Name: apical resection mouse model to study early mammalian heart regeneration
Uploaded: 2016-01-23T15:00:00
Description: Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

The authors&#160;thank Drs. James Hawkins, Zu-Xi Yu and Xuan Qu from the National Heart, Lung, and Blood Institute (NHLBI) for their assistance with mouse surgery and preparation and staining of paraffin sections. The authors are&#160;grateful to the NIH Fellows Editorial Board for editorial assistance.

All mouse experiments were approved by the Animal Care and Use Program at the National Institutes of Health (NIH) with protocol number H0083R3.&#160;The NHLBI IACUC approved the protocol without analgesics.
1. Hypothermia Anesthesia in Neonatal Mice
<ol>
	<li>Sterilize sponges and surgical equipment in an autoclave before surgery. Prepare all the surgical materials and switch on a hot bead sterilizer 15-20 min&#160;in advance to reach 240&#176;C to 270&#176;C.</li>
	<li>Transfer all C57BL/6 ...

<ol>
	<li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6, 231-239 (2014).</li><li>Xin, M., Olson, E. N., Bassel-Duby, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mending+broken+hearts:+cardiac+development+as+a+basis+for+adult+heart+regeneration+and+repair.">Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.</a> Nat Rev Mol Cell Biol. 14, 529-541 (2013).</li><li>Mahmoud, A. I., Porrello, E. 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I., Porrello, E. R., Kimura, W., Olson, E. N., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+models+for+cardiac+regeneration+in+neonatal+mice.">Surgical models for cardiac regeneration in neonatal mice.</a> Nat Protoc. 9, 305-311 (2014).</li><li>Allen, T. C., Prophet, E. B., Mills, B., Arrington, J. B., Sobin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin.">Hematoxylin and eosin.</a> Laboratory methods in Histotechnology. , 53-58 (1992).</li><li>Andersen, D. C., Ganesalingam, S., Jensen, C. H., Sheikh, S. 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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=c-kit++precursors+support+postinfarction+myogenesis+in+the+neonatal,+but+not+adult,+heart.">c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart.</a> Proc Natl Acad Sci U S A. 109, 13380-13385 (2012).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138, 1663-1674 (2011).</li></ol>

Cardiac regeneration shows potential for the treatment and prevention of heart failure1, 2. Animal models are indispensable and play a critical role in understanding how cardiac regeneration occurs3-6. Many amphibians and fish regenerate heart tissue in response to injury, providing insight into our understanding of human cardiovascular disease13, 14. In terms of evolution, however, the pathophysiology should be more conserved between a mouse model and humans. Heart regeneration in mammal...

Prolonged human life span leads to various aging- and lifestyle-related diseases, including heart failure, a leading cause of mortality. However, without replacement of lost or dysfunctional cardiac muscle cells, current therapeutics can only transiently improve cardiac function1, 2. Thus, it is necessary to discover and develop innovative strategies for cardiac regeneration and repair. The adult mammalian heart has limited regenerative potential. Studies from lower vertebrates, such as urodele amphibians and teleost fish, have provided unprecedented insights into the molecular and cellular mechanisms underlying heart regeneration3, 4. Recently, a neonatal mouse model of heart regeneration has emerged that might enable identification and characterization of more evolutionarily conserved pathophysiological events required for human heart regeneration5. 
Apical resection refers to the surgical removal of left ventricular apex. This procedure is restricted to 1- to 7-day-old (P1 to P7) mice due to its high lethality in older mice6. The cardiac regeneration process in neonatal mice after apical resection is expected to be as follows: (I) rapid and effective formation of a hematoma to seal the apex and prevent exsanguination; (II) cardiomyocyte regeneration and restoration of systolic function5, 7. Recent work has stimulated debate on the significance and efficiency of this model8-11. Thus, it is important to present the apical resection clearly and in detail. To this end, this protocol vividly and specifically describes a video of how I did apical resection based on a previous protocol6. 
Understanding the molecular mechanisms underlying cardiac regeneration is of importance for treatment of heart disease characterized by loss and/or injured cardiomyocytes, such as heart failure1, 2. Given the current and promising progress of apical resection in the research of cardiac regeneration, this study could promote the use of this technique and its uses in cardiac regeneration research.

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apical resection mouse model to study early mammalian heart regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

Mouse pups were euthanized 1, 2, and 21 days post-apical resection, and their hearts were collected for H&#38;E and Masson&#39;s Trichrome stain. Blue color in Masson&#39;s Trichrome stain indicates the deposition of epicardial extracellular matrix5. With successful apical resection, a blood clot is effectively formed to seal the LV one day post-apical resection, as shown in Figure 1A. A gradual resorption of the blood clot and early cardiac fibrosis is observed, and replacement by myocardial ...

Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

The overall goal of this procedure is to use apical resection in neonatal mice to study early mammalian heart regeneration. This matter can help answer key questions in the heart regeneration field such as what are the molecular and cellular mechanisms that underlie early mammalian cardiac regeneration. The main advantage of this technique is that apical resection mouse model provides a relatively simple and a direct approach for studying early mammalian heart regeneration.Before beginning the procedure, transfer all of the postnatal day one C57 black six pups from their nursing mother into a clean mouse cage with fresh bedding and nesting materials. Then for each neonate in turn, transfer the pup from an ice bed to the surgical area and immobilize the arms, legs, and tail in a supine position. Disinfect the chest with betadine followed by a gentle cleaning with a 70%alcohol prep pad.Now, make a transfer skin incision along the fourth inner costal area of the chest cavity. Use spring scissors to make an incision in the apex of the heart. To access the heart, use spring scissors to make an incision in the apex of the heart followed by a blunt dissection of the inner costal muscles.Use sterile 6-0 sutures to close the ribs and muscles together. Use skin glue to carefully close the skin incision and allow the neonate to recover under a heat lamp for about three minutes. Then use a 70%alcohol prep pad to clean all of the blood and glue traces from the pup and return the animal to its litter mates.After all of the surgeries have been performed, mix the pups with the mother's bedding and excrement before returning the neonates to the mother's nest. One day after surgery, count the number of sham and surgical pups to determine the survival rate within each group. At the appropriate time point after surgery, for each animal in turn, clean the chest with 70%ethanol and incise the midline skin and muscle of the chest.Remove the entire heart from the thoracic cavity and fix the tissue in five millimeters of four percent paraformaldehyde overnight at room temperature. The next day, transfer the samples to 70%ethanol for up to one week before paraffin embedding. After acquiring five micrometer thick paraffin sections through the entire ventricle, stain the samples with hematoxylin, and Eocene, and Masson's trichrome to examine the muscle replacement and fibrotic responses in the resected hearts respectively.Using a slide scanner, manually obtain at least three foci per sample. Then analyze the sample images at a 40X magnification according to the manufacturer's instructions using the default parameters of the scanner. With a successful apical resection, within one day of the apical resection, a blood clot is effectively formed to seal the left ventricle.A progressive resorption of the blood clot and early cardiac fibrosis is then observed with its gradual replacement by myocardial tissue until the complete regeneration of the myocardium by 21 days after the resection. Indeed, by three weeks after the surgery, no differences between the resected and sham operated hearts are apparent by morphological analysis. While attempting this procedure, it's important to remember to maintain sterile conditions.Following this procedure, other measures like labeling and the linear can be performed to answer additional questions about cardiomyocyte proliferation and apical resection. After watching this video, you should have a good understanding of how to perform apical resection in neonatal mice.

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