Summary
This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.
Abstract
The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.
Introduction
Acute coronary syndrome (ACS) is the leading causes of death in the Western world1,2. Acute occlusion of the coronary arteries leads to activation of ischemic cascade and necrosis of the affected cardiac tissue3. Damaged myocardium is gradually replaced by non-contractile scar tissue, which manifestz clinically as a heart failure4,5. Despite recent advances in the treatment of ACS, the prevalence of ACS and ACS-related heart failure is rising, and therapeutic options are limited6,7. Therefore, developing animal models to study ACS and its complications are of immense interest.
To date, the most widely used animal model to study ACS and ACS-induced myocardial remodeling is the ligation of the left descending coronary artery (LAD). Ligation of the LAD leads to acute ischemia of the myocardium, similar to human myocardial tissue during ACS. However, inconsistent infarct sizes remain the Achilles' heel of LAD ligation. Surgical variation and anatomical variability of the LAD lead to inconsistent infarct sizes and hinder the reproducibility and replicability of this procedure8,9,10. In addition, LAD ligation has a high intra- and postsurgical mortality. Despite recent endeavors to improve reproducibility and reduce mortality11,12, large numbers of animals are still needed to properly evaluate anti-remodeling therapies.
Alternative models of ACS have been proposed and studied over the recent years, including radio-frequency13, thermal14 or cryogenic injuries15,16,17,18. Current cryoinjury methods apply a metal rod pre-cooled in liquid nitrogen to damage the subject's cardiac tissue15,16. However, this procedure needs to be repeated several times to generate a sufficient infarct size. Due to the high conductivity and low heat capacity of the rod compared to the tissue, the probe warms quickly, and the tissue is cooled (and thus infarcted) heterogeneously. To overcome these limitations, we describe herein a cryoinfarction model utilizing a hand-held liquid nitrogen delivery system. This model is reproducible, easy to perform and can be established fast and reliably. A reproducible transmural infarct lesion independent of coronary anatomy is generated, which eventually leads to cardiac failure. This method is especially suitable to study the remodeling process for the evaluation of novel therapeutic pharmacological and tissue engineering-based strategies.
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Protocol
Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (the University of California San Francisco (UCSF) Institutional Animal Care and Use Committee).
1. Animal care
- Obtain mice at the age of 14 weeks weighing approximately 27 g (e.g., from the Institute of Laboratory Animals).
NOTE: BALB/c mice are used for this article. - Keep mice under conventional conditions in ventilated cabinets, feeding them standard mice chow and autoclaved water ad libitum.
2. Mouse preparation
- Use an induction chamber to anaesthetize mouse with isoflurane (3.5%).
- Remove the hair over the chest and neck using a hair trimmer.
- Place mouse in supine position on a heated pad and maintain anesthesia with a facemask covering mouth and nose of the mouse.
- Check for sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.
- Inject subcutaneous buprenorphine (0.03 mg/kg) for analgesia.
- Spread the hind and fore limbs and fix their position using tape.
- With povidone iodine, disinfect the shaved area, followed by scrubbing with 80% ethanol. Repeat this step twice.
- Use a small scissor to make a midline skin incision from the lower third of the sternum to the chin.
- Use curved forceps and carefully separate the muscles around the neck to expose the trachea.
- Use a micro-scissor to perform a tracheotomy between the second and third cartilage rings.
- Set the ventilator to a ventilation frequency of 110/min with a tidal volume of 0.5 mL.
- Remove the facemask and insert a plastic cannula (20 G), connected to the ventilator, into the trachea. Ventilate the animal.
NOTE: Ensure that the ventilation cannula is not inserted too deep by confirming bilateral lung ventilation. - Use cautery to detach the right pectoralis muscle from its sternal origin between the third and seventh ribs.
- Use side angled spring scissors to cut the fourth to sixth ribs as close as possible to the sternum.
- Cauterize the mammary artery, if bleeding is visible.
- Decrease isoflurane to 2.5%.
- Dissect underlying connective tissue to obtain a clear view into the chest cavity.
- Use blunt forceps to open the pericardium and expose the heart.
- Use a mini Goldstein retractor to spread the ribs and keep the chest cavity open.
- Lift the heart from the thoracic cavity with a blunt rod.
- Decrease the tension of the retractor to reduce chest opening and to keep the heart from falling back.
- Precool the cryoprobe (3 mm diameter) for 10 s.
- Apply the cryoprobe on the anterior left ventricle wall and freeze for 10 s to generate a left ventricular cryo-injury infarct.
NOTE: The cryoprobe can be applied to different heart walls depending upon the scientific question and need. - Irrigate the cryoprobe with room temperature saline to detach the probe from the left ventricular wall.
- Use the retractor to enlarge the chest opening.
- Gently return the heart to the thoracic cavity with a blunt rod.
- Remove the retractor and connect the sternotomy with a single knot using 6-0 suture.
- Close the chest cavity using 6-0 running suture. Use a 10 mL syringe to evacuate any remaining air from the chest before tying the knot.
- Adapt the skin at the caudal edge and suture it to the point of the tracheal opening with running suture (5-0).
- Set isoflurane to 1.5% and wait until the animal gains spontaneous breathing.
- Remove the tracheal catheter and reapply the facemask onto the animal mouth and nose to maintain anesthesia.
- Close the tracheal incision with one 8-0 suture.
- Reposition the ventral neck muscles back to their position to cover the trachea.
- Complete the skin suture.
- Add metamizole to the drinking water (50 mg metamizole per 100 mL) for pain analgesia for 3 days and monitor the animal daily.
NOTE: The observation period for this model is 8 weeks. Be sure to follow your institution's guidelines regarding analgesia regimen.
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Representative Results
The cryoinjury infarct model is suitable to study ACS and its complications. Low mortality rates and efficient postsurgical recovery is seen in this model. Cryoinjury induced myocardial damage leads to reduced cardiac function, electrical uncoupling, and transmural remodeling.
Echocardiography can be used to monitor cardiac function noninvasively in vivo. In cryo-injured hearts, echocardiography demonstrates significantly reduced ejection fraction and fractional area change (Figure 1a-c). Functional impairment continues from day 7 post-surgery until the observational endpoint of 56 days.
Detailed cardiac function can be assessed invasively through pressure volume loop (PV-loop) analysis. A 1.2 Fr conductance catheter is introduced into the left ventricle, and the left ventricular pressure is plotted against the left ventricular volume. Hemodynamic parameters such as stroke volume, stroke work, cardiac output, and preload-adjusted maximal power can be calculated. As shown in Figure 1d-h, cryoinfarction leads to impaired left ventricle (LV0 function, which is reflected as a decrease in stroke volume, stroke work, cardiac output and preload-adjusted maximal power.
To study cardiac electrophysiology, optical mapping can be performed ex vivo. Hearts are removed, perfused with Langendorff perfusion technique, and stained with a fluorescent voltage sensitive dye. Cryoinjured hearts demonstrate blockage of electrical conduction at the border of injury, indicating local electrical uncoupling (Figure 1i).
Histological staining with Masson's trichrome demonstrates transmural fibrotic tissue formation at the site of injury (Figure 2a). Infarct size can be calculated by measuring infarct scar area or midline scar length19 (Figure 2b). Immunofluorescence staining against alpha- sarcomeric actinin (cardiomyocyte marker) and collagen-I confirm fibrotic remodeling and loss of cardiomyocytes at the site of injury (Figure 2c).
Figure 1: Functional and electrophysiological analysis of cryoinjured heart. Representative two-dimensional echocardiography images taken pre-operatively (D0) and at post-operative day 7 (D7), 28 (D28), and 56 (D56). (a) The top panel shows the parasternal long-axis view at end-diastole and the bottom panel at end-systole. (b, c) Ejection Fraction (EF) and Fractional Area Change (FAC) decrease after cryo-infarction and remained diminished over time Cardiac function was assessed invasively by pressure volume curve analysis. (d-g) Day 56 post injury stroke volume (SV), stroke work (SW), cardiac output (CO), and preload-adjusted maximal power (PAMP) were significantly lower than in pre-operative native animals. (h) Representative PV-loops from native and 56 days post-surgery animals showed characteristic right shift and decline in amplitude of the pressure signal following thoracic vena cava (TVC) occlusion. (i) Isochrone map of cardiac optical mapping from native and cryoinjured hearts 14 days post-surgery. Top and bottom panels show hearts paced from the apex and base, respectively. Infarct area is marked by dashed white line. Intergroup differences were assessed by one-way analysis of variance (ANOVA) with Bonferroni's post-Hoc test or Student’s t-test. N = 3 animals. * indicates p < 0.05. The error bars represent the standard deviation (SD). ESPVR = end-systolic pressure volume relationship; EDPVR = end-diastolic pressure volume relationship. Please click here to view a larger version of this figure.
Figure 2: Histologic assessment of native and cryo-injured hearts. (a) Masson’s trichrome staining shows collagen deposition (green) in the infarcted area. The infarcted percentage of the left ventricle was measured as (b) area and (c) midline infarct length. (d) Immunofluorescence staining demonstrates increased collagen-I deposition with concomitant loss of cardiomyocytes in infarcted area. LV = left ventricle; RV = right ventricle; endo = endocardial; epi = epicardial. N = 3 animals. Error bars show SD. Please click here to view a larger version of this figure.
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Discussion
This article describes a mouse cryoinjury model to investigate ACS and related pharmacological and therapeutic options.
The most crucial step is the application of the cryoprobe on the cardiac tissue. Contact duration must be tightly controlled in order to obtain the optimal infarct size and to guarantee reproducible results. Prolonged cooling of the myocardium will lead to oversized infarcts or ventricular perforation. In contrast, shortened cooling time generates limited epicardial lesions and does not eliminate all resident cells. Hence, this can be confounding when studying regenerative cell transplantation.
Compared to other cryoinfarction methods20, the open chest approach described in this article has the advantage that the infarct can be induced freely on different positions of the heart. Moreover, this approach facilitates therapeutic cell injection or patch applications, as the infarct border is visible, and the site of cell transplantation can be chosen accordingly.
A drawback of this model is the etiology of myocardial injury. Cryoinjury results in cell death due to the generation of ice crystals disrupting the cell membrane rather than a direct ischemia. In addition, the direction of injury is usually from epicardium inwards, whereas ischemic infarcts tend to propagate outwards from the endocardial to the epicardial layer. Therefore, this model is limited to study the pathophysiological mechanisms of myocardial ischemia or to imitate the ischemia-reperfusion setting.
In conclusion, the model described here is inexpensive, easy to perform, can be established fast and reliably. Cardiomyocyte necrosis and subsequent scar formation develop over time, resulting in progressive impaired pump function and electrical conductance. Well-controllable infarct size, shape and location make this model ideal to evaluate experimental interventions aiming to restore cardiac function or cardiac regeneration. Successfully tested treatment options should be further confirmed in large animal studies.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
We thank Christiane Pahrmann for her technical assistance. D.W. was supported by the Max Kade Foundation. T.D. received grants from the Else Kröner Fondation (2012_EKES.04) and the Deutsche Forschungsgemeinschaft (DE2133/2-1_. S. S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; SCHR992/3- 1, SCHR992/4-1).
Materials
| Name | Company | Catalog Number | Comments |
| 10 ml Syringe | Thermo Scientific | 03-377-23 | |
| 5-0 prolene suture | Ethicon | EH7229H | |
| 6-0 prolene suture | Ethicon | 8706H | |
| 8-0 Ethilon suture | Ethicon | 2808G | |
| Absorption Spears | Fine Science Tools | 18105-01 | |
| BALB/c | The Jackson Laboratory | Stock number 000651 | |
| Bepanthen Eye and Nose ointment | Bayer | 1578675 | Eye ointment |
| Betadine Solution | Betadine Purdue Pharma | NDC:67618-152 | |
| Blunt Forceps | Fine Science Tools | 18025-10 | |
| Buprenex | Reckitt Benckiser | NDC Codes: 12496-0757-1, 12496-0757-5 | Buprenorphine |
| Cryoprobe 3mm | Brymill Cryogenic Systems | Cry-AC-3 B-800 | |
| Ethanol 70% | Th. Geyer | 2270 | |
| Forceps curved | S&T | 00284 | |
| Forceps fine | Fine Science Tools | 11251-20 | |
| Forceps standard | Fine Science Tools | 11023-10 | |
| Gross Anatomy Probe | Fine Science Tools | 10088-15 | |
| Hair clipper | WAHL | 8786-451A ARCO SE | |
| High temperature cautery kit | Bovie | 18010-00 | |
| ISOFLURANE | Henry Schein Animal Health | 029405 | |
| IV Catheter 20G | B. Braun | 603028 | |
| Mini-Goldstein Retractor | Fine Science Tools | 17002-02 | |
| NaCl 0.9% | B.Braun | PZN 06063042 Art. Nr.: 3570160 | saline |
| Needle holder | Fine Science Tools | 12075-14 | |
| Needle Holder, Curved | Harvard Apparatus | 72-0146 | |
| Novaminsulfon | Ratiopharm | PZN 03530402 | Metamizole |
| Operating Board | Braintree Scientific | 39OP | |
| Replaceable Fine Tip | Bovie | H101 | |
| Scissors | Fine Science Tools | 14028-10 | |
| Small Animal Ventilator | Kent Scientific | RV-01 | |
| Spring Scissors - Angled to Side | Fine Science Tools | 15006-09 | |
| Surgical microscope | Leica | M651 | |
| Transpore Surgical Tape | 3M | 1527-1 | |
| Vannas Spring Scissors | Fine Science Tools | 15400-12 | |
| Vaporizer | Kent Scientific | VetFlo-1205S |
References
- Writing Group. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 133 (4), 38-360 (2016).
- de Alencar Neto, J. N. Morphine, Oxygen, Nitrates, and Mortality Reducing Pharmacological Treatment for Acute Coronary Syndrome: An Evidence-based Review. Cureus. 10 (1), 2114 (2018).
- Detry, J. M. The pathophysiology of myocardial ischaemia. European Heart Journal. 17, Suppl G 48-52 (1996).
- Ertl, G., Frantz, S. Healing after myocardial infarction. Cardiovascular Research. 66 (1), 22-32 (2005).
- Jugdutt, B. I. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough. Circulation. 108 (11), 1395-1403 (2003).
- Velagaleti, R. S., Vasan, R. S. Heart failure in the twenty-first century: is it a coronary artery disease or hypertension problem. Cardiology Clinics. 25 (4), 487-495 (2007).
- Benjamin, E. J., et al. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation. 135 (10), 146-603 (2017).
- Morrissey, P. J., et al. A novel method of standardized myocardial infarction in aged rabbits. American Journal of Physiology-Heart and Circulatory Physiology. 312 (5), 959-967 (2017).
- Degabriele, N. M., et al. Critical appraisal of the mouse model of myocardial infarction. Experimental Physiology. 89 (4), 497-505 (2004).
- Chen, J., Ceholski, D. K., Liang, L., Fish, K., Hajjar, R. J. Variability in coronary artery anatomy affects consistency of cardiac damage after myocardial infarction in mice. American Journal of Physiology-Heart and Circulatory Physiology. 313 (2), 275-282 (2017).
- Reichert, K., et al. Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction. Journal of Visualized Experiments : JoVE. (122), e55353 (2017).
- Kim, S. C., et al. A murine closed-chest model of myocardial ischemia and reperfusion. Journal of Visualized Experiments : JoVE. (65), e3896 (2012).
- Antonio, E. L., et al. Left ventricle radio-frequency ablation in the rat: a new model of heart failure due to myocardial infarction homogeneous in size and low in mortality. J Card Fail. 15 (6), 540-548 (2009).
- Ovsepyan, A. A., et al. Modeling myocardial infarction in mice: methodology, monitoring, pathomorphology. Acta Naturae. 3 (1), 107-115 (2011).
- Ciulla, M. M., et al. Left ventricular remodeling after experimental myocardial cryoinjury in rats. Journal of Surgical Research. 116 (1), 91-97 (2004).
- Grisel, P., et al. The MRL mouse repairs both cryogenic and ischemic myocardial infarcts with scar. Cardiovascular Pathology. 17 (1), 14-22 (2008).
- Duerr, G. D., et al. Comparison of myocardial remodeling between cryoinfarction and reperfused infarction in mice. Journal of Biomedicine and Biotechnology. 2011, 961298 (2011).
- Ma, N., et al. Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells. Cardiovascular Research. 71 (1), 158-169 (2006).
- Takagawa, J., et al. Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches. Journal of Applied Physiology (1985). 102 (6), 2104-2111 (2007).
- 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. American Journal of Physiology-Heart and Circulatory Physiology. 289 (3), 1291-1300 (2005).
