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 techniques enable high-resolution 3D imaging of unsectioned transparent tissues to provide considerable insight into the cellular and structural microarchitecture of whole organs at micrometer resolution, thereby enabling the definition of anatomical connectivity networks.

Atherosclerosis involves three layers of the arterial wall, including the inner intima layer, the middle media layer, and the outer connective tissue layer, which is termed adventitia. Atherosclerotic plaques in the inner layer of arteries have been a conventional target of research for decades^1,2. However, the adventitia layer contains blood vessels, lymph vessels, and nerve fibers of the peripheral nervous system. Moreover, the adventitia is connected to the perivascular adipose tissue and neuronal tissue components, including peripheral nerves and perivascular ganglia^3,4. Peripheral nerves are known to use the adventitia as conduits to reach distant target tissues and, indeed, cells⁵. Our recent studies have advanced the progress in understanding the multi-layered interactions of the major biological systems, which include the immune system, the nervous system, and the cardiovascular system. We have termed these interactions neuroimmune-cardiovascular-interfaces^6,7. During atherogenesis, arterial wall components undergo robust restructuring and remodeling. For example, adjacent to atherosclerotic plaque progression in the intima, immune cell aggregates form, and neuronal axon neogenesis occurs in the murine aortic adventitia^6,8,9. As atherosclerosis progresses, immune cell aggregates develop into well-structured artery tertiary lymphoid organs (ATLOs) with distinct T cell, B cell, and plasma cell areas¹⁰. However, to delineate these changes in 3D, high-resolution imaging of the intact tissue has been challenging due to insufficient membrane permeabilization and inherent light scattering¹¹. Tissue-clearing approaches have overcome the major limitations of conventional histology approaches^{11,12,13,14,15} with enhanced penetration of antibodies to reach deep into intact tissues or organs by uniformly adjusting the refractive index (RI), leading to micrometer scale resolution images with higher imaging depth in voxels. RIs of samples can be matched either to glycerol (RI 1.46) or immersion oil (RI 1.52), thereby greatly reducing light scattering and spherical aberrations, enabling high resolution. Recent advances in the whole organ or whole-body tissue clearing techniques, such as aqueous-based 2,2-thiodiethanol (TDE) and immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO), respectively, together with volumetric imaging techniques (including confocal, multiphoton and light-sheet microscopy imaging) have allowed reconstructions of the microanatomy of the vascular architecture by constructing their connectivity atlas^11,16. Visualizing these cellular and structural connections in 3D can provide new insights to answer hitherto unanswered biological questions.

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 Xylazine (see Table of Materials) per mouse. Deeply anesthetize mice by intraperitoneal injection and confirm this by the lack of reaction to the deep pain test.
      NOTE: The concentration and volume of injection are as follows: Ketamine 100 mg/mL; Xylazine 20 mg/mL; Injection volume 3 µL/g per mouse for both the Ketamine and Xylazine.
   2. Place the mouse onto a foam plate covered with surgical paper towels, and fix arms and legs in a supine position with tapes. Disinfect the chest with 75% ethanol (see Table of Materials) and take blood via the left ventricle with a 1 mL disposable syringe to remove the blood for better perfusion.
      NOTE: Cardiac blood collection via thoracotomy can be used as well.
   3. Make a midline incision on the chest to expose the heart and a small incision in the right atrium to allow trans-cardiac perfusion. Perfuse the mouse via the left ventricle with 10 mL of 5 mM ethylenediaminetetraacetic acid (EDTA; see Table of Materials) in phosphate-buffered saline (PBS) for 5 min, followed by 20 mL of PBS for 5-10 min until the blood is flushed out. Finally, perfuse with 10 mL of 4% paraformaldehyde (PFA) for 20 min.
      NOTE: All the procedures can be performed with a peristaltic perfusion pump with 100-125 mm Hg pressure at room temperature (RT). The perfusion needle is inserted into the mouse heart through the same hole as the blood extraction.
   4. Remove internal organs, including the spleen, liver, lung, and gastrointestinal and reproductive organs, using surgical instruments. Keep the heart, aorta, and kidneys intact in situ.
      NOTE: Surgical instruments used for dissection and removal of organs are dissection scissors, fine iris scissors, spring scissors, blunt forceps, curved forceps, and curved fine forceps.
   5. Expose the whole aorta from the ascending aorta to the iliac bifurcation under the dissecting stereomicroscope (30-40x magnification). Carefully remove the thymus and adipose tissue without injury to the aorta.
      NOTE: Be careful not to cut the aorta. For Apoe^-/- mice, keep the minimal surrounded perivascular adipose tissue of the aorta for connected structure.
   6. Harvest the entire aorta into a Petri dish filled with PBS. Separate the aorta into different segments and split the entire aorta longitudinally using an iris or spring scissors to expose the intimal surface following the sequence and direction indicated in Figure 1A for TDE clearing.
   7. Pin the en face aorta onto the flat black wax plate in a Y-shape, as indicated in Figure 1A. Postfix it in 4% PFA overnight at 4 °C.
      NOTE: Pin the aorta to the wax plate before fixation to avoid folding and curling in the following steps.
   8. Unpin the aorta and transfer it into PBS. Thoroughly wash the aorta for 5 min for 5 times.
      NOTE: Transfer the aorta into different tubes filled with PBS each time for washing.
2. Antibody staining for en-face aorta
   NOTE: All incubation steps are performed in 2 mL safe-lock tubes with gentle rotation or shaking at RT.
   1. Transfer the fixed aorta into a blocking solution for 2 h for blocking and permeabilization.
      NOTE: Blocking solution varies based on the primary and secondary antibodies. For example, blocking solution: 0.5 mg/mL CD16/32 (1:100), 10% normal donkey serum, 2% Triton X-100 in PBS (see Table of Materials)
   2. Incubate the aorta with primary antibodies in the blocking solution above (step 1.2.1), for example, CD3e (1:100 dilution), NF200 (1: 200 dilution), and B220 (1:200 dilution) for 24 h (see Table of Materials). Wash the aorta in PBS for 5 min for 5 times.
   3. Incubate the aorta with secondary antibodies in 10% normal donkey serum (1:300 dilution) and 4',6-diamidino-2-phenylindole (DAPI; 1 mg/mL) for nuclear staining overnight (see Table of Materials). Wash the aorta in PBS for 5 min 5 times and store the aorta in PBS at 4 °C until the tissue-clearing procedure.
      NOTE: Transfer the aorta into different tubes filled with PBS each time for washing. Cover the tubes with aluminum foil to keep them in the dark for step 1.2.3.
3. TDE tissue clearing
   NOTE: All incubation steps are performed in 2 mL safe-lock tubes, gently rotating at RT, and covered with aluminum foil in the dark. TDE (see Table of Materials) working solutions are highly permeable and should be handled carefully in a fume hood. Collect and discard waste according to local regulations.
   1. Transfer the stained aorta into 20% TDE working solutions and incubate for 1 h.
   2. Transfer the aorta into 47% TDE working solutions and incubate for 12 h. Transfer the aorta into 60% TDE working solutions and incubate for 24-36 h until the samples become transparent in visible light to match the RI.
   3. Store the aorta in 60% TDE at RT in the dark until imaging.
      NOTE: Refresh working solutions every 30 min for the short-term of incubation (for example, step 1.3.1) and every 4 h for long-term of incubation (for example, step 1.3.2). Incubation time for each step can be adjusted depending on tissue size to get a better antibody penetration or clearing transparency. For Apoe^-/- mouse aorta surrounded with adipose tissue, the incubation time should be appropriately extended.
4. Imaging of TDE clearing aorta using confocal laser scanning microscope (CLSM)
   1. Use commercially available double-sided sticky rectangular imaging spacer wells with a thickness of 0.2 mm (see Table of Materials). Stick the well to a clean glass slide and transfer the cleared aorta. Make sure the en face aortic adventitia faces the coverslip.
      NOTE: Gently tap the en face aorta flat with the abluminal side of the en face aorta on the top using angled forceps. Stack multiple imaging spacer wells to match the tissue size if necessary.
   2. Mount the aorta with drops of 60% TDE solution. Carefully attach a coverslip to the well, avoiding air bubbles between the sample and the coverslip.
      NOTE: Carefully remove the bubbles with a needle before the coverslip. Do not use an excessive amount of mounting solution, as the sample may float around.
   3. Use an inverted CLSM instrument (see Table of Materials) equipped with a 20x immersion (multi) objective (NA: 0.75).
      NOTE: CLSM allows volumetric imaging up to 70 µm deep with a higher resolution, which is needed for the analysis of nerve diameter and the nerve-immune cell interaction in the aortic adventitia.
   4. Select the 20x/0.75 immersion (oil) objective. Tune the hybrid diode detectors based on the stained dyes. Adjust the display settings and select 1024 x 1024 pixel XY format for imaging.
      NOTE: An oil-immersion objective lens capture signals better than water.
   5. Drop immersion oil (with glycerol) evenly along the aorta on the coverslip. Move the 63x objective coarsely toward the sample until it touches the immersion oil/coverslip.
   6. Move the 63x objective finely under the microscope to locate the region of interest. Acquire Z-stack from the adventitia side of the en face aorta at 2-4 µm step-size up to 60 µm depth for 3D imaging.
   7. Name the file with sample details, scan details, and save the data.
5. Imaging of TDE clearing aorta using a multiphoton microscope
   1. Follow steps 1.4.1-1.4.2 to mount the en face aorta on the slide.
   2. Use an upright multiphoton microscope (see Table of Materials) equipped with a 20x objective (water-immersion, NA: 1.00, working distance = 2 mm), allowing volumetric imaging up to 1.5 mm deep.
   3. Acquire Z-stacks from the abluminal side at 10-15 µm step-size up to 700 µm depth. Name the file and save the data as in step 1.4.7.

2. Whole-body immunostaining and iDISCO tissue clearing

1. Tissue preparation
   1. Follow steps 1.1.1-1.1.3 above for anesthesia, fixing the mouse, and perfusion.
   2. Remove the skin and internal organs, including the spleen, liver, lung, and gastrointestinal and reproductive organs, using surgical instruments. Keep the heart, aorta, and kidneys intact in situ.
   3. Dissect the body part above the diaphragm level and fix the lower body part with 4% PFA for 1-2 days (s) at 4 °C.
      NOTE: Perform the fixation, washings, and incubations in 50 mL tubes on a gently rotating shaker. Refresh the solution in new tubes for 2-3 times.
   4. Thoroughly wash the sample for 10 min 3 times at RT.
      NOTE: Transfer the aorta into different tubes filled with PBS each time.
   5. Incubate the sample in 20% clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) solution for 48 h for decolorization.
      NOTE: For 20% CUBIC solution, dissolve 25 g of urea in 15 mL of Triton X-100 and 28 mL of Quadrol, and add PBS to the final volume of 100 mL. Prepare it in the hood since the ingredients are stimulating (see Table of Materials).
   6. Thoroughly wash the sample for 1 h five times in PBS at RT.
2. Antibody staining
   NOTE: All incubation steps are performed in 50 mL safe-lock tubes with gentle rotation or shaking at 4 °C.
   1. Incubate the sample in PBS-gelatin-Triton X-100-serum (PGST) solution overnight for permeabilization and blocking.
      NOTE: Blocking solution varies based on the antibodies. For example, blocking solution: PBS-gelatin-Triton X-100-serum (PGST, see Table of Materials) solution: 0.2% porcine skin gelatin, 0.5% Triton X-100, and 5% goat serum in PBS.
   2. Incubate the sample with primary antibodies in PGST solution, for example, NF200 (1: 200 dilution, see Table of Materials) at 4 °C, and gently shake for 10-12 days. Then, thoroughly wash the sample in PGST for 1 h 5 times at RT.
   3. Incubate the sample with secondary antibody solution (1:300 dilution) and DAPI (1 mg/mL) in PGST at 4 °C for 7 days.
   4. Thoroughly wash the sample in PGST for 1 h 5 times at RT. Transfer the sample into PBS and store the aorta in PBS at 4 °C for further steps.
      NOTE: Cover the tubes with aluminum foil to keep them dark for steps 2.2.3-2.2.4.
3. Modified iDISCO tissue clearing
   NOTE: During clearing, the tubes should be covered with aluminum foil to avoid signal bleaching/quenching due to direct natural/artificial light exposure. All organic reagents used in DISCO clearing are harmful, and all clearing steps should be done in a fume hood. Wear a lab coat, gloves, and a mask when dealing with the reagents to avoid inhalation and contact with skin and eyes. Collect and dump the clearing waste into appropriate waste containers in the hood.
   1. Transfer the stained samples from step 2.2.4 to a series of increased concentrations of tetrahydrofuran (THF, see Table of Materials) working solutions for dehydration, that is, 50%, 70%, 90%, and 100% (two times 100%). Incubate for 12 h per concentration.
      NOTE: Dilute the THF reagent (99%-100%) in distilled water for working solutions of different concentrations in the hood. Refresh working solutions every 6 h.
   2. Transfer the sample to absolute dichloromethane (DCM, see Table of Materials) solution for 3 h for lipid removal.
   3. Transfer the sample into RI matching benzyl-alcohol-benzyl-benzoate (BABB) working solution (see Table of Materials). Incubate for 3-6 h until the tissue is translucent and mostly transparent in visible light.
      NOTE: To prepare BABB working solution, mix 1 part of benzyl alcohol and 2 parts of benzyl benzoate (1:2) in a glass bottle. Gently shake it for 5 min in the hood.
4. Imaging of iDISCO clearing tissue using a light-sheet microscope
   NOTE: Image the cleared sample as soon as an optimal transparency is achieved. A light-sheet microscope, such as an ultramicroscope-II (see Table of Materials), is used for imaging. Fill the imaging reservoir with the final clearing solution, the BABB solution. Be careful not to spill BABB on the instrument to avoid instrument damage.
   1. Use a 1x air objective for low-magnification images covering the entire width of the whole sample. Gently transfer the sample from the clearing solution on a paper cloth and dry it shortly.
      NOTE: Use blunt forceps to avoid squeezing the sample.
   2. Select an appropriate-sized sample holder based on the sample/tissue size and attach the rear side of the sample to the sample holder with super-glue.
      NOTE: Wait for 1-2 min until the sample is firmly attached to the sample holder.
   3. Fill the imaging reservoir of the light-sheet microscope with BABB solution and gently place the sample into it. Adjust the sample holder to ensure that the sample is perpendicular to the light sheet and illuminated properly.
   4. Lower the objective to focus the sample and adjust the display settings while focusing to view the sample properly. Select the proper light sheet (s) in the microscope software and adjust its width to evenly illuminate the entire view field.
      NOTE: In an ultramicroscope system with both-side laser illumination, start by aligning and focusing the laser on one side. Once both sides are set, activate both side lasers to observe a merged scan image from both illuminations.
   5. Select a far-red channel (680 nm) to image NF200-stained neuronal structures and an autofluorescence channel (488 nm) to image unstained aorta and connective tissues. Adjust the laser power depending on the fluorescent signal intensity for an optimal signal-to-noise ratio, the exposure time, and the width of the light sheet (s).
   6. Select the x-y-z scan mode. Set the z-stack scan with a z-step of 2-8 µm and select 25%-60% overlap along the longitudinal y-axis of the tile scans (16 bit) covering ventral and dorsal surfaces of the samples to image the entire sample volume (up to 6-8 mm in depth).
   7. Name the scan with detailed information, including the date of scan, sample name, the antibody used, objective, zoom factor, z-steps, and lasers used. Save the data.
   8. Change to a higher magnification (2x or 4x) via zoom-in function to image specific body regions of interest.
   9. Follow steps 2.4.4-2.4.7 above for imaging and save the data.

3. Image processing and analyses

NOTE: A high-power processing workstation is needed for the processing. Ensure data backup immediately after processing due to the high volume of imaging (5-100 GB per image).

1. Image processing of CLSM and multiphoton images
   1. Load the Z-stack tiled images from CLSM and multiphoton microscopes to the image processing workstation equipped with Las X, Imaris (image analysis software), or Fiji software for 3D and two-dimensional (2D) visualization, segmentation, and quantification.
   2. To color code the volume depth of a cell or structure, use image restoration software (see Table of Materials) to deconvolute the raw image and generate a maximum intensity projection of the deconvoluted data using temporal color coding in Las X or Fiji.
2. Image processing of light-sheet images
   1. Load the Z-stack tiled TIFF image series to Fiji software. Stitch images with Fiji's stitching plugin and save stitched images in TIFF format.
      NOTE: If needed, compress the stitched images in LZW format to allow fast processing with different software.
   2. Load the stitched images into 3D visualization software (see Table of Materials) for image segmentation. Manually trace the neuronal and vascular structures in the x-y-z axis. For manual tracing of small nerve fibers, manually select the NF200⁺ signals pixel by pixel in every z-plane along the entire path of the nerve fiber between the aorta and ganglia.
   3. Load preprocessed images into image analysis software (see Table of Materials) for video generation, 2D, and 3D visualization of the images.
   4. Use autofluorescence to segment the aorta and connective tissues, including lymph nodes and muscle, in the image analysis software. Apply separate pseudocolors in the image analysis software to visualize distinct aortic segments such as aortic wall and plaque.
   5. Use the contrast-limited adaptive histogram equalization (CLAHE) function in Fiji software to enhance the local contrast over the background of the processed images.

Results

To demonstrate the microanatomy of the intact healthy and diseased aorta and to reveal the physical connectivities between the immune system, the nervous system, and the cardiovascular system in mouse models of atherosclerosis, we used two complementary tissue clearing approaches: TDE clearing of the isolated aorta, and iDISCO clearing of the whole mouse (Figure 1). After whole-mount immunostaining, the enface aorta was cleared with a series of TDE working solutions. The RI is match...

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