Three-dimensional (3D) reconstruction of cardiac damage in reperfused acute myocardial infarction (repAMI) enables faithful quantification and co-localization of associated disease-affecting patterns. Here, an automatable light sheet-guided imaging approach is provided for contemporaneous measurement of cardiac damage, area at risk, and immune cell response.
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Method Article
Simultaneous 3D Analysis of Cardiac Damage and Immune Response in Reperfused Acute Myocardial Infarction Using Light Sheet Fluorescence Microscopy
* These authors contributed equally
In This Article
Summary
Abstract
Complex cellular interactions determine functional and structural tissue remodeling during reperfused acute myocardial infarction (repAMI). These processes show distinct spatial distribution as the injured heart muscle is segmented into different areas (damage area, area at risk (AAR), and remote area). Three-dimensional (3D) visualization of these areas is essential for the analysis of various interactions between resident cardiac cells and infiltrating immune cells, enabling the identification of possible treatment targets. Here, a protocol is described for simultaneous and automatable 3D visualization and quantification of the cardiac damage area, AAR, and infiltrating immune cells (e.g., neutrophils) after repAMI. This includes intravital antibody-mediated staining of cardiac damage area (CD31neg) and neutrophil infiltration (Ly6G+) following ex vivo visualization of AAR by retrograde antibody perfusion and further non-toxic tissue clearing for light sheet fluorescence microscopy (LSFM) imaging. This technique allows the spatial analysis of target cells, e.g., infiltrating immune cells and damaged areas in an intact mouse heart after repAMI. Traditional histology and immunohistochemistry can be performed after non-toxic tissue clearing and image acquisition with computer-assisted post-processing. This allows multiplexing information gain within the same mouse heart, strengthening data robustness and being especially significant in a highly complex injury like repAMI.
Introduction
Tissue injury in reperfused acute myocardial infarction (repAMI) is the main driving factor for cardiac remodeling and development of heart failure1. Multiple local mediators and resident cardiac cells but also infiltrating immune cells affect its expansion in a complex interaction2,3. Understanding the spatial organization of these processes is essential to gain profound knowledge for the identification of new therapy targets to limit tissue injury after repAMI and improve cardiac function.
Since the heart muscle itself consists of different cell types, deta....
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Protocol
All experimental animal procedures were approved by the responsible local governmental agency (AZ 81-02.04.2023.A059 - Landesamt für Natur-, Umwelt- und Verbraucherschutz (LANUV)) and complied with all relevant ethical regulations for animal testing and research. Experiments were performed with male C57BL/6J mice (9-30 weeks of age). The animals were housed and used under specific pathogen-free (SPF) conditions at the local animal house on 12 h/12 h day and night cycle.
NOTE: All surgical interventions were performed under intraperitoneal induction of anesthesia using ketamine (100 mg/kg) and xylazine (10 mg/kg) with upkeep using isofl....
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Results
The presented approach using light sheet fluorescence microscopy (LSFM) allows simultaneous analysis of AAR, endothelial injury, and neutrophil infiltration after repAMI. Figure 1A illustrates the intravital and ex vivo antibody staining of C57BL/6J mice 24 h after induction of repAMI. Antibodies for staining of cardiac endothelial injury (anti-CD31 labeled with AlexaFluor 790) and neutrophil infiltration (anti-Ly6G labeled with AlexaFluor 647) were injected intravenously 10 min bef.......
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Discussion
There are several imaging entities to detect cardiac tissue injury during repAMI in mice. These techniques provide different advantages but also hold limitations. Staining decreased metabolic activity of cardiac cells using TTC is the most commonly used method for quantification of cardiac I/R injury and can be combined with Evan's blue staining for the distinction of AAR13,14,15. This is a fast and cost-effective method, bu.......
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Disclosures
Elias Haj-Yehia reports personal fees and others from AstraZeneca, which are outside the submitted work. Simon F. Merz and Lea Bornemann are currently employed by Miltenyi Biotec B.V. & Co. KG. Matthias Totzeck and Tienush Rassaf report personal fees, and others from Edwards and Novartis, Bristol Myers Squibb, Bayer, Daiichi Sankyo and Astra Zeneca, which are outside the submitted work. Tienush Rassaf and Ulrike B. Hendgen-Cotta cofounded Bimyo, a company focusing on the development of cardioprotective peptides. Matthias Gunzer received general research funding from Miltenyi BioTec B.V. & Co. KG. All other authors declare no conflict of interest.
Acknowledgements
The authors acknowledge the following funding sources: German Research Foundation (UMEA Clinician Scientist, FU 356/12-2, Elias Haj-Yehia; HE6317/2-1, Ulrike Hendgen-Cotta, RA969/12-1, Tienush Rassaf) and German Cardiac Society (DGK, Deutsche Gesellschaft für Kardiologie - Herz- und Kreislaufforschung e.V., DGK02/2022, Elias Haj-Yehia). Some of this work was performed at the Imaging Center Essen (IMCES), a Service Core Facility of the Faculty of Medicine of the University Duisburg-Essen, Germany. We thank the IMCES staff for their continuous technical support and the use of their instruments.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 27 G needle | Becton Dickinson | 305889 | BD Eclipse |
| 3D image processing & analysis software | Bitplane | Vers. 10.1.0 | Imaris |
| 5-0 suture | Serag-Wiessner | IC108000 | Silk |
| 6-0 suture | Ethicon | EH7814 | Prolene |
| AlexaFluor 546 antibody labeling kit | Invitrogen | A20183 | |
| AlexaFluor 790 antibody labeling kit | Invitrogen | A20189 | |
| Anti-mouse Ly6G antibody (AlexaFluor 647) | Biolegend | 127610 | Clone: 1A8 |
| Cell strainer (100 µm) | Corning | 431752 | |
| Dimethyl sulfoxide (DMSO) | Carl Roth | 7029.1 | |
| Ethanol | Carl Roth | 0911.4 | |
| Ethyl cinnemate (ECi) | Sigma-Aldrich | 112372 | |
| File converter software | Bitplane | Vers. 10.1.0 | Imaris |
| Gauze | Fuhrmann | 10021 | |
| Gellan gum powder | Sigma-Aldrich | P8169 | Phytagel |
| Heparin (25.000 IE/5 mL) | LEO Pharma | 15261203 | |
| Hydrogen peroxide (H2O2, 30%) | Carl Roth | 8070.2 | |
| Injection syringe | B Braun | 9161502S | Omnican F |
| Ketamin (100 mg/mL) | bela-pharma | 402581.00.00 | |
| Light sheet microscope system | Miltenyi Biotec | - | UltraMicroscope Blaze |
| Luer lock syringe | Becton Dickinson | 303172 | BD Plastipak |
| Magnetic stirrer | VWR | 444-0572 | |
| Neo sCMOS camera (4.2 MP) | Andor Technology | ZL41 Cell 4.2 | |
| Objective | Miltenyi Biotec | 130-133-625 | MI Plan 1.1x NA 0.1, WD 17.6 mm |
| Paraformaldehyde (PFA, 4%) | Sigma-Aldrich | 1.00496 | 10% formalin, neutral buffered |
| Petri dish | Greiner Bio-One | 632181 | |
| Phosphate buffered saline (PBS) | Gibco | 14190-094 | Without MgCl2/CaCl2 |
| Polypropylene tube (15 mL) | Greiner Bio-One | 188261 | |
| Polypropylene tube (5 mL) | Carl Roth | EKY9.1 | Black |
| Polypropylene tube (50 mL) | Greiner Bio-One | 227261 | |
| Preparation dish for mouse heart cannulation | Hugo Sachs Elektronik | 73-4327 | Cannula included |
| Purified anti-mouse CD31 antibody | Becton Dickinson | 53369 | Clone: MEC13.3 |
| Roller pump | Ismatec | ISM597-230 | |
| Sealing film | Bemis | PM-996 | Parafilm |
| Stereo microscope | Leica | S6D | |
| Tube rotator | Heidolph | Polymax 1040 | |
| White light laser | NKT photonics | SuperK extreme | 200 mW, 480–2400 nm |
| WT mouse strain | Janvier Labs | - | C57BL/6JRj |
| Xylazin (2%) | Ceva Tiergesundheit | 6324464.00.00 |
References
- Whelan, R. S., Kaplinskiy, V., Kitsis, R. N. Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu Rev Physiol. 72, 19-44 (2010).
- Frangogiannis, N. G. Inflammation in cardiac injury, repair and regeneration. Curr Opin Cardiol.
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