Current Issue

Volume 21, Number 1, Apr-Jun(Spring) 2019 , Serial Number: 81 Pages: 49-56

A Simple Technique for Three-Dimensional Imaging and Segmentation of Brain Vasculature Using Fast Free-of-Acrylamide Clearing Tissue in Murine


Arezoo Khoradmehr, M.Sc, 1, #, Fahime Mazaheri, M.Sc, 1, #, Morteza Anvari, Ph.D, 1, 2, *, Amin Tamadon, Ph.D, 3, *,
Research and Clinical Center for Infertility, Yazd Reproduction Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
*Corresponding Addresses: P.O.Box: 89195-999 Department of Biology and Anatomical Sciences Shahid Sadoughi University of Medical Sciences Yazd Iran P.O.Box: 3537 The Persian Gulf Marine Biotechnology Research Center The Persian Gulf Biomedical Sciences Research Institute Bushehr University of Medical Sciences Bushehr Iran Emails:moanvari@gmail.com,amintamaddon@yahoo.com

# The first two authors equally contributed to this work.

Abstract

Objective

Fast Free-of-Acrylamide Clearing Tissue (FACT) is a recently developed protocol for the whole tissue three-dimensional (3D) imaging. The FACT protocol clears lipids using sodium dodecyl sulfate (SDS) to increase the penetration of light and reflection of fluorescent signals from the depth of cleared tissue. The aim of the present study was using FACT protocol in combination with imaging of auto-fluorescency of red blood cells in vessels to image the vasculature of a translucent mouse tissues.

Materials and Methods

In this experimental study, brain and other tissues of adult female mice or rats were dissected out without the perfusion. Mice brains were sliced for vasculature imaging before the clearing. Brain slices and other whole tissues of rodent were cleared by the FACT protocol and their clearing times were measured. After 1 mm of the brain slice clearing, the blood vessels containing auto-fluorescent red blood cells were imaged by a z-stack motorized epifluorescent microscope. The 3D structures of the brain vessels were reconstructed by Imaris software.

Results

Auto-fluorescent blood vessels were 3D imaged by the FACT in mouse brain cortex. Clearing tissues of mice and rats were carried out by the FACT on the brain slices, spinal cord, heart, lung, adrenal gland, pancreas, liver, esophagus, duodenum, jejunum, ileum, skeletal muscle, bladder, ovary, and uterus.

Conclusion

The FACT protocol can be used for the murine whole tissue clearing. We highlighted that the 3D imaging of cortex vasculature can be done without antibody staining of non-perfused brain tissue, rather by a simple auto- fluorescence.