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.).

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&#39;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 (Table 1). The solution is filter-sterilized and stored at 4 &#176;C overnight. A surgical microscope (12.5x) is required for the heterotopic neonatal rat heart transplantation procedure.
1. Preparation and anesthesia
<ol>
	<li>Use male/female Lewis rats with a weight of around 150 g (5-6 weeks of age) as recipients.</li>
	<li>To start, generously shave the rat&#39;s abdomen with a razor.</li>
	<li>Place the rat into an isoflurane chamber, and turn on the oxygen flow at 2 L/min with 2% isoflurane until the animal is properly sedated but still spontaneously breathing. Inject 45 mg/kg ketamine and 5 mg/kg xylazine intraperitoneally (IP), as well as 300 U/kg heparin. Confirm proper anesthetization with a toe pinch test. 
	NOTE: Carefully monitor the spontaneous breathing and heart rate through palpation of the chest to assure a stable hemodynamic status throughout the entire process.</li>
	<li>For intubation, place the rat on an oblique shelf (Figure 1), secure the front teeth with a string, and place the head facing toward the surgeon.</li>
	<li>Place the light on the outside of the neck onto the area of the vocal cords, grab the tongue with two fingers, and slightly push it upward and to the left to provide optimal vision for intubation. Use an 18 G, 2 in cannula for a 100-150 g rat. Secure the intratracheal tube with tape. 
	NOTE: Surgical loups with 3.5x magnification are recommended for intubation.</li>
	<li>Connect the intubation cannula to the small animal ventilator, and adjust the settings according to the manufacturer&#39;s instructions based on the animal size. 
	NOTE: Use the following settings for a 150 g rat: volume mode; respiratory rate, 55/min; tidal volume, 1.3 mL 50 % I/E ratio, but this can be adjusted appropriately as needed. Assure proper bilateral and equal chest movement, and administer isoflurane continuously at 0.5%&#8211;2% through the ventilator.</li>
	<li>Place the rat on a heating pad (to maintain normal body temperature) in a supine position with the tail facing toward the surgeon. Sterilize the abdomen three times with betadine solution and 70% of ethanol alternatingly. Administer eye lube, and cover the rat with a sterile surgical drape, leaving the abdomen uncovered.</li>
</ol>
2. Surgical preparation and heterotopic transplantation of the neonatal donor heart in the recipient rat
<ol>
	<li>Perform a midline laparotomy using a 15 blade scalpel for the skin incision, and use scissors to open the anterior abdominal wall, followed by blunt exposure of the retroperitoneal abdominal aorta and inferior vena cava (IVC) with cotton tip applicators.</li>
	<li>Mobilize the intestines (including the descending colon), and place them toward the right upper quadrant. Cover the intestines with warm saline-soaked gauze. Use retractors to ensure optimal exposure of the IVC and abdominal aorta.</li>
	<li>Perform blunt dissection of the infrarenal IVC and abdominal aorta up toward the bifurcation. Ligate all the infrarenal branching arteries and veins (e.g., inferior mesenteric artery and lymph node arteries) with a 10-0 nylon suture. 
	NOTE: There is great variability in the anatomy of these side branches. Monitor the aorta&#39;s pulse and heart rate visually when no other hemodynamic monitoring is available. Assess the proper depth of anesthesia every 15 min through a toe pinch test. Adjust the isoflurane concentration accordingly.</li>
	<li>After the donor heart is harvested from a neonatal rat, deliver the excised heart in sterile conditions in a surgical basin containing Krebs-Henseleit buffer to the surgical field. Irrigate the donor heart intermittently with ice-cold cardioplegic solution. 
	NOTE: When a second surgeon is available, the heart should be prepared at the same time, as a second surgeon reduces the total anesthesia time of the recipient animal and the ischemia time of the donor heart. When a second surgeon is not available, cover the recipient&#39;s abdomen with warm saline, and monitor the animal during the harvesting procedure.</li>
	<li>Apply four small atraumatic vascular clamps to the distal and proximal segments of the infrarenal aorta and IVC. If needed, temporarily occlude an unfavorable renal vessel with a 7-0 silk suture, and release the suture after the procedure. Place a 10-0 nylon suture vertically onto the anterior wall of the aorta to facilitate the aortotomy. Perform an aortotomy with two small horizontal cuts (wedge-shaped) with microscissors by slightly pulling up the suture. 
	NOTE: To remove any blood clots, flushing of the aortic lumen with heparinized saline is recommended.</li>
	<li>Place the donor heart on the left side (from the animal&#8217;s perspective) of the aorta and secure the recipient&#8217;s infrarenal aorta and the donor&#8217;s ascending aorta end-to-side at the 12 o&#8217;clock and 6 o&#8217;clock positions of the aortotomy with sutures. Continue with the third and fourth sutures at the 3 o&#8217;clock and 9 o&#8217;clock positions, gently flipping the heart over to the right side of the aorta after the third suture. Complete the arterial anastomosis by adding one to two sutures to every interspace. 
	NOTE: Care should be taken to avoid touching either the donor&#39;s ascending aorta or the recipient&#39;s abdominal aorta with forceps when creating the anastomosis to avoid tissue damage.</li>
	<li>Rotate the rat counterclockwise, with the head facing toward the surgeon&#39;s left hand. Move the donor&#39;s aorta to the left side of the abdominal aorta to allow optimal sight onto the IVC.</li>
	<li>Perform a venotomy on the IVC, slightly proximal to the aortic anastomosis, using an 11 blade for puncture and microscissors for adequate size adjustment according to the diameter of the donor&#39;s pulmonary trunk. Again, flush the intracaval lumen with heparinized saline.</li>
	<li>Start with the venous anastomosis between the recipient&#39;s IVC and donor&#39;s pulmonary trunk, which is best achieved by placing interrupted 11-0 nylon sutures on the back wall of the vessel, starting at the 12 o&#39;clock and 6 o&#39;clock positions (related to the IVC), and then place a continuous 11-0 nylon suture on the front wall (from the 6 o&#39;clock toward the 12 o&#39;clock position).</li>
	<li>Cover the anastomoses with small strips of an absorbable gelatin sponge, and remove the microvascular clamps starting distally. Use a cotton tip applicator to lightly compress the sponges to obtain optimal hemostasis.</li>
	<li>Observe the graft&#39;s coronary vessels filling at the time of the release of the distal microvascular clamps, and make sure that the donor heart starts beating immediately when the proximal clamp is released. 
	NOTE: The graft&#39;s viability can be scored from 0 to 4 intraoperatively according to a modified Stanford score19 to confirm adequate graft function.</li>
	<li>Place the intestines back into the abdomen by ensuring not to distort the arterial and venous anastomosis.</li>
	<li>Administer meloxicam (1mg/kg) and ethiqa XR (0.65 mg/kg) subcutaneously while the animal is fully anesthezised to ascertain postoperative analgesia. Then, close the abdominal wall with a continuous 5-0 absorbable vicryl suture before closing the skin with a 6-0 absorbable vicryl suture intracutaneously. 
	&#8203;NOTE: Guidance regarding common failures and troubleshooting is presented in Table 2.</li>
</ol>
3. Harvesting of the neonatal donor heart
<ol>
	<li>Place the neonatal donor rat in a chamber insufflated with isoflurane (2%) for sedation. Administer ketamine (75 mg/kg) and xylazine (5 mg/kg), as well as heparin (300 U/kg) intraperitoneally.</li>
	<li>Confirm the depth of anesthesia by toe pinch, and place the rat in a supine position with the tail facing toward you. Sterilize the entire thorax and abdominal wall with betadine and 70% ethanol three times alternatively. Cover the rat with a sterile surgical drape.</li>
	<li>Using a 12.5x surgical microscope, remove the entire anterior thoracic wall by starting with a horizontal incision using a 15 blade scalpel at the xyphoid followed by vertical incisions laterally up to the axillae on both sides with scissors. The anterior thoracic wall can then be removed by continuing with another horizontal incision right beneath the neck.</li>
	<li>Dissect the IVC, right and left superior vena cavae, and pulmonary vessels with scissors, and then encircle and ligate all the vessels with a 7-0 silk suture. Administer 3 mL of ice-cold, high-potassium modified Krebs-Henseleit solution to the right atrium by puncturing the IVC with a 30 G needle and slightly pushing the diaphragm down with forceps.</li>
	<li>Cut the IVC, SVCs, pulmonary vessels, and aorta with scissors. Transect the pulmonary arteries as far as possible and the aorta distal to the brachiocephalic trunk to ensure proper length using a 11 blade scalpel.</li>
	<li>Separate the pulmonary trunk and ascending aorta with microscissors, and flush the heart with ice cold cardioplegic solution using a 3 mL syringe.</li>
</ol>
4. Recovery of the recipient and graft monitoring
<ol>
	<li>After surgery, give the rat ample time to wake up, which usually occurs in a 15 min time window, and let it recover on a heating pad. 
	NOTE: No antibiotics are necessary due to the very low risk of infection and in order to not compromise the experimental model, and no restriction to food or water is applied.</li>
	<li>Following transplantation, monitor the graft function by palpation of the transplanted heart daily, but consider that this can sometimes be difficult to assess due to intestine overlay. 
	NOTE: Abdominal echocardiography can more accurately measure the graft viability. For echocardiography, sedate the rat slightly with isoflurane (1-2%) inhaled through a nose cone, and position it on a heating pad. Echocardiography is usually performed on postoperative day (POD) 1, POD 7, and POD 14. To allow for the assessment of the heart rate and contractility, one can easily obtain long-axis and short-axis views (Figure 2A, B). To evaluate the anastomoses, use Doppler echocardiography (Figure 3A), and confirm the formation of EFE tissue as seen as an echo-bright endocardial layer within the left ventricular cavity (Figure 3B, C).</li>
</ol>

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

<ol>
	<li>Lurie, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+concepts+of+endocardial+fibroelastosis.">Changing concepts of endocardial fibroelastosis.</a> Cardiology in the Young. 20 (2), 115-123 (2010).</li><li>Crucean, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluation+of+hypoplastic+left+heart+syndrome+from+a+developmental+and+morphological+perspective.">Re-evaluation of hypoplastic left heart syndrome from a developmental and morphological perspective.</a> Orphanet Journal of Rare Diseases. 12 (1), 138 (2017).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+fibroelastosis+is+caused+by+aberrant+endothelial+to+mesenchymal+transition.">Endocardial fibroelastosis is caused by aberrant endothelial to mesenchymal transition.</a> Circulation Research. 116 (5), 857-866 (2015).</li><li>Eisenberg, L. M., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+atrioventricular+valvuloseptal+morphogenesis.">Molecular regulation of atrioventricular valvuloseptal morphogenesis.</a> Circulation Research. 77 (1), 1-6 (1995).</li><li>Illigens, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+prevents+endothelial-to-mesenchymal+transition+in+hypertrophy.">Vascular endothelial growth factor prevents endothelial-to-mesenchymal transition in hypertrophy.</a> Annals of Thoracic Surgery. 104 (3), 932-939 (2017).</li><li>Zeisberg, E. M., Potenta, S. E., Sugimoto, H., Zeisberg, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+kidney+fibrosis+emerge+via+endothelial-to-mesenchymal+transition.">Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.</a> Journal of the American Society of Nephrology. 19 (12), 2282-2287 (2008).</li><li>Zeisberg, E. M., Potenta, S., Xie, L., Zeisberg, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+endothelial+to+mesenchymal+transition+as+a+source+for+carcinoma-associated+fibroblasts.">Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts.</a> Cancer Research. 67 (21), 10123-10128 (2007).</li><li>Souilhol, C., Harmsen, M. C., Evans, P. C., Krenning, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-mesenchymal+transition+in+atherosclerosis.">Endothelial-mesenchymal transition in atherosclerosis.</a> Cardiovascular Research. 114 (4), 565-577 (2018).</li><li>Zeisberg, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-to-mesenchymal+transition+contributes+to+cardiac+fibrosis.">Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.</a> Nature Medicine. 13 (8), 952-961 (2007).</li><li>Rieder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-induced+endothelial-to-mesenchymal+transition:+A+novel+mechanism+of+intestinal+fibrosis.">Inflammation-induced endothelial-to-mesenchymal transition: A novel mechanism of intestinal fibrosis.</a> American Journal of Pathology. 179 (5), 2660-2673 (2011).</li><li>Johnson, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxia+as+a+cause+of+endocardial+fibroelastosis+in+infancy.">Anoxia as a cause of endocardial fibroelastosis in infancy.</a> AMA Archives of Pathology. 54 (3), 237-247 (1952).</li><li>Shimada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distention+of+the+immature+left+ventricle+triggers+development+of+endocardial+fibroelastosis:+An+animal+model+of+endocardial+fibroelastosis+introducing+morphopathological+features+of+evolving+fetal+hypoplastic+left+heart+syndrome.">Distention of the immature left ventricle triggers development of endocardial fibroelastosis: An animal model of endocardial fibroelastosis introducing morphopathological features of evolving fetal hypoplastic left heart syndrome.</a> Biomedical Research. 2015, 462-469 (2015).</li><li>Weixler, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+disturbances+and+the+development+of+endocardial+fibroelastosis.">Flow disturbances and the development of endocardial fibroelastosis.</a> Journal of Thoracic and Cardiovascular Surgery. 159 (2), 637-646 (2020).</li><li>Purevjav, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nebulette+mutations+are+associated+with+dilated+cardiomyopathy+and+endocardial+fibroelastosis.">Nebulette mutations are associated with dilated cardiomyopathy and endocardial fibroelastosis.</a> Journal of the American College of Cardiology. 56 (18), 1493-1502 (2010).</li><li>Friehs, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+endocardial+fibroelastosis.">An animal model of endocardial fibroelastosis.</a> Journal of Surgical Research. 182 (1), 94-100 (2013).</li><li>Emani, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staged+left+ventricular+recruitment+after+single-ventricle+palliation+in+patients+with+borderline+left+heart+hypoplasia.">Staged left ventricular recruitment after single-ventricle palliation in patients with borderline left heart hypoplasia.</a> Journal of the American College of Cardiology. 60 (19), 1966-1974 (2012).</li><li>Hickey, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+left+ventricular+outflow+tract+obstruction:+The+disproportionate+impact+of+biventricular+repair+in+borderline+cases.">Critical left ventricular outflow tract obstruction: The disproportionate impact of biventricular repair in borderline cases.</a> Journal of Thoracic and Cardiovascular Surgery. 134 (6), 1429-1436 (2007).</li><li>Oh, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+flow+conditions+promote+endocardial+fibroelastosis+via+endothelial-to-mesenchymal+transition,+which+is+responsive+to+losartan+treatment.">Abnormal flow conditions promote endocardial fibroelastosis via endothelial-to-mesenchymal transition, which is responsive to losartan treatment.</a> JACC: Basic to Translational Science. 6 (12), 984-999 (2021).</li><li>Blanchard, J. M., Pollak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+perfusion+and+storage+of+heterotopic+heart+transplants+in+mice.">Techniques for perfusion and storage of heterotopic heart transplants in mice.</a> Microsurgery. 6 (3), 169-174 (1985).</li><li>Kokudo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snail+is+required+for+TGFbeta-induced+endothelial-mesenchymal+transition+of+embryonic+stem+cell-derived+endothelial+cells.">Snail is required for TGFbeta-induced endothelial-mesenchymal transition of embryonic stem cell-derived endothelial cells.</a> Journal of Cell Science. 121 (20), 3317-3324 (2008).</li><li>Wu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+cells+form+the+coronary+arteries+by+angiogenesis+through+myocardial-endocardial+VEGF+signaling.">Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.</a> Cell. 151 (5), 1083-1096 (2012).</li><li>Clark, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+endocardial+fibroelastosis.">A mouse model of endocardial fibroelastosis.</a> Cardiovascular Pathology. 24 (6), 388-394 (2015).</li><li>Kovacic, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+to+mesenchymal+transition+in+cardiovascular+disease:+JACC+state-of-the-art+review.">Endothelial to mesenchymal transition in cardiovascular disease: JACC state-of-the-art review.</a> Journal of the American College of Cardiology. 73 (2), 190-209 (2019).</li><li>Derynck, R., Zhang, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad-dependent+and+Smad-independent+pathways+in+TGF-beta+family+signalling.">Smad-dependent and Smad-independent pathways in TGF-beta family signalling.</a> Nature. 425 (6958), 577-584 (2003).</li><li>Daugherty, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendation+on+design,+execution,+and+reporting+of+animal+atherosclerosis+studies:+A+scientific+statement+from+the+American+Heart+Association.">Recommendation on design, execution, and reporting of animal atherosclerosis studies: A scientific statement from the American Heart Association.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (9), e131-e157 (2017).</li></ol>

This animal model of heterotopic transplantation of a neonatal donor rat heart into the recipient&#39;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...

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 previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts through endothelial-to-mesenchymal transition (EndMT)3. EndMT can be detected using immunohistochemical double-staining for endothelial markers such as cluster of differentiation (CD) 31 or vascular endothelial (VE)-cadherin (CD144) and fibroblast markers (e.g., alpha-smooth muscle actin, &#945;-SMA). Furthermore, we also previously established the regulatory role of the TGF-&#223; pathway in this process with activation of the transcription factors SLUG, SNAIL, and TWIST3.
EndMT is a physiological process that occurs during embryonic cardiac development and leads to the formation of the septa and valves from endocardial cushions4, but it also causes organ fibrosis in heart failure, kidney fibrosis, or cancer and plays a key role in vascular atherosclerosis5,6,7,8. EndMT in cardiac fibrosis is mainly regulated through the TGF-&#946; pathway, as we and others have reported3,9. Various stimuli have been described to induce EndMT: inflammation10, hypoxia11, mechanical alterations12, and flow disturbances, including alterations of the intracavitary blood flow13, and EndMT may also be a consequence of a genetic disease14.
This animal model was developed using the key components of cardiac EFE development, which are immaturity and alterations of the intracavitary blood flow, specifically flow stagnation. Immaturity was fulfilled by using neonatal rat hearts as donors, since neonatal rats are known to be developmentally immature immediately after birth. Heterotopic heart transplantation offered the provision of intracavitary flow restriction15.
From a clinical point of view, this animal model allows for better investigating the impact of EndMT on the growing left ventricle (LV). The growth restriction imposed on the fetal and neonatal heart through EndMT-induced EFE formation16 precludes patients with left ventricular outflow tract obstructions (LVOTO) such as congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair17. This animal model facilitates the study of the cellular mechanisms and regulation of tissue formation through EndMT and allows for the testing of pharmacological treatment options3,18.
Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, EFE formation can be macroscopically observed within 3 days after transplantation. EFE tissue shows the same histopathological characteristics as human EFE tissue from patients with LVOTO.
Hence, this animal model, though developed for pediatric use in the spectrum of HLHS, can be applied when studying various diseases based on the molecular mechanism of EndMT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced Ventilator System For Rodents, SAR-1000</td>
			<td>CWE, Inc.</td>
			<td>12-03100</td>
			<td>small animal ventilator</td>
		</tr>
		<tr>
			<td>aSMA</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Axio observer Z1&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>inverted microscope</td>
		</tr>
		<tr>
			<td>Betadine&#160;Solution</td>
			<td>Avrio Health L.P.</td>
			<td>367618150092</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Invitrogen</td>
			<td>MA1-80069</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>DemeLON Nylon black 10-0</td>
			<td>DemeTECH</td>
			<td>NL76100065F0P</td>
			<td>10-0 Nylon suture</td>
		</tr>
		<tr>
			<td>ETFE IV Catheter, 18G x 2</td>
			<td>TERUMO SURFLO</td>
			<td>SR-OX1851CA</td>
			<td>intubation cannula</td>
		</tr>
		<tr>
			<td>Micro Clip 8mm</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-6471</td>
			<td>microvascular clamps</td>
		</tr>
		<tr>
			<td>Nylon black monofilament 11-0</td>
			<td>SURGICAL SPECIALTIES CORP</td>
			<td>AA0130</td>
			<td>11-0 Nylon</td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Embedding medium for frozen tissue specimen</td>
		</tr>
		<tr>
			<td>p-SMAD2/3</td>
			<td>Invitrogen</td>
			<td>PA5-110155</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Rodent, Tilting WorkStand</td>
			<td>Hallowell EMC.</td>
			<td>000A3467</td>
			<td>oblique shelf for intubation</td>
		</tr>
		<tr>
			<td>Silk Sutures, Non-absorbable, 7-0</td>
			<td>Braintree Scientific</td>
			<td>NC9201231</td>
			<td>Silk suture</td>
		</tr>
		<tr>
			<td>Slug/Snail</td>
			<td>Abcam</td>
			<td>ab180714</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Undyed Coated Vicryl 5-0 P-3 18&#34;</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>5-0 Vicryl</td>
		</tr>
		<tr>
			<td>Undyed Coated Vicryl 6-0 P-3 18&#34;</td>
			<td>Ethicon</td>
			<td>J492G</td>
			<td>6-0 Vicryl</td>
		</tr>
		<tr>
			<td>VE-Cadherin</td>
			<td>Abcam</td>
			<td>ab231227</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Zeiss OPMI 6-SFR</td>
			<td>Zeiss</td>
			<td></td>
			<td>Surgical microscope</td>
		</tr>
		<tr>
			<td>Zen, Blue Edition, 3.6</td>
			<td>Zen&#160;</td>
			<td></td>
			<td>inverted microscope software</td>
		</tr>
	</tbody>
</table>

a neonatal heterotopic rat heart transplantation model for the study of endothelial-to-mesenchymal transition

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&#39;s abdomen and connected to the recipient&#39;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.

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

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

Endocardial fibro elastosis hinders left ventricular growth and prevents biventricular surgical repair for patients with congenital critical aortic stenosis and hypoplastic left heart syndrome. While surgical resection is currently the only available treatment option, EFE frequently recurs. Therefore, we aimed to investigate the mechanisms behind EFE and develop new therapeutic strategies using an animal model.The main developments in the field of EndMT research are restricted to cell culture models which have inherent limitations, and research has been hampered due to a lack of in vivo models. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts, a process called endothelial to mesenchymal transition. This animal model allows for a more generalized examination of EndMT induced fibrosis in the heart, whereas other models are designed to induce EndMT through genetic modifications, hypertension, or dietary restrictions.As EFE often recurs after primary resection, sometimes with an even more infiltrative growth pattern, we are especially interested in the molecular interaction with the underlying myocardium.

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...

Watch this Scientific Journal Video about A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition at JoVE.com

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 ...

a-neonatal-heterotopic-rat-heart-transplantation-model-for-study

Research

JoVE Journal

Medicine

Surgical Preparation and Heterotopic Transplantation of the Neonatal Donor Heart in the Recipient Rat

Begin harvesting the neonatal donor heart by anesthetizing and sterilizing the rat, and making a horizontal incision at the xiphoid with a 15 blade scalpel under a 12.5x surgical microscope. Next, use scissors to create vertical incisions laterally up to the axillae, on both sides. Remove the anterior thoracic wall with another horizontal incision beneath the neck.Dissect the inferior vena cava, or IVC, right and left superior venae cavae, or SVC, and pulmonary vessels with scissors. Encircle and ligate dull vessels using a 7-0 silk suture. Slightly push the diaphragm down with forceps and administer three milliliters of ice-cold, high-potassium modified Krebs-Henseleit solution to the right atrium by puncturing the IVC with a 30-gauge needle.Cut the IVC, SVC, and pulmonary vessels using scissors. Ensure proper length by transecting the pulmonary arteries as far as possible, and the aorta, distal to the brachiocephalic trunk, using a number 11 blade scalpel. Flush the heart with the ice-cold cardioplegic solution using a three milliliter syringe.Place the anesthetized rat on an oblique shelf. Secure its front teeth with a string, and position the head towards the surgeon. Place the light on the outside of the neck onto the area of the vocal cords.Grasp the tongue with two fingers and gently push it upwards and to the left for optimal intubation visibility. Now, use an 18-gauge, two-inch cannula for a 100 to 150 gram rat and secure the intratracheal tube with tape. Connect the intubation cannula to the small animal ventilator, and adjust the settings based on the animal's size, following the manufacturer's instructions.After intubating the rat, perform a midline laparotomy using a 15 blade scalpel to make the skin incision. Open the anterior abdominal wall with scissors and mobilize the intestines toward the right upper quadrant. Next, expose the retroperitoneal abdominal aorta and IVC, using cotton tip applicators.Cover the intestines with warm, saline-soaked gauze and use retractors to maintain optimal exposure of the IVC and abdominal aorta. Now, perform blunt dissection of the infrarenal IVC and abdominal aorta up to the bifurcation. Ligate dull infrarenal branching arteries and veins with a 10-0 nylon suture.Transport the harvested donor's heart in a sterile surgical basin containing Krebs-Heneseleit buffer to the surgical field. Ensure to irrigate the donor heart intermittently with ice-cold cardioplegic solution. Next, separate the pulmonary trunk and descending aorta using micro scissors, and irrigate the donor heart with ice-cold cardioplegic solution.Next, apply four atraumatic vascular clamps to the distal and proximal segments of the infrarenal aorta and IVC. Perform an aortotomy using micro scissors to make two small horizontal cuts. Then, place the donor heart on the left side of the aorta.Secure the recipient's infrarenal aorta and the donor's ascending aorta and to side at the 12 and six o'clock positions of the aortotomy with sutures. Proceed with the third and fourth sutures at the three and nine o'clock positions, gently flipping the heart to the left side of the aorta after the third suture. Finalize the arterial anastomosis by adding one to two sutures to each interspace.Now, rotate the rat counterclockwise, so the head faces the surgeon's left hand. Shift the donor's heart to the left side of the abdominal aorta for an optimal view of the IVC. Perform a venotomy on the IVC, slightly proximal to the aortic anastomosis.Using an 11 blade for puncture and micro scissors to adjust the size according to the donor's pulmonary trunk diameter, flush the intracoval lumen with heparinized saline. Next, perform the venous anastomosis between the recipient's IVC and the donor's pulmonary trunk by placing interrupted 11-0 nylon sutures on the back wall of the vessel, starting at the 12 and six o'clock positions. Place a continuous 11-0 nylon suture on the front wall, place absorbable gelatin sponge strips over the anastomoses, and remove the microvascular clamps, beginning with the distal ones.Ensure that the coronary vessels fill and that the donor's heart begins beating shortly upon release of the proximal clamps. Apply light pressure using a cotton tip applicator on the sponges to achieve optimal hemostasis. Carefully place the intestines back into the abdomen, being cautious not to distort the arterial and venous anastomosis.Close the abdominal wall and allow the animal to recover. A horizontal slice of the donor graft resected at the mid ventricular level of the right and left ventricles is shown. Histological analysis indicated that the endocardial fibroelastosis tissue had high amounts of organized collagen and elastin fibers.Endocardial fibroelastosis tissue, double stained for VE-Cadherin and alpha-SMA. CD-31, phospho-SMAD2 and SMAD3, CD-31 and slug or snail indicated endothelial to mesenchymal transition.

810 Views.  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. 

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

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