional echocadiographic parameters had no significant difference between groups A and B,B and C(p>0.05).The conventional echocadiographic parameters (E/A was not included) had no significant difference between groups A and C(p>0.05),E/A was considerably lower in group C than that in group A(p<0.01).2. IBS There were no significant difference between groups B and A(p>0.05). IVS-IBS% and PW-IBS% were significantly higher in group C than those in groups A and B(p<0.01).IVS-CVIB and PW-CVIB were significantly lower in group C than those in groups A and B(p<0.01).3. QTVI There were no significant difference between groups A and B(p>0.05).Ve and Ve/Va were significantly lower in group C than those in groups A and B(p<0.01).Va was considerably higher in group C than that in groups A and B(p<0.01).Vs had no significant difference between groups A and C,groups B and C(p>0.05). 4. Tei index There were no significant difference between groups A and B(p<0.01),but the Tei indices obtained by QTVI were significantly higher in group C than those in groups A and B(p<0.01).ConclusionsPart one1. Conventional echocardiography can evaluate congestive heart failure due to adriamycin-induced myocardial damage in rabbits,but it was not able to assess adriamycin-induced myocardial damage during the subclinical stage in rabbits.2. IBS and QTVI can assess not only congestive heart failure but also adriamycin-induced myocardial damage during the subclinical stage in rabbits.They can show the degrees of pathological changes.3. QTVI can evaluate the left ventricular abnormal function through detecting the systolic mitral annular velocity in rabbits,especially abnormal left ventricular diastolic function.4. IBS and QTVI are valuable not only to assess adriamycin-induced myocardial damage model in rabbits during the subclinical stage but also to evaluate congestive heart failure induced by adriamycin in rabbits.And a good model in the same pathological changes can be established through dynamicly monitoring using IBS and QTVI.Part two1. TTI is a nonvasive and valuable method to assess the changes of left ventricular systolic function due to adriamycin-induced myocardial damage in rabbits.2. Adriamycin can lead to cardiomyocyte apoptosis which take part in the process of the adriamycin-induced myocardial damage in rabbits.3. Cardiomyocyte apoptosis due to adriamycin-induced myocardial damage in rabbits in groups C and D were closely correlated with the expression of Bcl-2 protein and the expression of Bax protein.4. Cardiomyocyte apoptosis due to adriamycin-induced myocardial damage in rabbits take part in the changes of decreasing left ventricular systolic function.Part three1. It was no valuable to assess epirubicin-induced myocardial damage during the subclinical stage in breast cancer patients after operation by conventional echocardiography.2. IBS and QTVI can nonvasively and quantitively assess epirubicin-induced myocardial damage during the subclinical stage in breast cancer patients after operation.3. The Tei indices obtained by QTVI can evaluate the left ventricular function of epirubicin-induced myocardial damage during the subclinical stage in breast cancer patients after operation.