Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

Summary

Here, we present optimized tissue-clearing protocols to image the murine aorta in three dimensions (3D). We delineate state-of-the-art procedures for immunostaining, optical clearing, and imaging with the intent to define the anatomical proximity of the peripheral nervous system with atherosclerotic plaques and the adventitia in atherosclerosis.

Abstract

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial wall, including the intima plaque, the media, and the adventitia, which forms the outer connective tissue coat of arteries. Our recent studies have suggested that the adventitia is used by the peripheral nervous system as a conduit for reaching all tissue cells. We also found that the peripheral nervous system, that is, the sensory and sympathetic nervous system, undergoes major remodeling processes involving the neogenesis of axon networks adjacent to atherosclerotic plaques. In this context, understanding the structure of the neural network and its interactions with vascular components of diseased arteries holds major promises for a better understanding of cardiovascular disease pathogenesis. To achieve these objectives, methods to visualize the subcellular architecture of the intact healthy and diseased arteries together with their surrounding perivascular compartments are needed. Tissue clearing allows intact deep-tissue imaging of larger tissue compartments that are otherwise inaccessible. It allows volumetric imaging of intact arteries through the integration of labeling, clearing, advanced microscopic imaging, and image processing tools. Here, we describe two distinct but complementary passive tissue clearing approaches, that is, aqueous-based 2, 2-thiodiethanol (TDE) clearing and solvent-based immunolabeling-enabled three-dimensional imaging of solvent-cleared organ (iDISCO) clearing to image isolated aortic segments or whole aorta in-situ in the whole mouse.

Introduction

Histological techniques provide a basic understanding of biological samples through the sectioning of tissues/organs. However, delineation of complex anatomical cell/cell and tissue/tissue interactions in three dimansion (3D) has been - until recently - difficult to achieve. This unmet need was particularly evident in the context of the cardiovascular system in healthy and diseased conditions. Imaging intact tissues has been challenging in the past due to light absorption and light scattering, making them intrinsically opaque. Tissue clearing makes the intact biological sample transparent by minimizing these limitations. Recent developments in tissue-clearing techniqu....

Protocol

The present study was performed according to the guidelines of the local and national animal use and care committee. Hyperlipidemic male Apoe^-/- mice on C57BL/6J background maintained on a standard rodent chow diet that spontaneously develop atherosclerosis during aging were used in the present study.

1. Whole-mount imaging of isolated aorta and TDE clearing

1. Tissue preparation
   1. Prepare the anesthetic at 150 mg/kg of Ketamine and 30 mg/kg of Xy.......

Discussion

Atherosclerosis can be viewed as a transmural inflammatory disease of arteries involving all three layers of the arterial wall. Moreover, arteries are surrounded by the perivascular adipose and neuronal tissues. During atherosclerosis progression, each of these tissues undergoes considerable cellular and structural alterations, which requires methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries. These methods are provided here to better understand cell-cell and cel.......

Materials

Name	Company	Catalog Number	Comments
2,2’-thiodiethanol (TDE)	Sigma	166782	Clearing reagent
Amira	Thermo Fisher Scientific		3D visualization software; Image processing software used for manual segmentation and tracing in 3D images
Benzyl alcohol	Sigma	W213713	Clearing reagent
Benzyl benzoate	Sigma	B6630	Clearing reagent
CD16/32	eBioscience	14-0161-82	Blocking solution
Confocal laser scanning microscope	Leica Microsystems	TCS- SP8 3X	Imaging device for multidimensional high-resolution imaging of intact biological tissues or sections with high specificity at subcellular resolution.
DAPI	Invitrogen	D3571	Nuclei marker
Dichloromethane (DCM)	Sigma	270997	Clearing reagent
Dissecting pan-black wax	Thermo Scientific	S17432	Aorta dissection and fixation
Dissection stereomicroscope	Leica Microsystems	Stemi 2000	Mouse organ dissection
Ethanol	Sigma	E7023	Defection
Ethylenediaminetetraacetic acid (EDTA)	Roth	8040.1	Perfusion buffer
Fiji	(ImageJ, NIH)		Open source image processing software for 2D and 3D images
Goat anti-Hamster IgG, Cy3	Dianova	127-165-099	Secondary antibody
Goat anti-Rabbit IgG, Alexa Fluor 680	Thermo Fisher Scientific / Invitrogen	A-21109	Secondary antibody
Goat anti-Rat IgG, Cy5	Dianova	712-175-150	Secondary antibody
Hamster Anti-CD3e	BD Bioscience	145-2C11	Pan-T cell marker
Huygens Professional	Scientific Volume Imaging, The Netherlands	Version 19.10	Image restoration software; Image processing software used mainly for deconvolution of 2D and 3D images
Image processing workstation	MIFCOM	MIFCOM X5	Image processing workstation equipped with all image processing software including Leica application suite X, Fiji, and Imaris for post-processing of images acquired by confocal, multiphoton and light sheet microscopes
Imaris	Bitplane	Version 8.4	Image analysis software; Image processing software used for automated segmentation of 3D images
Incubator and rotator	Marshall Scientific	Innova 4230	Incubation and rotation device during tissue clearing
iSpacer	Sunjin Lab	IS4020	Rectangular well as the sample holder
Ketamine	Livisto		Anesthetic
Leica Application Suite X (LAS-X)	Leica Microsystems	Version 3.5	Image processing software for the images acquired with Leica microscope
Light microscope	Leica Microsystems	DM LB	Imaging device for bright filed imaging
Light sheet  microscope	LaVision BioTech	Ultramicroscope II	Imaging technique for fast, high-resolution imaging of large biological specimens or whole mouse with low light exposure by rapidly acquiring images of thin optical sections.
Multiphoton microscopy	Leica Microsystems	TCS-SP5II MP	Imaging modality for multidimensional, high-resolution imaging of intact and viable biological tissues at sub-cellular and molecular level over prolonged periods of time, deep in the sample and with minimal invasion.
Normal goat serum	Sigma	G9023	Blocking solution
Paraformaldehyde (PFA)	Sigma	P-6148	Fixation
Phosphate-buffered saline (PBS)	Sigma	P4417-100TAB	Washing buffer
Porcine skin gelatin	Sigma	G1890	Incubation buffer
Quadrol	Sigma	122262	CUBIC clearing reagent
Rabbit Anti-NF200	Sigma	N4142	Pan-neuronal marker
Rat Anti-B220	BD Bioscience	RA3-6B2	Pan-B cell marker
Sucrose	Sigma	90M003524V	Dehydration
Sytox	Thermo Fisher Scientific	S11380	Nuclei marker
Tetrahydrofuran	Sigma	401757	Clearing reagent
Triton X-100	Roth	3051.1	Penetration
Urea	Sigma	U5128	CUBIC clearing reagent
Xylene	Fisher Chemical	x/0250/17	Anesthetic