Experimental Study On The Evaluation Of Doxorubicin Induced Cardiotoxicity By Two-Dimensional Speckle Tracking Echocardiography And Myocardial Contrast Echocardiography

BackgroundDoxorubicin（DOX）,a efficient and broad-spectrum anthracycline antibiotics,are widely used in the chemotherapy of various malignant tumors.DOX has significantly increased cancer survival rates,however,DOX can lead to cardiovascular complications,which affects the long-term prognosis of patients and limits the opportunity of tumor treatment.Left ventricular ejection fraction（LVEF）,usually acquired by convention al echocardiography,is a routinely indicator for monitoring cardiac toxicity during cancer treatment.However,LVEF often fails in detecting accurately early changes in DOX induced cardiotoxicity and cardiotoxicity can lead to irreversible damage to the h eart,there is no effective method to prevent cardiac toxicity.Accordingly,early detection and preemptive therapy of DOX induced cardiotoxicity are crucial.Along with the development of new ultrasound technology,two-dimensional speckle tracking imaging（2D-STI）allows to quantify regional and global myocardial deformation or strain in the longitudinal,circumferential and radial direction with less dependent on volume loading,size,scan angle and geometry of the left ventricular（LV）,and greater accuracy than the conventional echocardiography through tracking the displacement of endocardium,myocardium and epicardium.Myocardial contrast echocardiography（MCE）not only dynamically,continuously and accurately monitor the global and segmental myocardial microcirculation structure and function,but also allow qualitative and quantitative evaluation of myocardial microcirculation at rest and pressure by injecting microbubbles and performing high-energy blasting.Currently,there is a lack of research in the early dynamic assessment of myocardial strain and myocardial perfusion changes of DOX induced cardiotoxicity using 2D-STI and MCE.In order to further study the relationship between left ventricular function,myocardial strain and myocardial microcirculation and the occurrence and development of DOX induced cardiotoxicity,and to find the imaging indicators for early identification of cardiotoxicity,we applied 2D-STI and MCE to the rat model of DOX induced cardiotoxicity,and continuously and dynamically monitored the changes of series of ultrasonic parameters including myocardial function,myocardial strain and myocardial perfusion before and during the treatment to the process of obvious changes in LV systolic function,combined with histopathology.Objective（1）On the basis of conventional echocardiography,2D-STI was used to evaluate the rats model of DOX induced cardiotoxicity,and to explore the value of left ventricular global and lay-specific distribution of myocardial deformation in early dynamic monitoring of myocardial damage.（2）We applied MCE to evaluate the rats model of DOX induced cardiotoxicity to clarify the relationship between myocardial microcirculation and the occurrence and development of DOX induced cardiotoxicity,and to find the imaging indicators for early identification of cardiotoxicity.（3）Combined with histopathology changes of rats,we analyze the pathological basis of the ultrasonic findings of DOX induced cardiotoxicity.Methods1.Animal grouping and model preparation 40 adult male Sprague-Dawley（SD）rats（206.91±9.30 g）was included,forty rats were divided into control group（baseline,n=8）,Group 1（2 weeks,n=8）,Group 2（4 weeks,n=8）,Group 3（6 weeks,n=8）and Group 4（8weeks,n=8）by means of random number table.Group 1-4:a rat model of DOX-induced cardiotoxicity was established by intraperitoneal injection of DOX（2.5 mg/kg,once a week）,control group:8 rats received intraperitoneal injection of same dose of normal saline.2.The evaluation of DOX induced cardiotoxicity by conventional echocardiography in rats Left ventricular systolic function were determined from the M-mode tracings in a LV long-axis view at the level of the papillary muscles in all groups.Left ventricular diastolic function were measured on the four chamber cardiac plane,and the spectral Doppler and Tissue Doppler image was obtained.The ratio of E to e’（E/e’）and the Tei index were calculated.3.The evaluation of DOX induced cardiotoxicity by 2D-STI in rats In the parasternal long axis plane,the LV global longitudinal strain（GLS）,global longitudinal strain in the epicardial layer(GLS_epi),mid myocardial layer(GLS_mid)and endocardial layer(GLS_endo)were obtained with 2D-STI;the LV global circumferential strain（GCS）and global radial strain（GRS）were obtained in the parasternal papillary muscle short axis plane with 2D-STI.4.The evaluation of DOX induced cardiotoxicity by MCE in rats After the rats model of DOX induced cardiotoxicity was successfully established,MCE was performed in the left ventricular long axis section in all group and the myocardial microcirculation perfusion was quatitatively and quantitatively analyzed.The quantitative pa rameters of myocardial perfusion including myocardial blood volume（MBV）,myocardial blood flow velocity（β）and myocardial blood flow（MBF）were obtained.5.Histopathological examination After undergoing ultrasonic examination,the heart was removed immediately and fixed,embedded and sectioned.Histomorphological changes in cardiac tissue were examined using hematoxylin and eosin（H&E）staining.The development of fibrosis in cardiac tissue was visualized using Masson’s trichrome staining.And myocardial vessels was assessed using CD31 staining.Results1.The evaluation of DOX induced cardiotoxicity by 2D-STI in rats（1）After DOX injection,the LVEF of rats in each group showed a downward trend.In Group 4（8-week DOX treatment）,LVEF decreased significantly（P=0.008）,while the diastolic function indicator E/e’（P=0.038）and GLS（P=0.002）showed significant changes in Group 3（6-week DOX treatment）.Further analysis of the global longitudinal strain of the three layers of left ventricular myocardium in all groups showed that there was a gradient feature in the distribution:GLS_endo>GLS_myo>GLS_epi;GLS_endo decreased significantly in Group 3（6-week DOX treatment）（P=0.010）compared to control rats,but there was no remarkable change in GLS_myoand GLS_epi.2.The evaluation of DOX induced cardiotoxicity by MCE in rats MBF decreased significantly in Group 1（2-week DOX treatment）（P=0.048）than that of the control group,whileβand MBV were not statistically significant.And the myocardial blood flow velocityβ（P<0.001）and MBF（P<0.001）both decreased significantly in Group 2（4-week DOX treatment）,and the decrease was more obvious in Group 3（6-week DOX treatment）and Group 4（8-week DOX treatment）,while MBV began to decrease significantly in Group 3（6-week DOX treatment）（P=0.012）.3.Pathological changes The main pathological changes of rats in DOX groups were vacuolization and swelling of cardiac myocytes,interstitial edema,the deposition of collagen fibers gradually increased with the prolong of DOX treatment time,the perivascular fibrosis was more obvious;compared with the control group,myocardial microvessel diameter and microvessel density（MVD）were significantly decreased.Conclusion1.During the dynamic monitoring of DOX induced cardiotoxicity in rats,GLS is more sensitive than the LVEF acquired by conventional echocardiography,and GLS _endo may be the major reason of the change of GLS.2.Quantitative parameters of myocardial perfusion based on MCE can identify DOX induced cardiotoxicity in the earlier stage when left ventricular function appear normol,MCE may have potential as a non-invasive tool for the evaluation of early and dynamic cardiac changes.3.Myocardial perivascular fibrosis and decreased myocardial microcirculation diameter and density may be the pathological basis of myocardial microcirculation perfusion changes...