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


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.

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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.

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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.).

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Authors and Affiliations



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.

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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.

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Supplementary Figures 1 and 2 (PDF 309 kb)

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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).

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