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 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.
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.
All mouse experiments were approved by the Animal Care and Use Program at the National Institutes of Health (NIH) with protocol number H0083R3. The NHLBI IACUC approved the protocol without analgesics.
1. Hypothermia Anesthesia in Neonatal Mice
2. Thoracotomy
3. Surgical Amputation of the Heart Ventricular Apex
4. Suture and Recovery of Mice
5. Post-Surgical Analysis
Mouse pups were euthanized 1, 2, and 21 days post-apical resection, and their hearts were collected for H&E and Masson's Trichrome stain. Blue color in Masson'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 tissue occurs until complete regeneration of myocardium takes place 21 days after apical resection (Figure 1B and C). Morphological analysis shows no difference between resected and sham-operated hearts at 21 days after surgical operation (Figure 1C and D).
Figure 1. Representative H&E and Masson's Trichrome stain. Ventricular myocardium of neonatal mouse hearts 1 (A), 2 (B), or 21 (C) days after apical resection and 21 days after sham operation (D). For each subfigure, the scale bar of the left panel is 1 mm and the scale bar of the right panel is 100 µm. Please click here to view a larger version of this figure.
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 mammalian animal models is a relatively new but burgeoning research field for the therapeutics of heart failure3-6.
Recently, two surgical approaches, apical resection and myocardial infarction, have been established in the study of heart regeneration in neonatal mice6. Although apical resection provides a relatively easy and direct model for loss and/or injury of cardiomyocytes, the main limitation of apical resection compared to myocardial infarction is that this surgical method cannot be performed beyond P7 due to high mortality6. There are, however, a number of ways to reduce mortality with this method. For apical surgery on P7 pups, the pups are cooled to 16°C for optimum hypothermia anesthesia. Generally, sterile conditions and surgical proficiency are critical for successful apical resection surgery. To improve survival, the surgery should be performed as quickly and gently as possible and a minimally invasive operation will reduce bleeding. Additionally, ICR/CD-1 mice are thought as good foster mothers and can effectively avoid of severe maternal cannibalization. For example, placing the P0 C57BL/6 pups with an ICR/CD-1 mother increases the survival rates of P1 pups with apical resection approximately from 60% to 80%ref.6. In addition, cryoinjury is an alternative procedure for study of cardiac regeneration. Compared to apical resection, cryoinjury model represents a different regenerative response and may be a more reproducible model15, 16. Thus far, a comparison of apical resection with other models warrants further investigations.
Using phospho-histone H3 and 5-bromo-2-deoxyuridine staining, Porrello and colleagues showed that cardiomyocyte proliferation contributes to the heart regeneration of neonatal P1 mice after apical resection response. Moreover, no significant cardiac hypertrophy and increase of cell size have been observed in mice with apical resection relative to sham-operated controls. After apical resection of 7-day-old mice, there is no regenerative response for myocardium5. A more detailed time course analysis of morphology and cardiac fibrosis after apical resection has been demonstrated5. Of note, the usefulness of apical resection in neonatal mouse models for heart regeneration research has been questioned by Anderson and colleagues8. Although many independent groups have demonstrated the reproducibility of apical resection, variations in the details of surgical procedures and different amounts of resected myocardium might be responsible for confusing results10. Quantification of the genomic DNA in resected heart is helpful for control of minimal chamber exposition and reproducibility of this technique. Additional research will be needed to fully understand neomyogenesis and complete vs. incomplete heart regeneration in the apical resection model8-11.
The main aim of this study is to present the procedures of apical resection in a visualized way. This video of apical resection in the neonatal mouse will be helpful to increase the use of this method and teach it to heart regeneration researchers. In addition, it may promote the pursuit of better approaches for mammalian heart regeneration in animal models and facilitate the discovery and translation of the molecular and cellular mechanisms responsible for heart regeneration in humans.
The authors have nothing to disclose.
The authors 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 grateful to the NIH Fellows Editorial Board for editorial assistance.
Olsen-Hegar Needle Holders with Scissors,1.5mm | Fine Science Tools | 12002-12 | |
Vannas Spring Scissors – 2mm Cutting Edge | Fine Science Tools | 15000-03 | Iridectomy scissors |
Hot Bead Sterilizer | Fine Science Tools | 18000-45 | |
Iris Forceps, Straight, Serrated | Fine Science Tools | 11064-07 | |
Iris Forceps, Curved, Serrated | Fine Science Tools | 11065-07 | |
Shea Scissors – Curved/Blunt-Blunt/12cm | Fine Science Tools | 14105-12 | |
Round Handled Suture Tying Forceps, Straight | Fine Science Tools | 18025-12 | |
Round Handled Vannas Spring Scissors | Fine Science Tools | 15400-12 | |
Magnifying Lamp | Luxolamp Corp | IM120 | |
Heating lamp | Brandt Equipment llc | 51152/3 | |
6-0 Prolene sutures | Ethicon | 8889H | |
Skin glue-Vetbond Tissue Adhesive | 3M | 1469 | |
Sterile Cotton Tipped Applicators | Dynarex | 4305 | |
WEBCOL Alcohol Prep Pad | Covidien | 6818 | Medium 2 PLY, 200/BOX, Satured with 70% Isopropyl Alcohol |
Curity All Purpose Sponges | Covidien | 9024 | Non-woven 4 PLY, 4"x4" (10.2cm×10.2cm) |
Bench top protector sheet | KIMTECH SCIENCE | 7546 | 18" x 19.5" (45.72cm x 49.53cm) x 50 |
0.9% Sodium Chloride, 250ml | Hospira Inc. | NDC 0409-6138-22 | |
Betadine Solution Swabsticks | Purdue Products L.P. | NDC 67618-153-03 | |
Autoclave | TOMY Digital Biology | SX-700 | HIGH-PRESSURE STEAM STERILIZER |
Slide scanner | HAMAMATSU | NanoZoomer 2.0-RS |