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

Optogenetics is an emerging method that uses light to manipulate electrical activity in excitable cells exploiting the interaction between light and light-sensitive depolarizing ion channels, such as channelrhodopsin-2 (ChR2). Initially used in the neuroscience, it has been adopted in cardiac research where the expression of ChR2 in cardiac preparations allows optical pacing, resynchronization and defibrillation. Recently, optogenetics has been leveraged to manipulate cardiac electrical activity in the intact heart in real-time. This new approach was applied to simulate a re-entrant circuit across the ventricle. In this technical note, we describe the development and the implementation of a new software package for real-time optogenetic intervention. The package consists of a single LabVIEW program that simultaneously captures images at very high frame rates and delivers precisely timed optogenetic stimuli based on the content of the images. The software implementation guarantees closed-loop optical manipulation at high temporal resolution by processing the raw data in workstation memory. We demonstrate that this strategy allows the simulation of a ventricular tachycardia with high stability and with a negligible loss of data with a temporal resolution of up to 1 ms.

A Software Architecture to Mimic a Ventricular Tachycardia in Intact Murine Hearts by Means of an All-Optical Platform.

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.

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse's hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.

Autofluorescence enhancement for label-free imaging of myelinated fibers in mammalian brains.

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.

Francesco Giardini

European Laboratory for Non-linear Spectroscopy

Mesoscopic Optical Imaging of Whole Mouse Heart