Noninvasive small-animal imaging has taken on an increasing role in preclinical research as to become an independent sector. Today, the availability of advanced imaging techniques constitutes a key factor in the success and timeliness of research thanks to the possibility of conducting longitudinal studies on the same animal [1]. Among these techniques, μ-echocardiography is an inexpensive, repeatable, fast and noninvasive modality and as such it is particularly suited to this type of experimental projects.
Noninvasive imaging helps to render animal experiments more ethically acceptable as it is compliant with the principles of the 3Rs (Replacement, Reduction, Refinement) formulated in 1959, which consider the possibility of replacing [Replacement], where possible, the use and/or sacrifice of the animal with other equally effective methods, reducing [Reduction] the number of experiments, and refining [Refinement] techniques to minimize pain and distress of the animals [11, 12]. This study aimed to demonstrate that μ-echocardiography is a powerful tool in support of the 3R policy. Although the modality is well known as a non-invasive, reproducible and inexpensive diagnostic modality, only recently have been developed systems, which allow for a more detailed study of cardiac function in small animals. Thanks to high resolution images (40 MHz) acquisition, with a potential for recording more than 750 frames per second, these systems provide an analysis similar to that obtained in humans and therefore a more objective result of echocardiographic imaging [13, 14]. This latter aspect is crucial in order to reduce the number of sacrifices during the experiment, allowing several assessments during follow-up. This should not be regarded as a marginal issue particularly if we consider that, like humans, animals show individual variability in the course of the disease, and further assessment can improve our understanding of the evolution of the disease.
Echocardiography is widely used for the morphological and functional evaluation of the heart [15] as it permits an accurate analysis of cardiac anatomy and haemodynamics facilitating the understanding of the pathophysiological mechanisms underlying the disease being studied. This technique has been able to monitor the effects of gene therapy on the cardiac function of BIO 14.6 hamsters with dilated cardiomyopathy.
Only few ultrasound systems to date have been able to provide high-quality echocardiographic studies on hamsters, because of inadequate image resolution and an inability to process quantitative data. In the present study, despite the high hamsters heart rate, the μ-echocardiographic examination allowed to obtain a precise estimation of heart function parameters thanks to high-frequency transducers (40 MHz).
Indeed, μ-echocardiography is ideal for phenotyping and estimating left ventricular function; as a fast and noninvasive method, it can also be used in animals in suboptimal physical condition.
The lack of the gene coding for delta-SG in these animals entails a loss of sarcolemmal integrity with necrosis of the cardiomyocytes; this promotes the development of an inflammatory response with fibrotic degeneration. By limiting myocardial elasticity, fibrotic replacement reduces contractile function with progressive stretching and thinning of the fibrotic regions, leading to progressive dilation.
The increase in ventricular volume worsens the systolic dysfunction associated with mitral valve insufficiency, leading to low-output heart failure.
Serial echocardiography successfully documented this evolution in the untreated BIO 14.6 hamsters, but not in the treated hamsters. Gene therapy was in fact able to almost completely preserve cardiac function in the dystrophic hamsters, which showed similar morphology, echocardiographic parameters and survival to the wild type hamsters.
In order to confirm the role of this method in monitoring the evolution of cardiomyopathy, samples of myocardial tissue were studied by histological examination. The estimation of injury obtained at microscopic analysis correlated with that of the μ-echocardiographic study.
Our experience showed that μ-US imaging applied to the study of cardiomyopathy in BIO 14.6 hamsters, thanks to the high spatial and contrast resolution, provides an estimation of morphological changes which well correlate with the histological findings, and may help to reduce the animals sacrifice.
Even though histology remains indispensable for a definite diagnosis, echocardiography can be used to monitor the efficacy of treatment and/or the progression of dilated cardiomyopathy and constitutes an alternative tool for a repeatable and noninvasive assessment.
The implementation of power and color Doppler imaging, the use of contrast media and higher-frequency transducers will help to refine the characterization of sonographic patterns of disease also reducing both the time and cost of longitudinal studies.