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No more flat. Optical clearing to study rodent organs in three dimensions

6 Aug | By Marzena Stefaniuk
No more flat. Optical clearing to study rodent organs in three dimensions
A rat kidney before and after optical tissue clearing.
By: Lukasz Bozycki

Body is a sophisticated machine with many important elements. To learn how it works we need to peek inside and examine them thoroughly. Team of Professor Leszek Kaczmarek from the Nencki Institute recently developed a rapid protocol for the whole-body optical clearing of a rat. The method is described in one of the latest issues of Journal of Biophotonics.

Whole-organ and whole-body optical clearing is a new, rapidly developing methodology. It aims at preparing transparent biological specimens. As long as the tissue is transparent, it can be visualized in toto plane by plane, from top to the bottom, with a light-sheet fluorescence microscope (LSFM). It offers minimal phototoxicity and bleaching, fast scanning and high resolution. LSFM imaging of optically cleared samples allows studying cell distribution and architecture within the entire, unsectioned organs. “In the last few years, several methods capable of clearing of murine tissues were demonstrated, however very limited number of reports have been published in regard to the clearing of rat organs. We believe it is unfortunate, since due to its closer resemblance of physiology and behavior to humans compared with mice, rat is a valuable experimental model in toxicology, neurological studies and systemic diseases studies such as hypertension, diabetes or chronic inflammation”, says Stefaniuk.

How to make the rat organ disappear?

Tissue transparency can already be achieved by many distinct chemical approaches and relies on two general steps 1) lipid removal and 2) refractive index matching between processed tissue and imaging solution to minimize light scattering during the imaging process. While this was sufficient for mouse clearing, the approximately 10 times bigger and packed with endogenous fluorophores rat organs were challenging to make transparent. “Therefore, in our studies, we utilized formerly published CUBIC protocol, which was known to bear unique ability of tissue decolorization”, explains Stefaniuk. Even though 3 weeks of CUBIC reagent perfusion through rat circulatory system was not effective, sufficient transparency was achieved when additional step of organs dissection and further step with rotation in 37°C was introduced to the procedure. By optimizing the method, researchers were finally able to effectively decolorize and clear all of the rat organs within just as little as 4 days. Although rapid, presented protocol does not change general histological appearance of tissue neither structure of epitopes, as confirmed by successful detection of endogenous proteins using immunohistochemistry.

Screening for sites of tissue damage.

The only tissues that remain opaque after the optimized CUBIC clearing are bone and cartilage. However, this feature might easily become very advantageous, if one’s interest is to study distribution of bone or calcium deposits within limbs or peripheral organs. Therefore, researchers utilized alizarin red S and developed additional protocol for selective calcium staining of transparent organs. Taking into account that dystrophic calcification takes place in all areas of tissue necrosis independently from serum calcium level; such approach might be broadly used to detect prolonged tissue damage observed in a variety of animal models.

“We have already applied optimized CUBIC clearing and alizarin red S staining to detect sites of muscle dystrophy in mdx mouse model of Duchenne Muscular Dystrophy (lethal X-chromosome linked recessive disorder, affecting 1 in 3500-5000 male births). Thus far, progression of muscular dystrophy in mice models relied either on evaluation of histological sections taken from randomly chosen muscles or lower resolution and expensive MRI/CT scanning. Developed approach allowed us, for the first time, to visualize all of the necrotic sites in the entire mouse body suffering from this lethal, systemic disease. Results of this work will soon be available in Skeletal Muscle journal” - says Matryba.
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