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Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart
* These authors contributed equally
In This Article
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,<....
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 .......
Representative 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.......
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....
Materials
| Name | Company | Catalog Number | Comments |
| 1x Phosphate buffered saline | Sigma-Aldrich | P3813 | |
| 3D Viewer | Microsoft | ||
| 647 AffiniPure Donkey Anti-Rabbit IgG | Jackson ImmunoResearch Laboratories | 711-605-152 | |
| 647 AffiniPure Donkey Anti-Sheep IgG | Jackson ImmunoResearch Laboratories | 713-605-147 | |
| AF Micro-NIKKOR 200 mm f/4D IF-ED lens | Nikon | ||
| Anti-Actin, α-Smooth Muscle - Cy3 antibody | Sigma-Aldrich | C6198 | |
| Antigen Retrieval Buffer (100x EDTA Buffer, pH 8.0) | Abcam | ab93680 | |
| Anti-PGP9.5 (protein gene product 9.5) | Abcam | ab108986 | |
| Anti-TH (tyrosine hydrox ylase) | Abcam | ab1542 | |
| Anti-VAChT (vesicular acetylcholine transporter) | Synaptic Systems | 139 103 | |
| Benzyl ether | Sigma-Aldrich | 108014 | |
| Bovine serum albumin | Sigma-Aldrich | A4503-10G | |
| Cheetah 3D printer filament (95A), 1.75 mm | NinjaTek | ||
| Coverslip, 22 mm x 30mm, No. 1.5 | VWR | 48393 151 | |
| Cy3 AffiniPure Donkey Anti-Rabbit IgG | Jackson ImmunoResearch Laboratories | 711-165-152 | |
| Dichloromethane | Sigma-Aldrich | 270997-100ML | |
| Dimethyl sulfoxide | Sigma-Aldrich | D8418-500ML | |
| Ethanol, 100% | Decon laboratories | 2701 | |
| Glycine | Sigma-Aldrich | G7126-500G | |
| GNEXT PET/CT | SOFIE Biosciences | ||
| Heparin sodium salt from porcine intestinal mucosa | Sigma-Aldrich | H3149-50KU | |
| Histodenz | Sigma-Aldrich | D2158-100G | |
| Hydrogen peroxide solution | Sigma-Aldrich | H1009-500ML | |
| Imaging software | Zeiss | ZEN (black edition) | |
| Imaging software | Oxford Instruments | Imaris 10 | |
| iSpacer | Sunjin Labs | iSpacer 3mm | |
| KIRI Engine | KIRI Innovation | ||
| Laser scanning confocal microscope | Zeiss | LSM 880 | |
| LEAD-2 - Vertical & Multi-channels Peristaltic Pump | LONGER | ||
| Lightview XL | Brightech | ||
| Methanol (Certified ACS) | Fischer Scientific | A412-4 | |
| Nikon D850 | Nikon | ||
| NinjaTek NinjaFlex TPU @MK4 | NinjaTek | ||
| Normal donkey serum | Jackson ImmunoResearch Laboratories | 017-000-121 | |
| Original Prusa MK4 3D printer | Prusa Research | ||
| PAP pen | Abcam | ab2601 | |
| Paraformaldehyde, 32% | Electron Microscopy Sciences | 15714-S | |
| Polycam | Polycam | ||
| Primary antibody | |||
| PrusaSlicer 2.7.1 | Prusa Research | ||
| SARA-Engine | pita4 mobile LLC | ||
| Scaniverse | Niantic | ||
| Secondary antibody | |||
| SlowFade Gold Antiface Mountant | Invitrogen | S36936 | |
| Sodium azide, 5% (w/v) | Ricca Chemical Company | 7144.8-32 | |
| SOMATOM Definition AS | Siemens Healthcare | ||
| Standard Field Surgi-Spec Telescopes, | Designs for Vision | ||
| Stereomicroscope System SZ61 | OLYMPUS | ||
| StereoPhoto Maker | Free ware developed by Masuji Suto | ||
| Superfrost Plus Microscope Slides, Precleaned | Fisher Scientific | 12-550-15 | |
| Triton X-100 | Sigma-Aldrich | T8787-50ML | |
| Tween-20 | Sigma-Aldrich | P9416-100ML | |
| Xylene | Sigma-Aldrich | 534056-4L | |
| Ziostation2 | Ziosoft, AMIN |
References
- Tawara, S. Das reizleitungssystem des säugetierherzens. Eine anatomisch-histologische studie über das atrioventrikularbündel und die purkinjeschen fäden. Jena: Gustav fischer. , (1906).
- Stephenson, R. S., et al.
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