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Induction of murine experimental autoimmune myocarditis – technical details, implementation and evaluation Cover

Induction of murine experimental autoimmune myocarditis – technical details, implementation and evaluation

Romanian Journal of Cardiology
Volume 35 (2025): Issue 3 (September 2025)
By: Felicia-Luminita Dudu,  Razvan-Mihai Fueriu,  Gabriel Jakobsson,  Mihaela-Loredana Vlad,  Alexandra-Gela Lazar,  Simona-Adriana Manea,  Adrian Manea and  Alexandru Schiopu  
Open Access
|Aug 2025

Figures & Tables

Figure 1

Attaching the 2.5 mL Hamilton® syringes to the two-way stopcock with Luer-Lock to prepare the emulsion.
Attaching the 2.5 mL Hamilton® syringes to the two-way stopcock with Luer-Lock to prepare the emulsion.

Figure 2

Testing the consistency of the emulsion on the water surface: A) Representative image showing the emulsion after 1 hour of continuous mixing. Note the droplet dispersion, the resulting emulsion is not suitable for injection. B) Achieving stable emulsion consistency after 2.5 hours of continuous mixing. The emulsion remains compact and is ready to use.
Testing the consistency of the emulsion on the water surface: A) Representative image showing the emulsion after 1 hour of continuous mixing. Note the droplet dispersion, the resulting emulsion is not suitable for injection. B) Achieving stable emulsion consistency after 2.5 hours of continuous mixing. The emulsion remains compact and is ready to use.

Figure 3

Day 0 - Subcutaneous injection of 100 μL of CFA:α-MHC or CFA:PBS emulsion. A) Cranial, over right scapula. B) Caudal, over left lower flank. Day 7 - Subcutaneous injection of 100 μL of CFA:α-MHC or CFA:PBS emulsion. C) Cranial, over left scapula. D) Caudal, over right flank.
Day 0 - Subcutaneous injection of 100 μL of CFA:α-MHC or CFA:PBS emulsion. A) Cranial, over right scapula. B) Caudal, over left lower flank. Day 7 - Subcutaneous injection of 100 μL of CFA:α-MHC or CFA:PBS emulsion. C) Cranial, over left scapula. D) Caudal, over right flank.

Figure 4

Representative hematoxylin-eosin images taken at 5x, 10x, 20x and 40x magnification, depicting the immune cells infiltration in the myocardium of EAM mice. The area of immune cell infiltrate is marked with arrows.
Representative hematoxylin-eosin images taken at 5x, 10x, 20x and 40x magnification, depicting the immune cells infiltration in the myocardium of EAM mice. The area of immune cell infiltrate is marked with arrows.

Figure 5

Analysis of fibrosis within myocardium of EAM mice by Picrosirius Red staining. The images were taken at 5x, 10x, 20x and 40x magnification. The cardiomyocytes are stained in light orange and the collagen fibers in red. Arrows indicate the accumulation of collagen fibers.
Analysis of fibrosis within myocardium of EAM mice by Picrosirius Red staining. The images were taken at 5x, 10x, 20x and 40x magnification. The cardiomyocytes are stained in light orange and the collagen fibers in red. Arrows indicate the accumulation of collagen fibers.

Figure 6

Analysis of cardiac function by echocardiography on Day 21 after disease induction. The mice with induced EAM (N=15) have significantly reduced left ventricular ejection fraction compared to the non-immunized control group (N=9) and to the mice receiving CFA - α MHC (N=10). Cardiac output was also reduced in EAM mice compared to CFA controls. The mice did not suffer left ventricular dilation at this early stage of the disease compared to controls.
Analysis of cardiac function by echocardiography on Day 21 after disease induction. The mice with induced EAM (N=15) have significantly reduced left ventricular ejection fraction compared to the non-immunized control group (N=9) and to the mice receiving CFA - α MHC (N=10). Cardiac output was also reduced in EAM mice compared to CFA controls. The mice did not suffer left ventricular dilation at this early stage of the disease compared to controls.

Figure 7

Flow cytometry assessment of immune cell populations in mice with autoimmune myocarditis. (A-B) Gating strategy for myeloid cells and T cells in the heart. (C – D) Quantification of immune cell populations in the heart. (E) Gating strategy for myeloid cells in blood and spleen. (F-G) Quantification of myeloid immune cell populations in heart and spleen. CFA, Complete Freund’s Adjuvant (Control); CFA+MHC, Complete Freund’s Adjuvant + Myosin heavy chain peptide (EAM group), CM; central memory, EM; effector memory, moDCs; monocyte-derived dendritic cells. Statistical testing was performed with Student’s t-test. Data is represented as mean ± S.D. N=3-4 per group.
Flow cytometry assessment of immune cell populations in mice with autoimmune myocarditis. (A-B) Gating strategy for myeloid cells and T cells in the heart. (C – D) Quantification of immune cell populations in the heart. (E) Gating strategy for myeloid cells in blood and spleen. (F-G) Quantification of myeloid immune cell populations in heart and spleen. CFA, Complete Freund’s Adjuvant (Control); CFA+MHC, Complete Freund’s Adjuvant + Myosin heavy chain peptide (EAM group), CM; central memory, EM; effector memory, moDCs; monocyte-derived dendritic cells. Statistical testing was performed with Student’s t-test. Data is represented as mean ± S.D. N=3-4 per group.
DOI: https://doi.org/10.2478/rjc-2025-0026 | Journal eISSN: 2734-6382 | Journal ISSN: 1220-658X
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Language: English
Page range: 194 - 204
Published on: Aug 22, 2025
Published by: Romanian Society of Cardiology
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
experimental autoimmune myocarditis,
cardiac α-myosin heavy chain peptide,
inflammation,
immune response
Related subjects:
Medicine,
Clinical medicine,
Internal medicine,
Cardiology,
Surgery,
Cardiac surgery,
Pediatrics and juvenile medicine,
Paediatric cardiology

© 2025 Felicia-Luminita Dudu, Razvan-Mihai Fueriu, Gabriel Jakobsson, Mihaela-Loredana Vlad, Alexandra-Gela Lazar, Simona-Adriana Manea, Adrian Manea, Alexandru Schiopu, published by Romanian Society of Cardiology
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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