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Studying the Innate Immune Response to Myocardial Infarction in a Highly Efficient Experimental Animal Model

Razvan Gheorghita Mares
Razvan Gheorghita Mares
, 
Doina Manu
Doina Manu
, 
Istvan Adorjan Szabo
Istvan Adorjan Szabo
, 
Mihaela Elena Tomut
Mihaela Elena Tomut
, 
Gabriela Pintican
Gabriela Pintican
, 
Bogdan Cordos
Bogdan Cordos
, 
Gabriel Jakobsson
Gabriel Jakobsson
, 
Minodora Dobreanu
Minodora Dobreanu
, 
Ovidiu Simion Cotoi
Ovidiu Simion Cotoi
 y 
Alexandru Schiopu
Alexandru Schiopu
   | 05 may 2022
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Article Category: Original Article
Publicado en línea: 05 may 2022
Páginas: 573 - 585
DOI: https://doi.org/10.47803/rjc.2021.31.3.573
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© 2021 Razvan Gheorghita Mares et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Permanent coronary artery occlusion without mechanical ventilation in mice.Images show the schematic presentation of the surgical technique. A, Preparing the operating table. B, Anesthetizing the mouse in the induction chamber. C, Placing the unconscious mouse on a warm pad and removing the fur from the chest. D, Making a small skin cut in the middle position of precordial chest. E, Dissecting and retracting the pectoral major and minor muscles. F, Exposing the 4th intercostal space. G, Placing the curved mosquito clamp at the 4th intercostal space level with the help of a forceps and forcing the mosquito to open the pleural membrane and pericardium. H–J, Smoothly and gently lifting out the heart through the window, locating and ligating the LAD with silk suture. K, Placing the heart back into the thorax promptly after ligation, with manual evacuation of air by gentle but efficient side chest pressure. L, closure of the skin with Prolene suture.
Permanent coronary artery occlusion without mechanical ventilation in mice.Images show the schematic presentation of the surgical technique. A, Preparing the operating table. B, Anesthetizing the mouse in the induction chamber. C, Placing the unconscious mouse on a warm pad and removing the fur from the chest. D, Making a small skin cut in the middle position of precordial chest. E, Dissecting and retracting the pectoral major and minor muscles. F, Exposing the 4th intercostal space. G, Placing the curved mosquito clamp at the 4th intercostal space level with the help of a forceps and forcing the mosquito to open the pleural membrane and pericardium. H–J, Smoothly and gently lifting out the heart through the window, locating and ligating the LAD with silk suture. K, Placing the heart back into the thorax promptly after ligation, with manual evacuation of air by gentle but efficient side chest pressure. L, closure of the skin with Prolene suture.

Figure 2

Validation of LAD ligation by electrocardiographyElevations of the S-T segment appear a few minutes after LAD ligation, confirming the successful induction of ischemia.
Validation of LAD ligation by electrocardiographyElevations of the S-T segment appear a few minutes after LAD ligation, confirming the successful induction of ischemia.

Figure 3

Inflammatory infiltration post-AMI determined by histology and immunohistochemistryA, H&E at day 1 after AMI. B, H&E at day 3 after AMI. C, S100A8/A9 positive cells at day 1 after AMI. D, S100A8/A9 positive cells at day 3 after AMI. E, S100A8/A9 positive cells at day 7 after AMI. F, S100A8/A9 positive cells at day 14 after AMI. All 10X magnification.
Inflammatory infiltration post-AMI determined by histology and immunohistochemistryA, H&E at day 1 after AMI. B, H&E at day 3 after AMI. C, S100A8/A9 positive cells at day 1 after AMI. D, S100A8/A9 positive cells at day 3 after AMI. E, S100A8/A9 positive cells at day 7 after AMI. F, S100A8/A9 positive cells at day 14 after AMI. All 10X magnification.

Figure 4

Fibrous scar formation, neovascularization and myofibroblast presence at 7 and 14 days after AMIA, Masson's trichrome staining. B, CD31 positive cells. C, α-SMA positive cells. All 10X magnification.
Fibrous scar formation, neovascularization and myofibroblast presence at 7 and 14 days after AMIA, Masson's trichrome staining. B, CD31 positive cells. C, α-SMA positive cells. All 10X magnification.

Figure 5

Inflammatory cells dynamics after AMI and the flow cytometry gating strategyA, neutrophils in the infarcted myocardium at 1, 3 and 7 days after AMI. (a), (b) single cells selection using the forward scatter/side scatter plots. (c) myeloid cells (CD45+CD11b+) derived from the single cells gate. (d) Ly6G/CD11b plot to identify neutrophils (CD45+CD11b+Ly6G+). B, monocytes in blood at 1, 3 and 7 days after AMI. (a) cells selection using forward/side scatter plots, to eliminate debris. (b) myeloid cells. (c) CD115/CD11b plot identifying total monocytes (CD45+CD11b+CD115+ cells). (d) CD115/Ly6C plot to divide monocytes into inflammatory Ly6Chi monocytes (upper gate) and patrolling Ly6Clow monocytes (lower gate). C, macrophages in the infarcted myocardium at 1, 3 and 7 days after AMI. (a) cells selection using forward/side scatter plots. (b) myeloid cells. (c) F4/80/CD11b plot identifying total macrophages (CD45+CD11b+F4/80+ cells). (d) MerTK/Ly6C gate to identify the reparatory Ly6ClowMerTKhi macrophages (right lower quadrant) and inflammatory Ly6ChiMerTKlow macrophages (left upper quadrant).
Inflammatory cells dynamics after AMI and the flow cytometry gating strategyA, neutrophils in the infarcted myocardium at 1, 3 and 7 days after AMI. (a), (b) single cells selection using the forward scatter/side scatter plots. (c) myeloid cells (CD45+CD11b+) derived from the single cells gate. (d) Ly6G/CD11b plot to identify neutrophils (CD45+CD11b+Ly6G+). B, monocytes in blood at 1, 3 and 7 days after AMI. (a) cells selection using forward/side scatter plots, to eliminate debris. (b) myeloid cells. (c) CD115/CD11b plot identifying total monocytes (CD45+CD11b+CD115+ cells). (d) CD115/Ly6C plot to divide monocytes into inflammatory Ly6Chi monocytes (upper gate) and patrolling Ly6Clow monocytes (lower gate). C, macrophages in the infarcted myocardium at 1, 3 and 7 days after AMI. (a) cells selection using forward/side scatter plots. (b) myeloid cells. (c) F4/80/CD11b plot identifying total macrophages (CD45+CD11b+F4/80+ cells). (d) MerTK/Ly6C gate to identify the reparatory Ly6ClowMerTKhi macrophages (right lower quadrant) and inflammatory Ly6ChiMerTKlow macrophages (left upper quadrant).

Figure 6

Immune cell dynamics post-AMI in blood and myocardium during the first week post-AMI, determined by flow cytometryPercentages of: A, neutrophils in the infarcted myocardium out of total myeloid cells. B, circulating monocytes out of total myeloid cells in blood. C, inflammatory Ly6Chi monocytes out of total blood monocytes. D, macrophages in the infarcted myocardium out of total myeloid cells. E, reparatory Ly6CloMerTKhi macrophages in the infarcted myocardium out of total macrophages. F, Ly6ChiMerTKlo macrophages in the infarcted myocardium out of total macrophages.
Immune cell dynamics post-AMI in blood and myocardium during the first week post-AMI, determined by flow cytometryPercentages of: A, neutrophils in the infarcted myocardium out of total myeloid cells. B, circulating monocytes out of total myeloid cells in blood. C, inflammatory Ly6Chi monocytes out of total blood monocytes. D, macrophages in the infarcted myocardium out of total myeloid cells. E, reparatory Ly6CloMerTKhi macrophages in the infarcted myocardium out of total macrophages. F, Ly6ChiMerTKlo macrophages in the infarcted myocardium out of total macrophages.
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Temas de la revista:
Medicine, Clinical Medicine, Internal Medicine, Cardiology, Pediatrics and Juvenile Medicine, Paediatric Cardiology, Surgery, Cardiac Surgery
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