This work was funded by the German Research Foundation (DFG) SFB1123/Z1, German Centre for Cardiovascular Research (DZHK) DZHK 81X2600282, and a Corona foundation grant (S199/10087/2022) to SKM; and ERA-CVD (PLAQUEFIGHT) 01KL1808 and a government grant to AJRH at Easemedcontrol R &#38;D GmbH and Co. KG.

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
<ol>
	<li>Tissue preparation
	<ol>
		<li>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 50:50 by-volume mixture of ketamine and xylazine at 3 &#181;L/g.</li>
		<li>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.</li>
		<li>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%&#160;paraformaldehyde&#160;(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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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&#160;overnight at 4 &#176;C. 
		NOTE: Pin the aorta to the wax plate before fixation to avoid folding and curling in the following steps.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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)</li>
		<li>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.</li>
		<li>Incubate the aorta with secondary antibodies in 10% normal donkey serum (1:300 dilution) and 4&#39;,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 &#176;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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Transfer the stained aorta into 20% TDE working solutions and incubate for 1 h.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Imaging of TDE clearing aorta using confocal laser scanning microscope (CLSM)
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;m deep with a higher resolution, which is needed for the analysis of nerve diameter and the nerve-immune cell interaction in the aortic adventitia.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;m step-size up to 60 &#181;m depth for 3D imaging.</li>
		<li>Name the file with sample details, scan details, and save the data.</li>
	</ol>
	</li>
	<li>Imaging of TDE clearing aorta using a multiphoton microscope
	<ol>
		<li>Follow steps 1.4.1-1.4.2 to mount the en face aorta on the slide.</li>
		<li>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.</li>
		<li>Acquire Z-stacks from the abluminal side at 10-15 &#181;m step-size up to 700 &#181;m depth. Name the file and save the data as in step 1.4.7.</li>
	</ol>
	</li>
</ol>
2. Whole-body immunostaining and iDISCO tissue clearing
<ol>
	<li>Tissue preparation
	<ol>
		<li>Follow steps 1.1.1-1.1.3 above for anesthesia, fixing the mouse, and perfusion.</li>
		<li>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.</li>
		<li>Dissect the body part above the diaphragm level and fix the lower body part with 4% PFA for 1-2 days (s) at 4 &#176;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.</li>
		<li>Thoroughly wash the sample for 10 min 3 times at RT. 
		NOTE: Transfer the aorta into different tubes filled with PBS each time.</li>
		<li>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).</li>
		<li>Thoroughly wash the sample for 1 h five times in PBS at RT.</li>
	</ol>
	</li>
	<li>Antibody staining 
	NOTE: All incubation steps are performed in 50 mL safe-lock tubes with gentle rotation or shaking at 4 &#176;C.
	<ol>
		<li>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.</li>
		<li>Incubate the sample with primary antibodies in PGST solution, for example, NF200 (1: 200 dilution, see Table of Materials) at 4 &#176;C, and gently shake for 10-12 days. Then, thoroughly wash the sample in PGST for 1 h 5 times at RT.</li>
		<li>Incubate the sample with secondary antibody solution (1:300 dilution) and DAPI (1 mg/mL) in PGST at 4 &#176;C for 7 days.</li>
		<li>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 &#176;C for further steps. 
		NOTE: Cover the tubes with aluminum foil to keep them dark for steps 2.2.3-2.2.4.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Transfer the sample to absolute dichloromethane (DCM, see Table of Materials) solution for 3 h for lipid removal.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Select the x-y-z scan mode. Set the z-stack scan with a z-step of 2-8 &#181;m 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).</li>
		<li>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.</li>
		<li>Change to a higher magnification (2x or 4x) via zoom-in function to image specific body regions of interest.</li>
		<li>Follow steps 2.4.4-2.4.7 above for imaging and save the data.</li>
	</ol>
	</li>
</ol>
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).
<ol>
	<li>Image processing of CLSM and multiphoton images
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Image processing of light-sheet images
	<ol>
		<li>Load the Z-stack tiled TIFF image series to Fiji software. Stitch images with Fiji&#39;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.</li>
		<li>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.</li>
		<li>Load preprocessed images into image analysis software (see Table of Materials) for video generation, 2D, and 3D visualization of the images.</li>
		<li>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.</li>
		<li>Use the contrast-limited adaptive histogram equalization (CLAHE) function in Fiji software to enhance the local contrast over the background of the processed images.</li>
	</ol>
	</li>
</ol>

Optimized Tissue-Clearing Protocols for 3D Imaging of the Murine Aorta 

<ol>
	<li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+landscape+of+atherosclerosis.">The changing landscape of atherosclerosis.</a> Nature. 592 (7855), 524-533 (2021).</li><li>Mohanta, S., Yin, C., Weber, C., Hu, D., Habenicht, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aorta+atherosclerosis+lesion+analysis+in+hyperlipidemic+mice.">Aorta atherosclerosis lesion analysis in hyperlipidemic mice.</a> Bio Protoc. 6 (11), e1833 (2016).</li><li>Yin, C., Mohanta, S. K., Srikakulapu, P., Weber, C., Habenicht, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery+tertiary+lymphoid+organs:+Powerhouses+of+atherosclerosis+immunity.">Artery tertiary lymphoid organs: Powerhouses of atherosclerosis immunity.</a> Front Immunol. 7, 387 (2016).</li><li>Mohanta, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery+tertiary+lymphoid+organs+contribute+to+innate+and+adaptive+immune+responses+in+advanced+mouse+atherosclerosis.">Artery tertiary lymphoid organs contribute to innate and adaptive immune responses in advanced mouse atherosclerosis.</a> Circ Res. 114 (11), 1772-1787 (2014).</li><li>Carmeliet, P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+mechanisms+of+nerve+and+blood+vessel+wiring.">Common mechanisms of nerve and blood vessel wiring.</a> Nature. 436 (7048), 193-200 (2005).</li><li>Mohanta, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimmune+cardiovascular+interfaces+control+atherosclerosis.">Neuroimmune cardiovascular interfaces control atherosclerosis.</a> Nature. 605 (7908), 152-159 (2022).</li><li>Mohanta, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+brain+circuits.">Cardiovascular brain circuits.</a> Circ Res. 132 (11), 1546-1565 (2023).</li><li>Hu, D., Yin, C., Luo, S., Habenicht, A. J. R., Mohanta, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cells+contribute+to+atherosclerosis+immunity.">Vascular smooth muscle cells contribute to atherosclerosis immunity.</a> Front Immunol. 10, 1101 (2019).</li><li>Newland, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type-2+innate+lymphoid+cells+control+the+development+of+atherosclerosis+in+mice.">Type-2 innate lymphoid cells control the development of atherosclerosis in mice.</a> Nat Commun. 8, 15781 (2017).</li><li>Grabner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin+beta+receptor+signaling+promotes+tertiary+lymphoid+organogenesis+in+the+aorta+adventitia+of+aged+Apoe-/-+mice.">Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged Apoe-/- mice.</a> J Exp Med. 206 (1), 233-248 (2009).</li><li>Mai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+cellular+mapping+in+mouse+using+standard+igg+antibodies.">Whole-body cellular mapping in mouse using standard igg antibodies.</a> Nat Biotechnol. 42 (4), 617-627 (2024).</li><li>Molbay, M., Kolabas, Z. I., Todorov, M. I., Ohn, T. L., Erturk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guidebook+for+disco+tissue+clearing.">A guidebook for disco tissue clearing.</a> Mol Syst Biol. 17 (3), e9807 (2021).</li><li>Tainaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159 (4), 911-924 (2014).</li><li>Tian, T., Yang, Z., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+technique:+Recent+progress+and+biomedical+applications.">Tissue clearing technique: Recent progress and biomedical applications.</a> J Anat. 238 (2), 489-507 (2021).</li><li>Adolfs, Y., Raj, D. D. A., Brignani, S., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+tissue+clearing+and+3d+analysis+of+dopamine+neurons+in+the+developing+mouse+midbrain.">Protocol for tissue clearing and 3d analysis of dopamine neurons in the developing mouse midbrain.</a> STAR Protoc. 2 (3), 100669 (2021).</li><li>Power, R. M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+light-sheet+fluorescence+microscopy+for+multiscale+imaging.">A guide to light-sheet fluorescence microscopy for multiscale imaging.</a> Nat Methods. 14 (4), 360-373 (2017).</li><li>Sun, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+approaches+in+atherosclerosis.">Tissue clearing approaches in atherosclerosis.</a> Methods Mol Biol. 2419, 747-763 (2022).</li><li>Chistiakov, D. A., Ashwell, K. W., Orekhov, A. N., Bobryshev, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+the+arterial+wall+and+its+modification+in+atherosclerosis.">Innervation of the arterial wall and its modification in atherosclerosis.</a> Auton Neurosci. 193, 7-11 (2015).</li><li>Cao, Y., Wang, H., Wang, Q., Han, X., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+volume+fluorescence-imaging+of+vascular+plasticity+in+adipose+tissues.">Three-dimensional volume fluorescence-imaging of vascular plasticity in adipose tissues.</a> Mol Metab. 14, 71-81 (2018).</li><li>Kirst, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+fine-scale+organization+and+plasticity+of+the+brain+vasculature.">Mapping the fine-scale organization and plasticity of the brain vasculature.</a> Cell. 180 (4), 780-795.e25 (2020).</li><li>Todorov, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+learning+analysis+of+whole+mouse+brain+vasculature.">Machine learning analysis of whole mouse brain vasculature.</a> Nat Methods. 17 (4), 442-449 (2020).</li><li>Bhatia, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+proteomics+in+three-dimensional+intact+specimens.">Spatial proteomics in three-dimensional intact specimens.</a> Cell. 185 (26), 5040-5058.e19 (2022).</li><li>Mai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+tissue+labeling+and+clearing+of+intact+human+organs.">Scalable tissue labeling and clearing of intact human organs.</a> Nat Protoc. 17 (10), 2188-2215 (2022).</li></ol>

SKM, CJY, and AJRH are cofounders of Easemedcontrol R &amp;D GmbH and Co. KG.

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 cell-tissue interactions and to advance the understanding of cardiovascular disease pathogenesis. Tissue clearing and subsequent 3D microscopic imaging allow imaging of large-scale, hitherto inaccessible intact tissue compartments, revealing detailed reconstructions of arteries. Here, we introduce protocols for TDE and iDISCO tissue-clearing techniques, highlighting their effectiveness and simplicity in achieving near-complete tissue transparency, efficient imaging with confocal/multiphoton or light-sheet microscopy, and, at the same time, maintaining structural integrity.
TDE clearing uses a water-soluble reagent to achieve tissue transparency by adjusting the RI, thereby effectively preserving fluorescence signals in the tissue. It represents an efficient tissue-clearing method for smaller size blood vessels, that is micrometer to millimeter size tissue volume without tissue shrinkage. However, it requires longer processing time for larger tissues, that is, millimeter to centimeter-sized tissue volumes. Importantly, the procedure can be adapted to the user&#39;s need by adjusting the concentration of the reagents and the incubation time. On the other hand, iDISCO uses a series of organic solvents to remove lipids and water, leading to better tissue transparency for larger tissues, whole organs, or the whole mouse or larger human tissues. It offers the unique advantages of short processing times and suitability for large tissue volumes. However, it leads to shrinkage of the cleared samples, quenching of the fluorescence signals, and removal of lipids, which is a major limitation for lipid-rich atherosclerotic tissues. In addition, light-sheet microscopy, unlike confocal or multiphoton microscopy, cannot visualize subcellular details at the micrometer level and visualize small-diameter neuronal projections, such as single axons and their connections with other vascular cells.
Vascular innervation plays a significant role in the development of atherosclerosis6,7,18. Investigating the distribution of peripheral nerves along the arterial tree and their changes will help to understand disease progression. Due to its efficient processing capability, iDISCO is commonly used for whole vascular network imaging in atherosclerosis models in rats and mice12,19,20,21, providing high-resolution three-dimensional images. TDE, on the other hand, is suitable for studies requiring the preservation of fluorescent labels, such as observing specific neuronal markers, thereby accurately revealing the fine structure and changes in vascular innervation6. Both tissue-clearing protocols are required as TDE clearing does not involve shrinking and can image lipids, whereas iDISCO leads to tissue shrinking due to dehydration and delipidation. Moreover, modified iDISCO protocol can be used for tissue clearing of human cardiovascular tissues22,23. Both clearing techniques offer complementary advantages to studying arterial remodeling in atherosclerosis. By combining the strengths of both methods, a more comprehensive and in-depth understanding of the pathological mechanisms of atherosclerosis may be achieved.

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 decades1,2. 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 ganglia3,4. 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-interfaces6,7. 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 adventitia6,8,9. 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 approaches11,12,13,14,15 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 atlas11,16. Visualizing these cellular and structural connections in 3D can provide new insights to answer hitherto unanswered biological questions.

<table><tbody><tr><td>2,2&amp;rsquo;-thiodiethanol (TDE)</td><td>Sigma&amp;nbsp;</td><td>166782</td><td>Clearing reagent</td></tr><tr><td>Amira</td><td>Thermo Fisher Scientific</td><td></td><td>3D visualization software; Image processing software used for manual segmentation and tracing in 3D images</td></tr><tr><td>Benzyl alcohol</td><td>Sigma&amp;nbsp;</td><td>W213713</td><td>Clearing reagent</td></tr><tr><td>Benzyl benzoate</td><td>Sigma</td><td>B6630</td><td>Clearing reagent</td></tr><tr><td>CD16/32</td><td>eBioscience</td><td>14-0161-82</td><td>Blocking solution</td></tr><tr><td>Confocal laser scanning microscope&amp;nbsp;</td><td>Leica Microsystems</td><td>TCS- SP8 3X</td><td>Imaging device for multidimensional high-resolution imaging of intact biological tissues or sections with high specificity at subcellular resolution.</td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td>Nuclei marker</td></tr><tr><td>Dichloromethane (DCM)</td><td>Sigma&amp;nbsp;</td><td>270997</td><td>Clearing reagent</td></tr><tr><td>Dissecting pan-black wax&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>S17432</td><td>Aorta dissection and fixation&amp;nbsp;</td></tr><tr><td>Dissection stereomicroscope</td><td>Leica Microsystems&amp;nbsp;</td><td>Stemi 2000</td><td>Mouse organ dissection</td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>E7023</td><td>Defection&amp;nbsp;</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Roth&amp;nbsp;</td><td>8040.1</td><td>Perfusion buffer&amp;nbsp;</td></tr><tr><td>Fiji&amp;nbsp;</td><td>(ImageJ, NIH)</td><td></td><td>Open source image processing software for 2D and 3D images</td></tr><tr><td>Goat anti-Hamster IgG, Cy3</td><td>Dianova&amp;nbsp;</td><td>127-165-099</td><td>Secondary antibody</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 680</td><td>Thermo Fisher Scientific / Invitrogen</td><td>A-21109</td><td>Secondary antibody&amp;nbsp;</td></tr><tr><td>Goat anti-Rat IgG, Cy5</td><td>Dianova</td><td>712-175-150</td><td>Secondary antibody</td></tr><tr><td>Hamster Anti-CD3e&amp;nbsp;</td><td>BD Bioscience</td><td>145-2C11&amp;nbsp;</td><td>Pan-T cell marker</td></tr><tr><td>Huygens Professional</td><td>Scientific Volume Imaging, The Netherlands</td><td>Version 19.10</td><td>Image restoration software; Image processing software used mainly for deconvolution of 2D and 3D images</td></tr><tr><td>Image processing workstation</td><td>MIFCOM&amp;nbsp;</td><td>MIFCOM X5</td><td>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</td></tr><tr><td>Imaris</td><td>Bitplane</td><td>Version 8.4</td><td>Image analysis software; Image processing software used for automated segmentation of 3D images</td></tr><tr><td>Incubator and rotator&amp;nbsp;</td><td>Marshall Scientific&amp;nbsp;</td><td>Innova 4230</td><td>Incubation and rotation device during tissue&lt;br/&gt; clearing&amp;nbsp;</td></tr><tr><td>iSpacer</td><td>Sunjin Lab</td><td>IS4020</td><td>Rectangular well as the sample holder</td></tr><tr><td>Ketamine</td><td>Livisto</td><td></td><td>Anesthetic</td></tr><tr><td>Leica Application Suite X (LAS-X)&amp;nbsp;</td><td>Leica Microsystems</td><td>Version 3.5</td><td>Image processing software for the images acquired with Leica microscope</td></tr><tr><td>Light microscope</td><td>Leica Microsystems</td><td>DM LB</td><td>Imaging device for bright filed imaging</td></tr><tr><td>Light sheet&amp;nbsp; microscope</td><td>LaVision BioTech</td><td>Ultramicroscope II</td><td>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.</td></tr><tr><td>Multiphoton microscopy</td><td>Leica Microsystems</td><td>TCS-SP5II MP&amp;nbsp;</td><td>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.</td></tr><tr><td>Normal goat serum&amp;nbsp;</td><td>Sigma</td><td>G9023</td><td>Blocking solution</td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P-6148&amp;nbsp;</td><td>Fixation&amp;nbsp;</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma&amp;nbsp;</td><td>P4417-100TAB&amp;nbsp;</td><td>Washing buffer</td></tr><tr><td>Porcine skin gelatin</td><td>Sigma&amp;nbsp;</td><td>G1890</td><td>Incubation buffer</td></tr><tr><td>Quadrol</td><td>Sigma</td><td>122262</td><td>CUBIC clearing reagent</td></tr><tr><td>Rabbit Anti-NF200</td><td>Sigma</td><td>N4142</td><td>Pan-neuronal marker</td></tr><tr><td>Rat Anti-B220&amp;nbsp;</td><td>BD Bioscience</td><td>RA3-6B2</td><td>Pan-B cell marker</td></tr><tr><td>Sucrose</td><td>Sigma&amp;nbsp;</td><td>90M003524V&amp;nbsp;</td><td>Dehydration&amp;nbsp;</td></tr><tr><td>Sytox</td><td>Thermo Fisher Scientific</td><td>S11380</td><td>Nuclei marker</td></tr><tr><td>Tetrahydrofuran</td><td>Sigma&amp;nbsp;</td><td>401757</td><td>Clearing reagent&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Roth&amp;nbsp;</td><td>3051.1</td><td>Penetration</td></tr><tr><td>Urea</td><td>Sigma&amp;nbsp;</td><td>U5128</td><td>CUBIC clearing reagent</td></tr><tr><td>Xylene&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>x/0250/17&amp;nbsp;</td><td>Anesthetic&amp;nbsp;</td></tr></tbody></table>

three-dimensional imaging of aortic tissues in atherosclerosis

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.

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 matched at 1.4-1.5, depending on the TDE concentration of the final step, to achieve optimal transparency of the aortic tissue without changing its volume (Figure 1A). Unlike TDE clearing, the iDISCO clearing protocol includes decolorization and permeabilization steps before whole-mount immunostaining, followed by delipidation and dehydration using different chemical solvents, and the RI matching at 1.56 (Figure 1B). The iDISCO clearing allows visualization of large-size tissue and even the entire mouse body due to the shrinkage of the tissue.
Confocal and multiphoton 3D imaging of the TDE-cleared whole mount en face aorta allows the visualization of structures, including intimal plaques, adventitia cellularity, and adventitia neuronal projections among other structures, whereas light-sheet imaging of iDISCO cleared mouse allows the visualization and the reconstruction of arterial tree along with perivascular adipose tissue, renal lymph nodes and the associated neural components including nerves and ganglia (Figure 1C). For example, confocal imaging of TDE-cleared atherosclerotic aorta detected multiple CD3e stained T cells in plaque and extensive neogenesis of NF200 stained nerve fibers of different diameters in ATLOs, whereas multiphoton imaging of TDE cleared whole mount diseased abdominal aorta from aged Apoe-/- mice showed territoriality of NF200-expressing axon sprouting in B220 negative T cell areas within ATLOs (Figure 2A). Conversely, light-sheet imaging of iDISCO cleared whole mouse abdomen visualized the spatial relationship between the aorta and the adjacent sympathetic ganglia. 3D image segmentation and tracing of individual axons from the ganglia to the aortic surface revealed robust neuronal restructuring, including newly formed NF200 stained axons in the aorta adventitia adjacent to atherosclerotic plaque (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67400/67400fig01.jpg" /> 
Figure 1: Workflow of the study. (A) Aqueous-based TDE clearing of the en faced aorta. (B) Solvent-based iDISCO clearing of the whole mouse. (C). 3D volumetric imaging and image processing of optically cleared isolated aorta or whole mouse samples. This figure has been modified with permission from Sun et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67400/67400fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67400/67400fig02v2.jpg" /> 
Figure 2: Example of 3D images of TDE and iDISCO cleared tissues. (A) 3D imaging of TDE cleared en face aorta. Upper panel: Confocal images showed CD3e+ T cells (red, arrow) in intimal plaque and NF200+ nerve axons (green) in atherosclerotic aorta adventitia (ATLO); Lower panel: Multiphoton images showed NF200+ nerve axons (green, arrowheads), B220+ B cells (red, open triangle), collagen-derived second harmonic generation (SHG, white) in ATLOs of 78-weeks-old male Apoe-/- mice. Sytox (blue) and DAPI (blue) showed the nuclei. (B) 3D reconstructed and segmented images of iDISCO cleared whole mouse abdomen. Light-sheet 3D reconstruction, segmentation, and tracing of NF200+ nerves and axons (arrows, green) and their spatial relationship with sympathetic ganglia (arrowheads) of the intact abdominal aorta. Open triangle indicated a newly formed axon adjacent to atherosclerotic plaques. Autofluorescence (auto) was used to image the aorta and connective tissue, including plaque (cyan) and renal lymph node (RLN, blue). CG, celiac ganglia, SycG, sympathetic chain ganglia. This figure has been modified with permission from Mohanta et al.6 and Sun et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67400/67400fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

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.

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial ...

three-dimensional-imaging-of-aortic-tissues-in-atherosclerosis

Nanyang Technological University

Research

JoVE Journal

Biology

781 Views.  The First Affiliated Hospital of Sun Yat-sen University. Ons onderzoek richt zich op het begrijpen van de rol van het zenuwstelsel bij de progressie van atherosclerose. Specifiek bij elke interactie met de kam willen we beantwoorden hoe de zenuw verandert tijdens atherosclerose en hoe de interactie de ziekteprogressie beïnvloedt. Verschillende geavanceerde technologieën worden momenteel gebruikt om het onderzoek naar atherosclerose vooruit te helpen en het begrip van ziektemechanismen te verbeteren.Deze om...

Video: Driedimensionale beeldvorming van aortaweefsels bij atherosclerose

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Medicine

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Bioengineering

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach warrants good early results, but adequate follow-up imaging after EVAR is imperative to maintain long-term positive outcomes. Potential graft-related complications are graft migration, infection, fraction, and endoleaks, with the last one being the most common. The most frequently used imaging after EVAR is computed tomography angiography (CTA) and duplex ultrasound. Dynamic, time-resolved computed tomography angiography (d-CTA) is a reasonably new technique to characterize the endoleaks. Multiple scans are done sequentially around the endograft during acquisition that grants good visualization of the contrast passage and graft-related complications. This high diagnostic accuracy of d-CTA can be implemented into therapy via image fusion and reduce additional radiation and contrast material exposure.
This protocol describes the technical aspects of this modality: patient selection, preliminary image review, d-CTA scan acquisition, image processing, qualitative and quantitative endoleak characterization. The steps of integrating dynamic CTA into intra-operative fluoroscopy using 2D-3D fusion-imaging to facilitate targeted embolization are also demonstrated. In conclusion, time-resolved, dynamic CTA is an ideal modality for endoleak characterization with additional quantitative analysis. It can reduce radiation and iodinated contrast material exposure during endoleak treatment by guiding interventions.

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

Dynamic computed tomography angiography (CTA) imaging provides additional diagnostic value in characterizing aortic endoleaks. This protocol describes a qualitative and quantitative approach using time-attenuation curve analysis to characterize endoleaks. The technique of integrating dynamic CTA imaging with fluoroscopy using 2D-3D image fusion is illustrated for better image guidance during treatment.

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach ...

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...

Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video