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Planar imaging for preliminary assesement of optical clearing effect in tumor bearing mice

Aysiay Saydasheva, FRC of Biotechnology of the RAS, Sechenov medical university
Kazachkina Natalia. I. FRC of Biotechnology of the RAS
Zherdeva Victoria.V FRC of Biotechnology of the RAS
Bogdanov Alexei Jr. FRC of Biotechnology of the RAS
Savitsky Alexander P.FRC of Biotechnology of the RAS


Optical clearing (OC) allows observation of events occurring in opaque tissues at a depth of some millimeters. The OC effect is based, in particular, on the equalization of the refractive index in various tissues.
Recently, we have shown that gadobutrol (Gadovist), a contrast agent for magnetic resonance imaging, is a promising agent for optical clearing of tissues in vivo (Tuchina et al. 2020). The aim of this study was to investigate the effect of gadobutrol on the fluorescence intensity of tumors expressing colored proteins.
The experiments were carried out in nu/nu mice bearing Hep-2 tumor expressing the red color protein TagRFP. Gadobutrol was used in the form of a 1.0 M aqueous solution or a 0.7 M aqueous solution with the addition of 5% dimethyl sulfoxide (DMSO). Gadobutrol was applied to the tumor growth area and kept for 15 minutes then the residue was removed.
Tissue fluorescence was studied by in vivo planar imaging.
Fluorescent images were obtained on an iBox automated system (UVP, USA) before and 15, 30, 60 min after application. Fluorescence was excited with light in the wavelength range of 502-547 nm and registered in the range of 570-640 nm.
Image analysis was performed using the ImageJ software. The average fluorescence intensity of the tumor was normalized to the average fluorescence intensity of different skin areas during mathematical processing.
It was shown that the fluorescence of tumors increased by 1.1 - 1.5 times in varyous animals under the influence of gadobutrol. The increase in intensity was more pronounced when using 0.7 M gadobutrol with the addition DMSO (Fig. 1). This is apparently due to the greater depth of its penetration compared to 1.0 M gadobutrol, which was facilitated by the presence of DMSO. Skin penetration of the OC solution was further independently confirmed by magnetic resonance imaging, capable of detecting the penetration of paramagnetic contrast agents into the subcutaneous space by changing the T1-weighted signal.
Thus, the topical application of gadobutrol as an OC agent makes it possible to enhance the fluorescence yield of subcutaneous tumors expressing fluorescent proteins, and the presence of paramagnetic properties allows parallel detection of the OP agent in tissues to determine the depth of its penetration through the skin of experimental animals.

This work was supported by the Government of the Russian Federation (grant no 14.W03.31.0023).

Tuchina, D. K., I. G. Meerovich, O. A. Sindeeva, et al. 2020. Magnetic resonance contrast agents in optical clearing: Prospects for multimodal tissue imaging. J Biophotonics:e201960249.

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Asiya Saydasheva
FRC of Biotechnology of the RAS


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