Abstract 645: CUBIC as an Alternative “Clearing” Method for Creating Hydrogel-based Structure in Organs

Abstract

Cardiac nerves have a major influence on cardiac health, heart failure, and sudden cardiac death. Understanding the impact of the cardiac nervous system requires in depth cell-specific analysis of the heart, which can be done using tissue clearing followed by 3D microscopic imaging. “Clearing” is the creation of hydrogel-based structures of organs where the lipids are removed without damaging the proteins of the organ. We are interested in using clearing to analyze cardiac neural anatomic pathways with high accuracy. Recently the CUBIC clearing method has gained momentum since it can clear tissue quicker than other methods. While previous research has shown qualitative images and descriptions to describe CUBIC efficacy, quantification of the clearing performance is lacking. The purpose of this study was to compare sequential images of cardiac tissue immersed in CUBIC solution alongside tissue absorbance spectra to measure the efficiency of the CUBIC method. To visualize cardiac neurons, we selectively expressed a fluorescent probe: an enhanced yellow fluorescent protein (EYFP) conjugated with the light-activated channel (channelrhodopsin (ChR2). Mice were crossbred to express EYFP+ChR2 in parasympathetic cardiac neurons which was driven by a choline acetyltransferase (ChAT) promoter using the Cre-Lox system. Hearts from these mice were excised and perfused with PFA as a fixative and perfused with the CUBIC solution. The tissue then was immersed in CUBIC solution and kept in an insulated shaker at 37°C. Each day, the tissue holder that held the cardiac tissue was taken out of the CUBIC solution and a white light from an LED was directed through the heart and the light intensity was recorded by a spectrometer. Beer-Lambert’s Law related light intensity and transmittance to absorbance to measure daily changes in the absorbance spectrum. Finally, we imaged the right atria of a cleared heart to capture the parasympathetic neural anatomic pathways using 2-photon confocal microscopy. We found that, that over the course of 14 days, the absorbance spectrum of the heart sample decreased over time, with the greatest reduction occurring between days 1 and 4. Future work would include decreasing environmental factors when measuring light intensity.