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Abstract

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Medicine

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Published: July 21st, 2023

DOI:

10.3791/65426

1Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.

To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.

A neonatal rat heart is transplanted into an adolescent rat's abdomen and connected to the recipient's infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.

This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Endocardial fibroelastosis (EFE), defined by the accumulation of collagen and elastic fibers in the subendocardial tissue, presents as a pearly or opaque thickened endocardium; EFE undergoes most active growth during the fetal period and early infancy1. In an autopsy study, 70% of cases with hypoplastic left heart syndrome (HLHS) were associated with the presence of EFE2.

Cells expressing markers for fibroblasts are the main cell population in EFE, but these cells also concomitantly express endocardial endothelial markers, which is an indication of the origin of these EFE cells. Our group prev....

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All the animal procedures were conducted in accordance with the National Research Council. 2011. Guide for the Care and Use of Laboratory Animals: Eighth Edition. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital.

Prior to surgery, all the surgical instruments are steam-autoclaved, and modified Krebs-Henseleit buffer, with a final concentration of 22 mmol/L KCl, is prepared as a cardioplegic solution (Ta.......

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Graft viability and beating
In this work, the graft viability was visually assessed after all the clamps had been removed, and an approximate reperfusion time of 10-15 min was allowed with an open abdomen for observation of the graft. The same scoring system to objectively verify graft viability was used for visual assessment at the end of surgery and for the echocardiography on POD 1, POD 7, and POD 14.

0 = no organ function; 1 = (rest) organ function, only minimal cont.......

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This animal model of heterotopic transplantation of a neonatal donor rat heart into the recipient's abdomen creates the possibility to study EndMT-derived fibrosis through detailed histological tissue evaluation, identify regulatory signaling pathways, and test treatment options. Since EndMT is the underlying mechanism for fibrotic diseases of the heart, this model has great value in the field of pediatric cardiac surgery and beyond. In this model, many factors can negatively influence the outcome of the procedure. T.......

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This research was funded by Additional Ventures - Single Ventricle Research Fund (SVRF) and Single Ventricle Expansion Fund (to I.F.) and a Marietta Blau scholarship of the OeAD-GmbH from funds provided by the Austrian Federal Ministry of Education, Science and Research BMBWFC (to G.G.).

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NameCompanyCatalog NumberComments
Advanced Ventilator System For Rodents, SAR-1000CWE, Inc.12-03100small animal ventilator
aSMASigmaA2547Antibody for Immunohistochemistry
Axio observer Z1 Carl Zeissinverted microscope
Betadine SolutionAvrio Health L.P.367618150092
CD31InvitrogenMA1-80069Antibody for Immunohistochemistry
DAPIInvitrogenD1306Antibody for Immunohistochemistry
DemeLON Nylon black 10-0DemeTECHNL76100065F0P10-0 Nylon suture
ETFE IV Catheter, 18G x 2TERUMO SURFLOSR-OX1851CAintubation cannula
Micro Clip 8mmRoboz Surgical Instrument Co.RS-6471microvascular clamps
Nylon black monofilament 11-0SURGICAL SPECIALTIES CORPAA013011-0 Nylon
O.C.T. CompoundTissue-Tek4583Embedding medium for frozen tissue specimen
p-SMAD2/3InvitrogenPA5-110155Antibody for Immunohistochemistry
Rodent, Tilting WorkStandHallowell EMC.000A3467oblique shelf for intubation
Silk Sutures, Non-absorbable, 7-0Braintree ScientificNC9201231Silk suture
Slug/SnailAbcamab180714Antibody for Immunohistochemistry
Undyed Coated Vicryl 5-0 P-3 18"EthiconJ493G5-0 Vicryl
Undyed Coated Vicryl 6-0 P-3 18"EthiconJ492G6-0 Vicryl
VE-CadherinAbcamab231227Antibody for Immunohistochemistry
Zeiss OPMI 6-SFRZeissSurgical microscope
Zen, Blue Edition, 3.6Zen inverted microscope software

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