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Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish


The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than on tissue damage. We recently characterized the cellular response and regenerative capacity of the zebrafish heart after cryoinjury (CI), an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). After anesthesia, localized CI with a liquid nitrogen–cooled copper probe induced damage in 25% of the ventricle, in a procedure requiring <5 min. Here we present a detailed description of the technique, which provides a valuable system for the study of the mechanisms of heart regeneration and scar removal after MI in a versatile vertebrate model.

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Figure 1: Detailed description of cardiac cryoinjury in an adult zebrafish.
Figure 2: Constructing a cryoprobe.
Figure 3: Dissection of the adult zebrafish heart.
Figure 4: Effect of cryoinjury on the zebrafish heart ventricle.


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We thank D. Bartolomé and M.Á. Ricote for constructing the cryoprobe, E. Díaz and others at the CNIC animal facility for zebrafish husbandry, and S. Bartlett (CNIC) for text editing. The Tg(cmlc2:GFP) line was provided by A. Raya (Institute for Bioengineering of Catalonia (IBEC), Spain) and the Tg(fli1a:GFP)y1 line was from the Zebrafish International Resource Center, which is supported by grant no. P40 RR012546 from the US National Institutes of Health National Center for Research Resources (NIH-NCRR). Funding was from the Fundación CNIC Carlos III, the Fundación ProCNIC, the Comunidad de Madrid (FIBROTEAM S2011/BMD-2321) and the Spanish Ministry of Economy and Competitiveness (FPU fellowship to J.M.G.-R., RYC-2006-001694 and BFU-2008-0012BMC to N.M.).

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J.M.G.-R. performed the experiments. J.M.G.-R. and N.M. designed the experiments and prepared the manuscript.

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Correspondence to Nadia Mercader.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Cryoinjury using a platinum cryoprobe. (a and b) Whole mount views of bright field (a) and fluorescence (b) images of freshly cryoinjured dissected Tg(fli1a:GFP) zebrafish hearts. Anterior is to the top, dorsal to the left. The injured area is visible as a pale area in a and as the region lacking coronary vasculature in b. (c) Graph showing the percentage of the ventricle occupied by the injured area at 1 hour postinjury in cryoinjured hearts using a 0.3 mm copper filament (red dots) or a 0.5 mm platinum filament (blue squares). Mean and standard deviation for copper filament was 28% ± 8% (mean ± S.D.) and for platinum 35% ± 11%. at, atrium; ba, bulbus arteriosus; IA, injury area; v, ventricle. Scale bars, 100 µm. (TIFF 19289 kb)

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

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