Spatial deconvolution of marker expression in whole heart subjected to ischemia-reperfusion

Please describe your research into the ischemia-reperfusion mechanism.
As with many teams worldwide, we are studying the molecular mechanisms controlling cell death over ischemia-reperfusion. Although we are mainly focusing on what happens in the mitochondria of cardiomyocytes, we are also interested in the participation of immune cells at the onset of reperfusion.
Thanks to the high throughput methods coupled to systems biology, we have isolated expression markers from heart biopsies. However, we would like to understand where these markers are really expressed: at the necrotic core, in the periphery of the ischemic area, or in the healthy area.

How has the X-CLARITY Tissue Clearing System helped to quantify the living and necrotic tissue in your subjects?
Thanks to the X-CLARITY Tissue Clearing System, we can now clear mouse hearts prior to imaging them with a fluorescence microscope. This enables the 3D rendering of fluorescent markers in the heart volume.

How do you label the necrotic and living tissue?
We first inject Unisperse Blue into the healthy area in order to increase the contrast between healthy and ischemic area.
We also tested different methods to label the damaged area. This is the most exciting part of our work.

What advantages are there to whole sample tissue clearing when compared to previous methodologies?
Previous methods are less spatially resolved and limited by the number of labels. Our research with clear hearts has been to detect the ischemic area, while being able to specifically label 2-3 markers of interest. Thanks to the revolution brought by light sheet microscope technology, we can now reconstruct the heart volume in 3D with unprecedented spatial resolution.

What does the future hold for your research? How do you hope your research will be applied in the future?
I hope that the chain-of-methods, including use of the X-CLARITY, will provide another quantitative tool for our colleagues studying ischemia-reperfusion. I also hope that these methods will provide us new valuable information in our lab’s aim to find out new molecular targets to improve cell survival after ischemia-reperfusion.

What future applications do you see for the X-CLARITY Tissue Clearing System within your group’s research?
We aim to perform spatial deconvolution of gene expression in healthy, peripheral, and ischemic areas. This “spatial” information will enrich our database of gene expression of mouse myocardium over the ischemia-reperfusion sequence, which we hope will provide us new molecular targets to heal myocardial infarction.

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