A Cryoinjury Model to Study Myocardial Infarction in the Mouse

Podsumowanie

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Streszczenie

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.

Wprowadzenie

Acute coronary syndrome (ACS) is the leading causes of death in the Western world^1,2. 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 failure^4,5. Despite recent advances in the treatment of ACS, the prevalence of ACS and ACS-related heart failure is rising, and therapeutic options are limited^6,7. 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 procedure^8,9,10. In addition, LAD ligation has a high intra- and postsurgical mortality. Despite recent endeavors to improve reproducibility and reduce mortality^11,12, 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 injuries^15,16,17,18. Current cryoinjury methods apply a metal rod pre-cooled in liquid nitrogen to damage the subject's cardiac tissue^15,16. 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.

Protokół

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

1. 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.
2. Keep mice under conventional conditions in ventilated cabinets, feeding them standard mice chow and autoclaved water ad libitum.

2. Mouse preparation

1. Use an induction chamber to anaesthetize mouse with isoflurane (3.5%).
2. Remove the hair over the chest and neck using a hair trimmer.
3. Place mouse in supine position on a heated pad and maintain anesthesia with a facemask covering mouth and nose of the mouse.
4. Check for sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.
5. Inject subcutaneous buprenorphine (0.03 mg/kg) for analgesia.
6. Spread the hind and fore limbs and fix their position using tape.
7. With povidone iodine, disinfect the shaved area, followed by scrubbing with 80% ethanol. Repeat this step twice.
8. Use a small scissor to make a midline skin incision from the lower third of the sternum to the chin.
9. Use curved forceps and carefully separate the muscles around the neck to expose the trachea.
10. Use a micro-scissor to perform a tracheotomy between the second and third cartilage rings.
11. Set the ventilator to a ventilation frequency of 110/min with a tidal volume of 0.5 mL.
12. 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.
13. Use cautery to detach the right pectoralis muscle from its sternal origin between the third and seventh ribs.
14. Use side angled spring scissors to cut the fourth to sixth ribs as close as possible to the sternum.
15. Cauterize the mammary artery, if bleeding is visible.
16. Decrease isoflurane to 2.5%.
17. Dissect underlying connective tissue to obtain a clear view into the chest cavity.
18. Use blunt forceps to open the pericardium and expose the heart.
19. Use a mini Goldstein retractor to spread the ribs and keep the chest cavity open.
20. Lift the heart from the thoracic cavity with a blunt rod.
21. Decrease the tension of the retractor to reduce chest opening and to keep the heart from falling back.
22. Precool the cryoprobe (3 mm diameter) for 10 s.
23. 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. 
24. Irrigate the cryoprobe with room temperature saline to detach the probe from the left ventricular wall.
25. Use the retractor to enlarge the chest opening.
26. Gently return the heart to the thoracic cavity with a blunt rod.
27. Remove the retractor and connect the sternotomy with a single knot using 6-0 suture.
28. 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.
29. Adapt the skin at the caudal edge and suture it to the point of the tracheal opening with running suture (5-0).
30. Set isoflurane to 1.5% and wait until the animal gains spontaneous breathing.
31. Remove the tracheal catheter and reapply the facemask onto the animal mouth and nose to maintain anesthesia.
32. Close the tracheal incision with one 8-0 suture.
33. Reposition the ventral neck muscles back to their position to cover the trachea.
34. Complete the skin suture.
35. 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. 

Wyniki

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 (

Dyskusje

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 a...

Materiały

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