Optical clearing in cardiac imaging: A comparative study.

The optical clearing of the cardiac tissue has always been a challenging goal to obtain successful three-dimensional reconstructions of entire hearts. Typically, the developed protocols are targeted at the clearing of the brain; cardiac tissue requires proper arrangements to the original protocols, which are usually tough and time-consuming to figure out. Here, we present the application of three different clearing methodologies on mouse hearts: uDISCO, CLARITY, and SHIELD. For each approach, we describe the required optimizations that we have developed to improve the outcome; in particular, we focus on comparing the features of the tissue after the application of each methodology, especially in terms of tissue preservation, transparency, and staining. We found that the uDISCO protocol induces strong fiber delamination of the cardiac tissue, thus reducing the reliability of structural analyses. The CLARITY protocol confers a high level of transparency to the heart and allows deep penetration of the fluorescent dyes; however, it requires long times for the clearing and the tissue loses its robustness. The SHIELD methodology, indeed, is very promising for tissue maintenance since it preserves its consistency and provides ideal transparency, but further approaches are needed to obtain homogeneous staining of the whole heart. Since the CLARITY procedure, despite the disadvantages in terms of tissue preservation and timings, is actually the most suitable approach to image labeled samples in depth, we optimized and performed the methodology also on human cardiac tissue from control hearts and hearts with hypertrophic cardiomyopathy.

Mesoscopic Optical Imaging of Whole Mouse Heart.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls.

Systemic arterial hypertension is a highly prevalent chronic disease associated with hypertensive cardiomyopathy. One important feature of this condition is remodelling of intramural small coronary arteries and arterioles. Here, we investigated the implications of this remodelling in the downstream vascular organization, in particular at the capillary level. We used Spontaneously Hypertensive Rats (SHR) exhibiting many features of the human hypertensive cardiomyopathy. We generated 3D high-resolution mesoscopic reconstructions of the entire network of SHR hearts combining gel-based fluorescent labelling of coronaries with a CLARITY-based tissue clearing protocol. We performed morphometric quantification of the capillary network over time to assess capillary diameter, linear density, and angular dispersion. In SHRs, we found significant remodelling of the capillary network density and dispersion. SHR capillary density is increased in both ventricles and at all ages, including before the onset of systemic hypertension. This result suggests that remodelling occurs independently from the onset of systemic hypertension and left ventricular hypertrophy. On the contrary, capillary angular dispersion increases with time in SHR. Consistently, our multicolor imaging underlined a strong correlation between vascular dispersion and cellular disarray. Together our data show that 3D high-resolution reconstruction of the capillary network can unveil anatomic signatures in both physiological and pathological cardiac conditions, thus offering a reliable method for integrated quantitative analyses.