Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A cryoinjury model in neonatal mice for cardiac translational and regeneration research

Abstract

The introduction of injury models for neonatal mouse hearts has accelerated research on the mechanisms of cardiac regeneration in mammals. However, some existing models, such as apical resection and ligation of the left anterior descending artery, produce variable results, which may be due to technical difficulties associated with these methods. Here we present an alternative model for the study of cardiac regeneration in neonatal mice in which cryoinjury is used to induce heart injury. This model yields a reproducible injury size, does not induce known mechanisms of cardiac regeneration and leads to a sustained reduction of cardiac function. This protocol uses reusable cryoprobes that can be assembled in 5 min, with the entire procedure taking 15 min per pup. The subsequent heart collection and fixation takes 2 d to complete. Cryoinjury results in a myocardial scar, and the size of injury can be scaled by the use of different cryoprobes (0.5 and 1.5 mm). Cryoinjury models are medically relevant to diseases in human infants with heart disease. In summary, the myocardial cryoinjury model in neonatal mice described here is a useful tool for cardiac translational and regeneration research.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Main steps of the construction of cryoprobes and experimental setup.
Figure 2: Detailed description of cardiac cryoinjury in a neonatal mouse.
Figure 3: Resection of heart after cryoinjury at day of life 1 (P1).
Figure 4: Cryoinjury in neonatal mice induces tissue damage.
Figure 5: Cryoinjury in neonatal mice induces apoptosis in myocardial cells.
Figure 6: Cryoinjury induces scar formation in neonatal mice.

Similar content being viewed by others

References

  1. Naqvi, N. et al. A proliferative burst during preadolescence establishes the final cardiomyocyte number. Cell 157, 795–807 (2014).

    Article  CAS  Google Scholar 

  2. Wulfsohn, D., Nyengaard, J.R. & Tang, Y. Postnatal growth of cardiomyocytes in the left ventricle of the rat. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 277, 236–247 (2004).

    Article  CAS  Google Scholar 

  3. Li, F., Wang, X., Capasso, J.M. & Gerdes, A.M. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J. Mol. Cell. Cardiol. 28, 1737–1746 (1996).

    Article  CAS  Google Scholar 

  4. Mollova, M. et al. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc. Natl. Acad. Sci. USA 110, 1446–1451 (2013).

    Article  CAS  Google Scholar 

  5. Bergmann, O. et al. Evidence for cardiomyocyte renewal in humans. Science 324, 98–102 (2009).

    Article  CAS  Google Scholar 

  6. Beinlich, C.J., Rissinger, C.J. & Morgan, H.E. Mechanisms of rapid growth in the neonatal pig heart. J. Mol. Cell. Cardiol. 27, 273–281 (1995).

    Article  CAS  Google Scholar 

  7. Mahmoud, A.I., Porrello, E.R., Kimura, W., Olson, E.N. & Sadek, H.A. Surgical models for cardiac regeneration in neonatal mice. Nat. Protoc. 9, 305–311 (2014).

    Article  CAS  Google Scholar 

  8. Porrello, E.R. et al. Transient regenerative potential of the neonatal mouse heart. Science 331, 1078–1080 (2011).

    Article  CAS  Google Scholar 

  9. Porrello, E.R. et al. Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proc. Natl. Acad. Sci. USA 110, 187–192 (2013).

    Article  CAS  Google Scholar 

  10. Haubner, B.J. et al. Complete cardiac regeneration in a mouse model of myocardial infarction. Aging 4, 966–977 (2012).

    Article  CAS  Google Scholar 

  11. Jesty, S.A. et al. c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart. Proc. Natl. Acad. Sci. USA 109, 13380–13385 (2012).

    Article  CAS  Google Scholar 

  12. Burns, K.M. et al. New mechanistic and therapeutic targets for pediatric heart failure: report from a National Heart, Lung, and Blood Institute Working Group. Circulation 130, 79–86 (2014).

    Article  Google Scholar 

  13. Bryant, D.M. et al. A systematic analysis of neonatal mouse heart regeneration after apical resection. J. Mol. Cell. Cardiol. 79, 315–318 (2015).

    Article  CAS  Google Scholar 

  14. Andersen, D.C., Ganesalingam, S., Jensen, C.H. & Sheikh, S.P. Do neonatal mouse hearts regenerate following heart apex resection? Stem Cell Rep. 2, 406–413 (2014).

    Article  CAS  Google Scholar 

  15. Konfino, T., Landa, N., Ben-Mordechai, T. & Leor, J. The type of injury dictates the mode of repair in neonatal and adult heart. J. Am. Heart Assoc. 4, e001320 (2015).

    Article  Google Scholar 

  16. van den Bos, E.J., Mees, B.M., de Waard, M.C., de Crom, R. & Duncker, D.J. A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation. Am. J. Physiol. Heart Circ. Physiol. 289, H1291–H1300 (2005).

    Article  CAS  Google Scholar 

  17. Robey, T.E. & Murry, C.E. Absence of regeneration in the MRL/MpJ mouse heart following infarction or cryoinjury. Cardiovasc. Pathol. 17, 6–13 (2008).

    Article  CAS  Google Scholar 

  18. van Amerongen, M.J., Harmsen, M.C., van Rooijen, N., Petersen, A.H. & van Luyn, M.J. Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice. Am. J. Pathol. 170, 818–829 (2007).

    Article  CAS  Google Scholar 

  19. Polizzotti, B.D. et al. Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window. Sci. Transl. Med. 7, 281ra245 (2015).

    Article  Google Scholar 

  20. Darehzereshki, A. et al. Differential regenerative capacity of neonatal mouse hearts after cryoinjury. Dev. Biol. 399, 91–99 (2015).

    Article  CAS  Google Scholar 

  21. Strungs, E.G. et al. Cryoinjury models of the adult and neonatal mouse heart for studies of scarring and regeneration. Methods Mol. Biol. 1037, 343–353 (2013).

    Article  CAS  Google Scholar 

  22. Leferovich, J.M. et al. Heart regeneration in adult MRL mice. Proc. Natl. Acad. Sci. USA 98, 9830–9835 (2001).

    Article  CAS  Google Scholar 

  23. Gonzalez-Rosa, J.M., Martin, V., Peralta, M., Torres, M. & Mercader, N. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development 138, 1663–1674 (2011).

    Article  CAS  Google Scholar 

  24. Gonzalez-Rosa, J.M. & Mercader, N. Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish. Nat. Protoc. 7, 782–788 (2012).

    Article  CAS  Google Scholar 

  25. Schnabel, K., Wu, C.C., Kurth, T. & Weidinger, G. Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS ONE 6, e18503 (2011).

    Article  CAS  Google Scholar 

  26. Chablais, F., Veit, J., Rainer, G. & Jazwinska, A. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev. Biol. 11, 21 (2011).

    Article  Google Scholar 

  27. Poss, K.D., Wilson, L.G. & Keating, M.T. Heart regeneration in zebrafish. Science 298, 2188–2190 (2002).

    Article  CAS  Google Scholar 

  28. Sadek, H.A. et al. Multi-investigator letter on reproducibility of neonatal heart regeneration following apical resection. Stem Cell Rep. 3, 1 (2014).

    Article  Google Scholar 

  29. Sen, S. & Sadek, H.A. Neonatal heart regeneration: mounting support and need for technical standards. J. Am. Heart Assoc. 4, e001727 (2015).

    Article  Google Scholar 

  30. Wald, R.M. et al. Effects of regional dysfunction and late gadolinium enhancement on global right ventricular function and exercise capacity in patients with repaired tetralogy of Fallot. Circulation 119, 1370–1377 (2009).

    Article  Google Scholar 

  31. Babu-Narayan, S.V. et al. Late gadolinium enhancement cardiovascular magnetic resonance of the systemic right ventricle in adults with previous atrial redirection surgery for transposition of the great arteries. Circulation 111, 2091–2098 (2005).

    Article  Google Scholar 

  32. Babu-Narayan, S.V. et al. Ventricular fibrosis suggested by cardiovascular magnetic resonance in adults with repaired tetralogy of Fallot and its relationship to adverse markers of clinical outcome. Circulation 113, 405–413 (2006).

    Article  CAS  Google Scholar 

  33. Xin, M., Olson, E.N. & Bassel-Duby, R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat. Rev. Mol. Cell Biol. 14, 529–541 (2013).

    Article  CAS  Google Scholar 

  34. Mahmoud, A.I. et al. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature 497, 249–253 (2013).

    Article  CAS  Google Scholar 

  35. Xin, M. et al. Hippo pathway effector Yap promotes cardiac regeneration. Proc. Natl. Acad. Sci. USA 110, 13839–13844 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Sadek and M. Ahmad (University of Texas Southwestern Medical Center) for training in mouse surgery, and members of the Kühn laboratory for helpful suggestions and discussions. We thank M.A. Missinato, University of Pittsburgh, for sharing her cryoinjury learning curve experience. We apologize to researchers whose relevant work could not be discussed or referenced because of the limitations of the scope of this paper. This research was supported by the Department of Cardiology and the Translational Research Program at Boston Children's Hospital and US National Institutes of Health (NIH) grants R01HL106302 and K08HL085143 (to B.K.). B.D.P. was supported by the Office of Faculty Development (Boston Children's Hospital) and by grant no. T32HL007572 from the NIH. B.G. and B.K. are supported by the Richard King Mellon Institute for Pediatric Research (Children's Hospital of Pittsburgh of UPMC). B.G., B.J.H., J.M.P. and B.K. were supported by Transatlantic Network of Excellence grants by the Fondation Leducq (no. 15CVD03 to B.G. and B.K.).

Author information

Authors and Affiliations

Authors

Contributions

B.D.P., B.G., B.J.H. and B.K. designed the research. B.D.P., B.G. and B.J.H. performed and analyzed the experiments. B.J.H. and J.M.P. provided data for Figures 4a,c and 5. B.D.P., B.G. and B.K. wrote the manuscript, and all authors edited the manuscript.

Corresponding author

Correspondence to Bernhard Kühn.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of the presented protocol with examination of learning curve.

The graphs show the survival at 2 hr (left panel) and 24 hr (right panel) of batches of surgery. A batch of mice operated by one researcher in a day usually consisted of 1-3 litters of ICR mice. Researcher 1 developed and optimized the protocol, while researchers 2 and 3 (with no prior mouse surgical experience) followed the suggested sequence of steps (Table 3) to learn the cardiac cryoinjury technique.

Supplementary Figure 2 Cryoinjury induces scar formation in neonatal mice.

Mice underwent sham surgery or cryoinjury on day of life 1 with a 0.5 mm probe. Scar size at 7 and 30 dpi was quantified by thresholding in Metamorph. Statistical significance was tested with analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. *P< 0.05, **P< 0.01. Cryo, Cryoinjury.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 309 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Polizzotti, B., Ganapathy, B., Haubner, B. et al. A cryoinjury model in neonatal mice for cardiac translational and regeneration research. Nat Protoc 11, 542–552 (2016). https://doi.org/10.1038/nprot.2016.031

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.031

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing