A modified apical resection model with high accuracy and reproducibility in neonatal mouse and rat hearts

Figures

• 

  Fig. 1. A schematic flow chart illustrating the major operation process of modified apical resection model in 1-day-old neonatal rodents. A modified pipette tip cut to a given internal diameter is prepared for the modified apical resection model. After hypothermia anesthesia and thoracotomy, the modified pipette tip, which is connected to a vacuum pump working at 0.06 ± 0.005 MPa, is gently kept in touch with the apex for 12 s. Apical resection is performed by a single incision adjacent to the pipette tip. Finally, chest closure is performed and rodents are returned together to mother’s cage.

• 

  Fig. 2. Cardiac structural and functional recovery after modified apical resection surgery in 1-day-old neonatal mice. a A stereoscope, a desktop vacuum pump, and a modified 10 μL pipette tip with its tip cut to 0.48 mm in internal diameter using a sharp blade, were prepared for modified apical resection (AR) method. b Heart weight (HW), body weight (BW), and heart weight/body weight ratio (HW/BW) at 21 days post resection (dpr) (n = 7-8). c Echocardiography for left ventricle ejection fraction and fractional shortening at 21 dpr (n = 7-8). d Representative images for hematoxylin-eosin (HE) and Masson’s trichrome staining for AR hearts at 7 dpr and 21 dpr. Scale bar = 200 μm. ns not significant. All data are expressed as means ± SD.

• 

  Fig. 3. Homogeneity and reproducibility of modified and conventional apical resection methods in 1-day-old neonatal mice. a Representative images of resected left ventricle (LV) apex using modified apical resection (AR) method (left panel, n = 5) and conventional AR method (right panel, n = 5) by one operator. b Corresponding variation of resected LV apex areas (left panel) and mean value of resected apex areas using modified versus conventional AR methods (right panel) by the same operator. c Relative standard deviation (RSD) analysis for resected LV apex areas by modified versus conventional AR methods. **P < 0.01; ns not significant. All data are expressed as means ± SD.

• 

  Fig. 4. Regenerative responses after modified apical resection surgery in 1-day-old neonatal mice. Co-immunofluorescent staining for α-actinin and EdU (a) or phospho-histone H3 (pHH3) (b) in sham and apical resection (AR) hearts at 7 days post resection (7 dpr) (n = 6-7). Scale bar = 20 μm. A selected area with a higher magnification (Scale bar = 5 μm) for each representative image was presented. c Co-immunofluorescent staining for α-actinin and Aurora B at 7 dpr to evaluate Aurora B-kinase expression at the cleavage furrow between two cardiomyocytes (n = 4). The representative asymmetrical (in the apex and remote region) and symmetrical (in the border region) Aurora B positive cardiomyocytes were indicated by white arrows. Scale bar = 20 μm. d Quantitative PCR for inflammatory and immunomodulatory factors and myocardial pro-proliferative factors in sham and AR hearts at 1 dpr and 7 dpr (n = 4). **P < 0.01; ***P < 0.001; ns not significant. All data are expressed as means ± SD.

• 

  Fig. 5. Cardiac structural and functional recovery after 5% modified apical resection surgery in 1-day-old neonatal rats. a A stereoscope, a desktop vacuum pump, and a 200 μL pipette tip with its tip cut to 0.63 mm in internal diameter using a sharp blade, were prepared for modified apical resection (AR) method. b Heart weight (HW), body weight (BW), and heart weight/body weight ratio (HW/BW) at 42 days post resection (dpr) (n = 7-8). c Echocardiography for left ventricle ejection fraction and fractional shortening at 42 dpr (n = 7-8). d Representative images for hematoxylin-eosin (HE) and Masson’s trichrome staining for AR hearts at 7, 14, 28, and 42 dpr. Scale bar = 200 μm. ns not significant. All data are expressed as means ± SD.

• 

  Fig. 6. Regenerative responses after 5% modified apical resection surgery in 1-day-old neonatal rats. Co-immunofluorescent staining for α-actinin and EdU (a) or phospho-histone H3 (pHH3) (b) in sham and apical resection (AR) hearts at 7 days post resection (7 dpr) (n = 6). Scale bar = 20 μm. A selected area with a higher magnification (Scale bar = 5 μm) for each representative image was presented. c Co-immunofluorescent staining for α-actinin and Aurora B at 7 dpr to evaluate Aurora B-kinase expression at the cleavage furrow between two cardiomyocytes (n = 4). The representative asymmetrical Aurora B positive cardiomyocytes were indicated by white arrows. Scale bar = 20 μm. d Quantitative PCR for inflammatory and immunomodulatory factors and myocardial pro-proliferative factors in sham and AR hearts at 1 dpr and 7 dpr (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. All data are expressed as means ± SD.

• 

  Fig. 7. Long-term cardiac function under hemodynamic stress after 5% modified apical resection surgery in 1-day-old neonatal rats. a Echocardiography for left ventricle ejection fraction and fractional shortening at baseline before phenylephrine injection in rats at 60 days post resection (dpr) (n = 9). b Tail cuff blood pressure measurements including systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) (n = 9). Data were presented as change from baseline (%) once SBP increased over 40%. c Echocardiography for heart rate, left ventricle ejection fraction, and fractional shortening when SBP increased over 40% from baseline after phenylephrine injection (n = 9). Data were presented as change from baseline (%). ns not significant. All data are expressed as means ± SD.