Method Article

Simultaneous 3D Analysis of Cardiac Damage and Immune Response in Reperfused Acute Myocardial Infarction Using Light Sheet Fluorescence Microscopy

DOI:

10.3791/68347

⸱

September 26th, 2025

 ,  ,  ,  ,  ,  ,  ,  , 
1Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, University Hospital Essen, 2Miltenyi Biotec B.V. & Co. KG, 3Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg-Essen

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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

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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, detailed analysis of cardiac damage is challenging as every cell type shows its own specific resistance capacity during ischemia and reperfusion injury4,5. One important hallmark in the context of repAMI is a major loss of vasculature accompanied by deterioration of endothelial cell function6. The most established marker used for the depiction of vascular structure and analysis of endothelial dysfunction in mice and humans is CD31 (also known as PECAM-1), an adhesion molecule expressed on the endothelial cell surface7. In different ischemia/reperfusion models, loss of CD31 showed to be a good surrogate marker for the distinction of endothelial tissue damage8,9,10. Furthermore, three major injury compartments are distinguished throughout the whole heart during repAMI. Cardiac tissue not affected by ischemia/reperfusion (I/R) injury represents the remote area. Myocardium downstream of a vessel occlusion is defined as an area at risk (AAR)11,12. After the onset of reperfusion, a distinct area of cardiac tissue can be differentiated inside the AAR, which was damaged by preceding ischemia (damage area)13. Conventional assessment of cardiac I/R injury in mice uses triphenyl tetrazolium chloride (TTC) in serial thick sections displaying decreased metabolic activity of damaged cardiac cells13,14. Simultaneously, ex vivo Evan's blue staining can be added for the determination of AAR15. However, these established methods hold several limitations, including the reduced possibility of co-assessment of immune cell infiltration, distinction of cell-type specific injury, and accurate 3D tissue reconstruction.

Light sheet fluorescence microscopy (LSFM) enables the possibility to visualize intact cleared whole mouse organs with fluorescence signal resolution down to the cellular level. Recently, an improved non-toxic tissue-clearing method was introduced using ethyl cinnamate (ECi) for 3D visualization of intact mouse hearts8. To address the limitations of TTC/Evan's blue staining, intravital antibody-based CD31 fluorescence staining was used, revealing cardiac endothelial injury as areas without CD31 staining (CD31neg) during repAMI8. Additional administration of ex vivo retrograde aortic injection with a fluorophore-conjugated anti-CD31 antibody with another fluorescence spectrum as for intravital staining accounts for contemporaneous 3D depiction of AAR. Furthermore, this method can be extended by adding neutrophil staining, allowing cell distribution analysis separated by remote area, AAR, and cardiac endothelial injury volume to improve understanding of neutrophil function during repAMI.

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Protocol

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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 isoflurane (1.5 Vol%). To diminish animal suffering, all animals received subcutaneous injections of 0.1 mg/kg buprenorphine pre- and post-anesthesia during the day in combination with buprenorphine containing drinking water overnight. The surgical protocol for inducting repAMI has been previously described elsewhere16,17. Before starting with intravital antibody injection, prepare two 6 cm Petri dishes that are used for ex vivo retrograde antibody injection in the later steps described below. One shallow petri dish with PBS on ice is needed for the crude removal of excessive surrounding tissue from the heart (it is recommended to use cut-to-size pieces of gauze inside the dish to ease the later handling process). Another dish is needed for aortic cannulation with a 20 G blunt steel cannula needle submersed in PBS on ice with a loose knot of 5-0 suture over the cannula notch. Avoid or remove air bubbles inside the cannula, as they can block coronary vessels and prevent total perfusion.

1. Intravital antibody injection

  1. Anesthetize the mouse using ketamine (100 mg/kg) and xylazine (10 mg/kg).
  2. Inject 10 µg each of directly labeled mouse anti-CD31 (clone: MEC13.3; fluorophore: AlexaFluor 790) and mouse anti-Ly6G (clone: 1A8; fluorophore: AlexaFluor 647) antibodies in PBS with a total volume of 5 µL per g bodyweight (100 µL for a 20 g mouse) via intravenous tail vein injection.
    NOTE: This antibody injection will label all the endothelium inside the heart and is meant to quantify repAMI injury size.
  3. Let the injected antibodies circulate for 10 min before continuing the procedure.

2. Heart harvesting

NOTE: Sections 2 and 3 are time-critical and need to be completed in less than 6 min.

  1. Sacrifice the animal by cervical dislocation 10 min after antibody injection. Check death by assessing reflexes.
    NOTE: If blood samples are needed, take them before proceeding to the next step.
  2. Place the mouse under a stereo microscope.
  3. Open the chest cavity and incise the exposed superior vena cava close to the exit of the vessel from the rib cage to the neck.
    NOTE: Keep the aorta intact as it is needed for ex vivo retrograde antibody injection.
  4. Perfuse hearts in situ via right and left atrial auricle with PBS containing heparin (20 IE/mL) using a 27 G needle with a roller pump (70 µL/s) for a total of 3 min. Sufficient cardiac blood removal becomes noticeable by the fading of superficial vascular drawing of the heart and whitening of the lung and liver.
    NOTE: Avoid heart perfusion via the right or left ventricle as it may cause imaging artifacts during light sheet fluorescence microscopy.
  5. Excise the heart and at least 5 mm of remaining aorta together with surrounding lung tissue.
    NOTE: Do not hold the heart muscle directly during excision to avoid tissue damage and imaging artifacts during light sheet fluorescence microscopy.

3. Ex vivo retrograde antibody injection

  1. Transfer the heart to a 6 cm Petri dish with ice-cold PBS and gauze prepared earlier.
  2. Cut off the remaining surrounding lung, fat, and vessel tissue until only the heart with approximately 5 mm aorta remains.
  3. Transfer heart to prepared dish for aortic cannulation.
  4. Pull the remaining aorta over the 20 G blunt steel cannula and ligate it with the pre-knot 50 suture to the cannula notch.
    NOTE: The cannula tip should be pushed close to the aortic valve to ensure no leakage of perfusion fluid.
  5. Re-ligate the left anterior descending artery (LAD) permanently with a 6-0 suture at the exact location used for induction of repAMI previously.
  6. Inject 400 µL of PBS containing 20 µg mouse anti-CD31 (clone: MEC13.3) antibody, labeled with AlexaFluor 546, retrogradely via inserted cannula.
    NOTE: Close the junction between the injection syringe and the inserted cannula with sealing film to ensure complete antibody perfusion via coronary arteries. This antibody injection will only stain endothelium outside the AAR and can thus be used for AAR quantification.
  7. Pull out the inserted cannula from the aorta.
  8. Transfer the heart to a 15 mL polypropylene reaction tube containing 10 mL of 4% paraformaldehyde (PFA) and incubate without agitation overnight at 4 °C in the dark.

4. Sample processing and clearing

  1. Wash fixated heart in three changes of 5 mL PBS for 10 min each at room temperature (RT) in the dark always agitating with near overhead rotation in 5 mL polypropylene reaction tubes.
  2. Dehydrate fixated heart in 5 mL ascending ethanol series at 50%, 70%, and 100% in deionized water, each for at least 2 h at RT in the dark always agitating with near overhead rotation in 5 mL polypropylene reaction tubes.
    NOTE: Incubation in 50% and 70% ethanol can be prolonged up to the next day, while incubation in 100% ethanol can be prolonged up to 3 days.
  3. Bleach dehydrated heart in 15 mL polypropylene reaction tubes containing 10 mL of 100% ethanol with 5% DMSO and 5% H2O2 without agitation for 4 h at 4 °C in the dark.
    NOTE: Pressure might be building inside the tube during the bleaching process; take care when opening the tube afterward.
  4. Wash bleached heart in 5 mL of 100% ethanol three times for 12 h each at RT in the dark always agitating with near overhead rotation in 5 mL polypropylene reaction tubes.
    NOTE: Each incubation step can be prolonged for up to 3 days.
  5. Transfer the heart to a 5 mL polypropylene reaction tube containing 4 mL of Ethyl cinnemate (ECi) for tissue clearing and incubate without agitation for 3 days at RT in the dark.
    NOTE: Cleared heart can remain in ECi for at least 3 months without a decrease in fluorescence intensity under correct storage in the dark at RT. Do not store ECi or samples stored in ECi below 6 °C, as ECi freezes at this temperature, harming the sample in the process.

5. Preparation of gellan gum blocks

  1. Mix 1 g of gellan gum powder in 100 mL of heated tap water (50-60 °C).
  2. Let mix until resolved completely using a magnetic stirrer.
    NOTE: If the powder is not resolving properly, heat the solution until boiling in a microwave.
  3. Filter the gellan gum solution through a 100 µm cell strainer.
  4. Plate filtered solution into a shallow 10 cm Petri dish.
  5. Let cool until the gel is hardened.
  6. Cut gel into blocks of 15 mm 15 mm 5 mm.
  7. Dehydrate blocks in 50 mL of ascending ethanol series at 50% and 70% in deionized water, each for at least 2 h at RT always agitating with near overhead rotation in one 50 mL polypropylene reaction tube.
    NOTE: Fill the reaction tube with ethanol after adding gel blocks. Only fill the reaction tube up to half with gel blocks.
  8. Incubate dehydrated blocks in 50 mL of 100% ethanol three times for 12 h each at RT always agitating with near overhead rotation in one 50 mL polypropylene reaction tube.
    NOTE: Fill the reaction tube with ethanol after adding gel blocks. Only fill the reaction tube up to half with gel blocks. Gel blocks can remain in the last reaction tube with 100% ethanol for extended periods until cleared in ECi.
  9. Transfer blocks to a 50 mL polypropylene reaction tube containing 25 mL of ECi and incubate without agitation for 3 days at RT.
    NOTE: Put only a few blocks in one reaction tube with ECi to avoid clearing artifacts. Cleared blocks can remain in ECi for at least 6 months and stored at RT.

6. Imaging

  1. Cut cleared gellan gum blocks to the size needed to stabilize the cleared heart in the microscope sample holder.
    NOTE: Gellan gum blocks can be re-used multiple times.
  2. Detect fluorescence signals of interest (Autofluorescence, AAR staining [anti-CD31 antibody labeled with AlexaFluor 546], AlexaFluor647-coupled anti-Ly6G antibody and CD31neg staining [anti-CD31 antibody labeled with AlexaFluor 790]) with appropriate excitation and emission filter settings. For acquisition, use a 0.1 NA objective lens with a working distance of 17.6 mm.
    NOTE: Detect signals with a white light laser (200 mW; 480-2400 nm). For detection of connective-tissue derived autofluorescence, use a 470/30 nm bandpass excitation filter and a 525/50 nm bandpass emission filter; CD31 staining with an AlexaFluor 546-coupled antibody is detected with a 520/40 nm bandpass excitation filter and a 585/40 nm bandpass emission filter; Ly6G staining with an AlexaFluor 647-coupled antibody is detected with a 630/30 nm bandpass excitation filter and a 680/30 nm bandpass emission filter; CD31 staining with an AlexaFluor 790-coupled antibody is detected with a 740/40 nm bandpass excitation filter and a 824/55 nm bandpass emission filter. Illumination times and laser attenuation percentage have to be adjusted according to signal quality.
  3. Choose the following settings for light sheet microscopy imaging:
    1. Choose sheet NA of 0.079, resulting in a light sheet thickness of 5 µm.
    2. Choose 10 µm as the distance between two individual z-planes.
      NOTE: This results in undersampling and subsequent loss of precision for tracing and volume measurements. To avoid this, a distance between two individual z-planes should equal half the light sheet thickness, so in this case, 2.5 µm. This study chose to undersample the data, as the benefits gained by reduced imaging time and data stack size outweigh the minimal loss of analysis precision.
    3. Use a sheet width of 100% to get a homogenous illumination of the sample.

7. Image post-processing

NOTE: The acquired digital image data were further processed using a scientific 3D image processing and analysis software listed in the Table of Materials.

  1. Start the Imaris file converter 10.1 software and load the raw dataset into the software by either drag and drop the folder with the image files onto the Imaris file converter window or clicking Browse and navigating to the folder with the image files.
  2. Select the output folder via Select Folder.
  3. Click Start All and wait for the files to be converted into .ims Imaris files.
  4. Start the software and select Surpass in the upper-left corner.
  5. Open image stacks by choosing Open and select the according .ims data file.
  6. To create an automatically generated surface of the whole heart, select 3D View and Surfaces.
  7. Choose Segment only a Region of Interest, Process entire Image finally, and Object-Object Statistics while disabling Classify Surfaces in the Algorithm Settings section.
    NOTE: Enabling Object-Object Statistics allows for the overlay calculation of different surfaces and spot functions.
  8. Click the blue arrow button in the bottom-right corner of the Create sheet to continue with the next steps.
  9. Determine the three-dimensional region of interest using the manipulators appearing inside the image view.
  10. Proceed with the next step by choosing the source channel (Autofluorescence channel).
  11. Enable Smooth and choose the default setting of 11.8 µm.
  12. Continue with the next step and determine the threshold needed to create an optically satisfying three-dimensional surface appearing inside the image view.
  13. In the last step, adjust the surface filter, choosing Number of Voxels as the filter type and determining 10 as the threshold.
  14. Create a surface by clicking the green arrow button in the bottom-right corner of the Create section. By using the Color sheet, change the material and color of the created surface as required.
  15. Generate two additional manually created surfaces for AAR (CD31neg volume in the AlexaFluor 546 channel) and cardiac endothelial injury volume (CD31neg volume in the AlexaFluor 790 channel) by choosing 3D View and Surfaces once more.
  16. Choose Segment only a Region of Interest, Process entire Image finally, and Object-Object Statistics while disabling Classify Surfaces in the Algorithm Settings section and click Skip automatic creation, edit manually.
  17. Inside the Board sheet, select XY for orientation, None for visibility and manually adjust the resolution controller to maximum.
  18. Go to the Model sheet, click the Distance icon, and set vertex spacing to 100 µm.
  19. Enable Draw and encircle CD31neg areas in the AlexaFluor 546 channel or the AlexaFluor 790 channel every 10 image slices.
  20. After finishing encircling all according areas, choose Autofit and Perform Autofit.
  21. Click Create Surface and adjust the material and color of the generated surfaces in the Color sheet.
  22. To display neutrophils as Ly6G+ spots, generate a spot function by choosing 3D View and Spots.
  23. Select Segment only a Region of Interest, Process entire Image finally, and Object-Object Statistics while disabling Classify Spots in the Algorithm Settings section.
  24. Proceed to the next steps using the blue arrow button in the bottom-right corner of the Create sheet.
  25. Determine the three-dimensional region of interest using the manipulators appearing inside the image view as mentioned above.
  26. Continue with the next step by using the source channel (AlexaFluor 647 channel).
  27. Set the estimated XY Diameter to 10 µm, enable Model PSF-elongation along Z-axis, and set the estimated Z Diameter to 20 µm.
  28. Disable Background Subtraction.
  29. Proceed with the next step, select Intensity Min as a filter, and set an appropriate threshold.
    NOTE: For estimation of the threshold level, click Slice in the upper area of the program window and select the AlexaFluor 647 channel. Move the cursor over different spots in the raw image slice representing a positive Ly6G staining and read the intensity value in the bottom-left corner of the program window.
  30. Generate spots by clicking the green arrow button in the bottom-right corner of the Create sheet.
  31. Set the Radius Scale to 3.0 in the Points Style/Quality section of the Create sheet.
  32. Got to the Color sheet for adjustment of spot color as wanted.

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Results

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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

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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

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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

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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

List of materials used in this article
NameCompanyCatalog NumberComments
27 G needleBecton Dickinson305889BD Eclipse
3D image processing & analysis softwareBitplaneVers. 10.1.0Imaris
5-0 sutureSerag-WiessnerIC108000Silk
6-0 sutureEthiconEH7814Prolene
AlexaFluor 546 antibody labeling kitInvitrogenA20183
AlexaFluor 790 antibody labeling kitInvitrogenA20189
Anti-mouse Ly6G antibody (AlexaFluor 647)Biolegend127610Clone: 1A8
Cell strainer (100 µm)Corning431752
Dimethyl sulfoxide (DMSO)Carl Roth7029.1
EthanolCarl Roth0911.4
Ethyl cinnemate (ECi)Sigma-Aldrich112372
File converter softwareBitplaneVers. 10.1.0Imaris
GauzeFuhrmann10021
Gellan gum powderSigma-AldrichP8169Phytagel
Heparin (25.000 IE/5 mL)LEO Pharma15261203
Hydrogen peroxide (H2O2, 30%)Carl Roth8070.2
Injection syringeB Braun9161502SOmnican F
Ketamin (100 mg/mL)bela-pharma402581.00.00
Light sheet microscope systemMiltenyi Biotec-UltraMicroscope Blaze
Luer lock syringeBecton Dickinson303172BD Plastipak
Magnetic stirrerVWR444-0572
Neo sCMOS camera (4.2 MP)Andor TechnologyZL41 Cell 4.2
ObjectiveMiltenyi Biotec130-133-625MI Plan 1.1x NA 0.1, WD 17.6 mm
Paraformaldehyde (PFA, 4%)Sigma-Aldrich1.0049610% formalin, neutral buffered
Petri dishGreiner Bio-One632181
Phosphate buffered saline (PBS)Gibco14190-094Without MgCl2/CaCl2
Polypropylene tube (15 mL)Greiner Bio-One188261
Polypropylene tube (5 mL)Carl RothEKY9.1Black
Polypropylene tube (50 mL)Greiner Bio-One227261
Preparation dish for mouse heart cannulationHugo Sachs Elektronik73-4327Cannula included
Purified anti-mouse CD31 antibodyBecton Dickinson53369Clone: MEC13.3
Roller pumpIsmatecISM597-230
Sealing filmBemisPM-996Parafilm
Stereo microscopeLeicaS6D
Tube rotatorHeidolphPolymax 1040
White light laserNKT photonicsSuperK extreme200 mW, 480–2400 nm
WT mouse strainJanvier Labs-C57BL/6JRj
Xylazin (2%)Ceva Tiergesundheit6324464.00.00

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Light Sheet MicroscopyCardiac Damage AnalysisImmune Response HeartMyocardial Infarction 3DTissue ClearingNeutrophil InfiltrationArea At RiskAntibody StainingEndothelial Injury3D Heart Imaging

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