Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart
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
Representative Results
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…
Divulgaciones
The authors have nothing to disclose.
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
| 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 |
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