To the best of our knowledge this is the first evidence that UTC, by using CC-IBS, can detect the initial myocardial lesion (collagen deposition) secondary to DXR infusion early than clinical significant LVEF decrease. Our results are congruent with previously published data  showing that this experimental animal model of DXR infusion reproduces pathologic myocardial alterations similar to those found in human disease.
At the end of the DXR infusion protocol, LVEF decrease was about only 6% at 8 mg/Kg DXR and about 10% at 12 mg/Kg. This result is in agreement with previously published data showing that the LVEF decreases slightly at 12 weeks of DXR infusion [17–20].
Information regarding myocardial ultra-structure and composition can be provided by ultrasonic tissue characterization, a non-invasive technique capable of detecting and quantifying acoustic properties of myocardial tissue (4;5). The IBS intensity is related to physical and structural properties of the myocardium and is particularly influenced by tissue collagen content and spatial distribution of this component . In addition, IBS intensity also depends on ultrasound system settings and on ultrasound attenuation, which has been a major limitation for the current use of this index. In order to overcome this limitation, several methods have been employed to calibrate the absolute value of IBS according to a reference value, derived from the ventricular cavity , the pericardium  or from a gray scale phantom  such as the one used in this investigation. Although absolute IBS values derived from ventricular cavity or pericardium could reduce the influence of the ultrasound system settings, there are limitations for their use so as to correct the effects of ultrasound attenuation; This, associated to the fact that the pericardium membrane (and consequently its IBS intensity) could be affected by DXR toxicity, influenced our choice of a rubber phantom referential for this investigation.
There is little published data about CC-IBS in this animal model, but Mimbs, studying rabbits, has already demonstrated CC-IBS increments at the end of the total cumulative dose of DXR infusion. In this study we reproduced their findings, but we were also able to demonstrate a dose–response relationship beginning at an 8mg/Kg cumulative dose. Also, CC-IBS was the only index with a significant correlation with collagen deposition, secondary to DXR infusion. The capability of IBS in detecting fibrosis content of the myocardium was already described in several diseases, including dilated cardiomyopathy [22, 24].
During cardiac contraction and relaxation, a cardiac cycle-dependent variation of myocardial IBS can be documented. The magnitude of cyclic variation of the IBS, measured as peak-to-peak variation of the IBS curve (MCV), is an additional acoustic parameter, independent on ultrasound system settings and responsible for its wider application. However, this index is more strictly correlated with the myocardial contractile function than the collagen myocardial content. Previously published data showed a decrease in MCV with DXR infusion [11, 20, 25], even when LVEF was preserved. However, Ha et al., using the same experimental animal model described in our study, failed to demonstrate the decrease in MCV with DXR infusion . Considering that our investigation evaluated MCV through the whole infusion DXR protocol, our results are the first to demonstrate that MCV decrease occurs at initial cumulative DXR dosages (8mg/Kg). MCV of IBS could be used, in addition to CC-IBS, to detect early myocardial DXR toxicity. MCV of IBS may be more related to the myocardium contractile function than collagen content. If so, the reduction of MCV from initial dosages of DXR infusion may accompany initial contraction impairment, as suggested in recent studies [26–29] using regional contraction analysis techniques like “strain rate”. Maybe cellular loss, which is the initial myocardium damage before full collagen deposition, could be detected with these tools (MCV of IBS and by myocardium deformation analysis data, like strain, strain rate and speckle tracking techniques) in a similar way.
The good accuracy of CC-IBS, as demonstrated here, suggests that this parameter could be a non-invasive tool to detect initial collagen deposition secondary to DXR infusion, and it may have the potential to be used as an alternative tool for serial evaluation of myocardial lesions in the clinical setting.
Clinical applicability: this work confirms the capability of UTC using CC-IBS to detect the initial myocardial DXR lesion earlier than the decrease of LVEF in rats. The UTC technique using the CC-IBS is feasible in serial myocardial assessments in humans. As such, this study brings a perspective in the use of UTC as a non-invasive method in collagen myocardium detection, and as an alternative tool for endomyocardial biopsies, with good accuracy, but human studies are needed.
Limitations: the anatomo-pathological studies were limited to the rats that completed the protocol phase and which had not suffered spontaneous deaths. In fact, if those who died were included more striking differences could have been obtained, but adding some confounding factors. The high mortality rates at final cumulative dosages of DXR limited the number of animals analyzed at this point. This study did not include other pathologic myocardium analysis methods, such as myocardial fibers diameter quantification, ultra-structural myocardial analysis, or analysis of the tridimensional myocardial arrangement that could be better correlated to UTC indices like MCV. Another significant limitation is the lack of diastolic data. Since our acoustic window was limited and did not permit a collection of distinct validated diastolic dysfunction parameters, we preffer to exclude it from the protocol. A specific protocol can be developed from this work to address this issue. In summary, UTC was able to identify the DXR myocardial lesion earlier when compared to LVEF, showing good accuracy to detect the initial collagen deposition in this experimental animal model.