mesoscopic-optical-imaging-of-whole-mouse-heart

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 ...

European Laboratory for Non-linear Spectroscopy

The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro-cardiac coupling at specific junctional sites (neuro-cardiac junctions). The ventricular sympathetic network topology is well-defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long-term modulation of cardiac structure is achieved via direct sympatho-cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro-cardiac junctions.

Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery.

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.

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

Although neuronal density analysis on human brain slices is available from stereological studies, data on the spatial distribution of neurons in 3D are still missing. Since the neuronal organization is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than relying on sparse sampling methods. To achieve this goal, we implement a new tissue transformation protocol to clear and label human brain tissues and we exploit the high-resolution optical sectioning of two-photon fluorescence microscopy to perform 3D mesoscopic reconstruction. We perform neuronal mapping of 100mm human brain samples and evaluate the volume and density distribution of neurons from various areas of the cortex originating from different subjects (young, adult, and elderly, both healthy and pathological). The quantitative evaluation of the density in combination with the mean volume of the thousands of neurons identified within the specimens, allow us to determine the layer-specific organization of the cerebral architecture.

Large-scale, cell-resolution volumetric mapping allows layer-specific investigation of human brain cytoarchitecture.

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.

Erica Lazzeri

European Laboratory for Non-linear Spectroscopy

Mesoscopic Optical Imaging of Whole Mouse Heart