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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

Abstract

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results to illustrate progress in the research of human cardiac anatomy.

Introduction

As function follows form, understanding the architecture of the heart is fundamental for appreciation of its physiology. Although numerous investigations have revealed cardiac anatomy from micro- to macroscales1,2,3, multiple questions remain unresolved, especially those related to human cardiac anatomy. This is in part because basic studies focusing on functional anatomy generally utilized animal hearts4,5,6, which are often distinct from human hearts1,7,8. Furthermore, each individual study, even those using human heart samples, tends to focus on very specific structures, which renders it difficult to apply the findings in the context of the whole heart. This is even more so if the focused structures are at micro- or mesoscales, such as the perinexus9 and ganglionated plexuses10.

In this context, systemic structural study of the human heart rejected for transplantation provides a unique and rare opportunity to obtain a comprehensive atlas of cardiac structures in focus across microscopic and macroscopic scales11. Microscopic examination protocols include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs, iDISCO+)12,13, and immunohistochemical staining. Mesoscopic examination protocols include stereoscopic dissection, macro photography, and micro-computed tomographic (CT) scanning. Macroscopic examination protocols include gross dissection14, photography (including anaglyphs and photogrammetry)15,16,17, CT, virtual dissection18, and 3D printing of the physically or virtually dissected or whole heart17. In preparation for macroscopic examination, pressure-perfusion fixation is performed to maintain the 3D architecture and physiologically relevant morphology of the heart14,19,20,21. The combined application of these techniques is unique and crucial to correlate distinct anatomic features in the context of the 3D architecture of the human heart.

As the opportunity to obtain a non-pathological human heart sample is extremely limited, a multi-scale approach described herein maximizes the use of the sample. By applying various procedures described below, representative results will illustrate to the reader how the findings can be utilized for multiple purposes, including discovery in scientific research11 (comprehensive analyses of cardiac innervation, distribution of ganglionated plexuses), improvement of clinical procedures (simulation for surgical and interventional approaches), and anatomical education (real 3D demonstration of cardiac anatomy).

Protocol

This study used de-identified tissue samples collected from non-failing donor human hearts and was approved by the Institutional Review Board of the University of California, Los Angeles (UCLA). Samples were obtained from non-failing hearts that were rejected for transplantation. The hearts were pressure-perfused, fixed in 4% paraformaldehyde (PFA), and imaged before tissue processing per the following methods. Figure 1 summarizes the flow chart of the order of the study. The details of the reagents and the equipment used in the study are listed in the Table of Materials.

1. Micro-scale examination

  1. Tissue clearing using modified immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO+) protocol.
    1. Dissect 4% PFA-fixed tissue with a scalpel to fit within the 3 mm × 16 mm × 25 mm chamber for confocal microscopy. To image thicker tissues, additional chambers and/or spacers may be stacked on the slide.
    2. Dehydrate specimens using graded methanol (MeOH) series (20%, 40%, 60%, 80%, and 100% MeOH in deionized H2O [vol/vol]) for 1 h each at room temperature (RT) with agitation.
    3. Wash with 100% MeOH for 1 h at RT and immerse in 66% dichloromethane/33% MeOH at RT with agitation overnight.
    4. The next day, wash twice in MeOH (100%) for 1 h at RT, chill at 4 °C, and treat with 5% H2O2 in MeOH (vol/vol) overnight at 4 °C.
    5. Rehydrate with graded MeOH series (80%, 60%, 40%, and 20% MeOH) and wash in 0.01 mol/L PBS for 1 h each at RT with agitation.
    6. Wash the tissues twice in 0.01 mol/L PBS with 0.2% Triton X-100 for 1 h at RT.
    7. Prepare for immunolabeling by permeabilizing in 0.01 mol/L PBS, 20% dimethyl sulfoxide (DMSO), 0.2% Triton X-100, and 0.3 mol/L glycine for 2 days at 37 °C with agitation.
    8. Block in 0.01 mol/L PBS with 10% DMSO, 0.2% Triton X-100, and 5% normal donkey serum for another 2 days at 37 °C with agitation.
    9. Label with a primary antibody that is compatible with MeOH conjugated to fluorophores diluted in 0.01 mol/L PBS with 10 mg/mL heparin (PTwH), 0.2% Tween-20, 5% DMSO, and 3% normal donkey serum for 3-4 days at 37 °C with agitation.
    10. Replenish the antibody solution and incubate for another 3-4 days at 37 °C with agitation.
    11. After 1-week incubation in primary antibody solution, wash 4 to 5 times in PTwH overnight at RT.
    12. Incubate with secondary antibody conjugated to fluorophores diluted in PTwH, 3% normal donkey serum for 3 days at 37 °C with agitation.
    13. Replenish the secondary antibody solution incubate for another 3 days at 37 °C with agitation.
    14. After 6-day incubation in secondary antibody solution, wash in PTwH 4-5 times overnight at RT.
    15. Dehydrate with a graded MeOH series (20%, 40%, 60%, 80%, 100%, and 100% MeOH). The sample may be stored overnight at RT.
    16. Incubate in 66% dichloromethane/33% MeOH for 3 h at RT with agitation.
    17. Wash twice in 100% dichloromethane for 15 min at RT with agitation.
    18. Incubate and store specimens in benzyl ether. Fill the tube to minimize air from oxidizing the sample.
  2. Imaging tissue-cleared specimen
    1. Affix a chamber containing adhesive to a slide and apply nail polish around the perimeter of the chamber. For thicker tissues, additional chambers and/or spacers may be stacked on the slide.
    2. Place cleared tissue in the chamber, fill with benzyl ether, and apply a coverslip.
    3. Apply nail polish around the coverslip to create a seal.
    4. Obtain tilescan and Z stack images using an upright laser scanning confocal microscope with a 5x or 10x lens to image at a depth up to the working distance of the lens.
    5. Image at a resolution of 1024 x 1024 using laser lines appropriate for emission spectra of fluorophores used. Muscle autofluorescence is visible using the 488 nm laser line.
    6. Ensure that the z-axis step size is commensurate with Nyquist sampling based on the numerical aperture of the specified objective11.
    7. Stitch images and use software for 3D visualization.
    8. Create figures using maximum intensity projection (MIP) images of Z stacks for individual and merged channels (Figure 2).
  3. Immunohistochemistry
    NOTE: After the tissue is paraffin-embedded22, the following procedure is used to create slides for immunohistochemical study.
    1. Preparation of refractive index matching solution (RIMS).
      1. Prepare 0.1 mol/L phosphate buffer by adding 10.9 g of Na2HPO4 (anhydrous) and 3.1 g of NaH2PO4 (monohydrate) to deionized H2O to a total volume of 1 L (pH 7.4). Filter-sterilize the solution and store it at RT.
      2. Dilute the phosphate buffer to 0.02 mol/L.
      3. Dissolve Histodenz in 30 mL of 0.02 mol/L phosphate buffer by stirring the solution for 10 min with a magnetic stir bar in the final storage bottle that may be sealed to minimize evaporation and contamination.
      4. Add sodium azide to a total concentration of 0.01% (w/v) and adjust the pH to 7.5 with NaOH.
      5. Adjust the RI by varying the final concentration of Histodenz.
      6. Store the RIMS at RT for months. Discard if microbial contamination is noted.
        NOTE: Do NOT autoclave any solutions containing sodium azide.
    2. Preparation of slides for immunohistochemical study
      1. Create sections of 5 µm thickness with microtome. Apply tissue section to charged slides.
      2. Remove paraffin by incubating the slides in >75% xylene for 10 min. Move slides to a second container with xylene for an additional 10 min.
      3. Remove xylene by immersing the slides in 100% EtOH for 10 min, then in 95% EtOH for 5 min and 70% EtOH for 5 min.
      4. Rinse the slides with deionized H2O for 5 min.
      5. Immerse slides in antigen retrieval buffer for 25 min at 90-95°C.
      6. Allow the container to cool to RT for 1 h with agitation.
      7. Immerse the slides in soaking buffer (0.01 mol/L PBS + 0.4% Triton X-100) for 30 min at 4°C.
      8. Encircle the tissue with a PAP pen. Add PBS to each slide and place it in a humidified chamber to prevent desiccation.
      9. Wash slides with PBS at RT with agitation for 5 min.
      10. Block with blocking buffer (0.01 mol/L PBS + 10% Donkey Serum + 0.1% TX-100) for 1 h with agitation.
      11. Incubate with a primary antibody diluted in blocking buffer overnight at 4°C.
      12. The next day, allow slides to warm to RT for 15 min.
      13. Wash the slides 3 times with 0.01 mol/L PBS + 0.2% TritonX-100 for 5 min.
      14. Incubate with a secondary antibody diluted in blocking buffer for 1 h at RT with agitation.
      15. Wash the slides 3 times with 0.01 mol/L PBS + 0.2% TritonX-100 for 5 min.
      16. Wash the slides 3 times with 0.01 mol/L PBS for 5 min.
      17. Place 1 drop of RIMS with a dropper and apply a coverslip.
      18. Apply nail polish around the coverslip to create the seal.
      19. As a negative control, run a sample without the primary antibody to demonstrate the absence of specific staining.
    3. Imaging immunostained slides
      1. Image the slides with a laser scanning confocal microscope with 10x, 20x and 40x objective lenses.
      2. Image at a resolution of 1024 x 1024 using laser lines appropriate for the emission spectra of the secondary antibodies used.
      3. Create figures using maximum intensity projection (MIP) images of Z stacks for individual and merged channels (Figure 3).

2. Meso-scale examination

  1. Stereoscopic dissection
    1. Perform delicate dissections focusing on tiny or thin structures, such as atrioventricular node, atrioventricular node artery, and cardiac nerve plexus (submillimeter to millimeters in scale) with either a magnifying desk lamp with clamp, surgical telescopes, or stereomicroscope.
  2. Micro-CT scanning
    NOTE: CT imaging is performed after pressure perfusion and fixation and at any stages of dissection using a micro-positron emission tomography (PET)/CT scanner (Figure 4).
    1. Warm up the CT X-ray source for 25 min before sample imaging.
    2. Place the heart sample on the scanner bed.
    3. Move the scanner bed to a horizontal position of 544 mm and a vertical position of 14 mm to center the heart in the CT field of view (FOV).
    4. Acquire CT image at 80 kVp, 150 µA, with 720 projections during 1 min scan time at a spatial resolution of 200 µm.
    5. Reconstruct the CT data with a 12 cm x 12 cm x 10 cm field of view and a matrix of 600 x 600 x 500 voxels, and save as a DICOM file.

3. Macro-scale examination

  1. Pressure perfusion and fixation
    NOTE: The authors modify previously described pressure perfusion and fixation techniques and apply them to the non-failing human hearts rejected for transplantation14,19,20,21.
    1. Use high-flow pumps for perfusion fixation. Use either 100% ethanol14, 4% PFA, or 10% formalin for the fixative.
      NOTE: The heart is recovered with the ascending aorta, pulmonary trunk, and both vena cavae, and pulmonary veins resected as distally as possible and delivered in University of Wisconsin solution23.
    2. Use two 20-24 Fr surgical cannulas for right- and left-heart perfusion. For right-heart perfusion, cannulate the superior vena cava, and place a vent at the pulmonary trunk or pulmonary artery with half-cut 12-30 mL sized plastic syringes with Luer-Lock tips attached to three-way stopcocks.
    3. Occlude the inferior vena cava and the other pulmonary artery with twine after putting an appropriately sized locked half-cut plastic syringe or 1.5-5.0 mL centrifuge tube.
      1. For antegrade perfusion of the left heart, cannulate one of the pulmonary veins and place a vent at the distal cut end of the aorta with the half-cut 12-30 mL sized plastic syringes with Luer-Lock tips attached to three-way stopcocks.
      2. For retrograde perfusion of the left heart, cannulate one of the aortic arch branches and place a vent at another branch of the aortic arch. Place the tips of the cannulas in both ventricles.
    4. Occlude other vessel orifices with twine after inserting an appropriately sized locked half-cut plastic syringe or 1.5-5.0 mL microcentrifuge tube. Use a thin gauze to cover the inserting portion of the syringes/ tubes/ cannulas to prevent leakage and slipping. Fix large leaks using suture, banding, or clumping. Small leaks are permissible.
    5. Suspend the heart in a plastic container.
    6. Connect 22-24 Fr soft plastic tubing to each cannula and insert the other end of the tube into the container filled with fixative.
    7. Circulate fixative through the right- and left-heart circuits using a high-flow pump set at approximately 100-300 mL/min for the right heart and 200-400 mL/min for the left heart to achieve approximately 20 mmHg in the right ventricle and 80 mmHg in the left ventricle, respectively.
    8. Maintain perfusion at 4 °C for 24 h.
    9. Wash the heart with 0.01 mol/L PBS for 30 min with agitation four times.
    10. Store heart in 0.01 mol/L PBS/0.02% sodium azide at 4 °C.
      NOTE: The pressure-perfusion fixation is only effective for a fresh heart, not for a heart recovered from an embalmed cadaver.
  2. Gross dissection
    1. Perform progressive dissection with photographic recordings at each stage of dissection.
    2. To maintain clinical relevance, pay special attention to avoid distorting/deforming any structures to maintain the physiological morphology of the heart.
    3. Image target structures using clinically relevant orientation, such as right anterior oblique orientation.
  3. Photography
    1. Place the pressure-perfused and fixed heart on a tripod with a platform mounted with multiple prongs and the ability to rotate 360o.
    2. Photograph the heart using a digital single-lens reflex camera (Figure 5)24 while using multiple light-emitting diode light panels set on the C-Stands and wide black duvetyn background cloth.
    3. Photograph using the lens with a long focal length (200 mm) for a working distance of 4-6 ft to minimize distortion of the subject14.
  4. Anaglyphs
    1. To display anaglyphic images, reconstruct a pair of photographs or volume-rendered images from CT datasets with a 10° difference in the rotation angle on the horizontal plane.
    2. Convert a set of these two-dimensional (2D) images, referred to as a stereogram, into anaglyphs using freeware16.
    3. To view an anaglyph, use red/cyan glasses.
  5. Photogrammetry
    NOTE: Photogrammetry is the applied science of generating a three-dimensional surface-rendered reconstruction from multiple two-dimensional photographs taken at varying angles17.
    1. Suspend the sample on C-Stands or place it on the rotation table to obtain hundreds of multi-directional photographs with a smartphone.
    2. Generate the 3D model in FBX format using commercially available software.
  6. CT scanning
    NOTE: CT scanning may be performed after pressure perfusion and fixation and at any stage of dissection.
    1. Suspend the heart sample from a bar placed across the top of the container. To prevent the heart from swinging during the scan, support the base of the heart with plastic prongs fixed at the bottom of the container. Thus, the air will serve as a negative contrast.
    2. Perform the CT scan using a commercially available multi-detector row CT scanner with the following parameters: tube voltage of 120 kV, tube current of 800-900 mA, and a gantry rotation of 280 ms. The dose length product is generally 500-1200 mGy.cm.
    3. Reconstruct axial image data using the following parameters: a section thickness, 0.6 mm; an incremental interval, 0.3 mm; a field of view, as small as possible (generally 100-200 mm); and a matrix, 512 × 512.
  7. Virtual dissection
    1. Analyze the CT scan images using commercially available software to generate virtual dissection images.
      NOTE: Virtual dissection is a modification of the volume-rendering process wherein the focus is shifted to the walls of the cardiac chambers and vessels18. In this process, manual thresholding virtually removes the enhanced chamber from the original datasets.
    2. Visualize the non-enhanced walls, septa, and valves with virtual dissection to produce images similar to gross dissection. Unlike gross dissection of the heart specimens, the cut planes during virtual dissection are practically unlimited. Almost any view can be recreated to visualize the structures of interest as needed.
  8. 3D printing
    1. Open the compatible file of the heart sample in 3D printer software.
    2. Use the profile of 0.10 mm Fast DETAIL for the print Settings in the 3D printer and reduce the print speed to 20 mm/s. Enable Generate support material.
    3. Use the profile of TPU​ filament for "Filament Settings" in the 3D printer.
    4. Use the profile of the Original Prusa MK4 Input Shaper 0.4 nozzle for "Printer Settings" in the printer.
    5. After slicing is completed, save the BGCODE file in a USB flash drive for 3D printing.
    6. Use the 1.75 mm TPU filament for 3D printing the human heart sample. Before 3D printing, dry the TPU filament for 6 h using a filament dryer.
    7. To reduce filament tension during 3D printing, place the filament spool on a spool holder with a built-in bearing to facilitate filament spool rotation. Perform 3D printing using a commercially available 3D printer with a textured powder-coated steel sheet.
    8. Carefully remove support materials when 3D printing is complete.

Results

Microscale examinations
Applying tissue clearing allows imaging of larger volumes of tissue in 3D using confocal microscopy. In the heart, ganglia containing cardiac neurons and the neural patterning of myocardial innervation can be visualized (Figure 2). Figure 3 shows a confocal image of the human left ventricle myocardium immunostained for nerves and smooth muscle cells. Blood vessels are noted to traverse the myocardium, and numerous n...

Discussion

The present study demonstrates the comprehensive pipeline to analyze samples obtained from whole human hearts. Representative results show micro- to macroscale anatomical examinations carried out routinely for a single heart. As a human heart sample is extremely precious, a multi-scale approach is ideal and effective so as not to waste any parts of the sample by applying multiple protocols for various purposes, including discovery in scientific research, improvement of clinical procedures, and anatomi...

Disclosures

None.

Acknowledgements

We thank the individuals who have donated their bodies for the advancement of education and research. We are grateful to the OneLegacy Foundation, which formed the basis for obtaining donor hearts for research. We are also grateful to Anthony A. Smithson and Arvin Roque-Verdeflor of the UCLA Translational Research Imaging Center (Department of Radiology) for their support in CT data acquisition. This project was supported by the UCLA Amara Yad Project. We are thankful to Drs. Kalyanam Shivkumar and Olujimi A. Ajijola for establishing and maintaining a human heart pipeline for research. We appreciate our Research Operations Manager, Amiksha S. Gandhi for her dedication to support our projects. This work was made possible by support from NIH grants OT2OD023848 & P01 HL164311 and Leducq grant 23CVD04 to Kalyanam Shivkumar, the American Heart Association Career Development Award 23CDA1039446 to PH, and the UCLA Amara-Yad Project (https://www.uclahealth.org/medical-services/heart/arrhythmia/about-us/amara-yad-project). The GNEXT microPET/CT scanner used in this study was funded by an NIH Shared Instrumentation for Animal Research Grant (1 S10 OD026917-01A1).

Materials

NameCompanyCatalog NumberComments
1x Phosphate buffered salineSigma-AldrichP3813
3D ViewerMicrosoft
647 AffiniPure Donkey Anti-Rabbit IgGJackson ImmunoResearch Laboratories711-605-152
647 AffiniPure Donkey Anti-Sheep IgGJackson ImmunoResearch Laboratories713-605-147
AF Micro-NIKKOR 200 mm f/4D IF-ED lensNikon
Anti-Actin, α-Smooth Muscle - Cy3 antibodySigma-AldrichC6198
Antigen Retrieval Buffer (100x EDTA Buffer, pH 8.0)Abcamab93680
Anti-PGP9.5 (protein gene product 9.5)Abcamab108986
Anti-TH (tyrosine hydrox ylase)Abcamab1542
Anti-VAChT (vesicular acetylcholine transporter)Synaptic Systems139 103
Benzyl etherSigma-Aldrich108014
Bovine serum albuminSigma-AldrichA4503-10G
Cheetah 3D printer filament (95A), 1.75 mmNinjaTek
Coverslip, 22 mm x 30mm, No. 1.5VWR48393 151
Cy3 AffiniPure Donkey Anti-Rabbit IgGJackson ImmunoResearch Laboratories711-165-152
DichloromethaneSigma-Aldrich270997-100ML
Dimethyl sulfoxideSigma-AldrichD8418-500ML
Ethanol, 100%Decon laboratories2701
GlycineSigma-AldrichG7126-500G
GNEXT PET/CTSOFIE Biosciences
Heparin sodium salt from porcine intestinal mucosaSigma-AldrichH3149-50KU
HistodenzSigma-AldrichD2158-100G
Hydrogen peroxide solutionSigma-AldrichH1009-500ML
Imaging softwareZeissZEN (black edition)
Imaging softwareOxford InstrumentsImaris 10
iSpacerSunjin LabsiSpacer 3mm
KIRI EngineKIRI Innovation
Laser scanning confocal microscopeZeissLSM 880
LEAD-2 - Vertical & Multi-channels Peristaltic PumpLONGER
Lightview XL Brightech
Methanol (Certified ACS)Fischer ScientificA412-4
Nikon D850Nikon
NinjaTek NinjaFlex TPU @MK4NinjaTek
Normal donkey serumJackson ImmunoResearch Laboratories017-000-121
Original Prusa MK4 3D printerPrusa Research
PAP penAbcamab2601
Paraformaldehyde, 32%Electron Microscopy Sciences15714-S
PolycamPolycam
Primary antibody
PrusaSlicer 2.7.1Prusa Research
SARA-Enginepita4 mobile LLC
ScaniverseNiantic
Secondary antibody
SlowFade Gold Antiface MountantInvitrogenS36936
Sodium azide, 5% (w/v)Ricca Chemical Company7144.8-32
SOMATOM Definition ASSiemens Healthcare
Standard Field Surgi-Spec Telescopes, Designs for Vision
Stereomicroscope System SZ61OLYMPUS
StereoPhoto MakerFree ware developed by Masuji Suto
Superfrost Plus Microscope Slides, PrecleanedFisher Scientific12-550-15
Triton X-100Sigma-AldrichT8787-50ML
Tween-20Sigma-AldrichP9416-100ML
XyleneSigma-Aldrich534056-4L
Ziostation2Ziosoft, AMIN

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