Mouse Model of Coronary Collateral Growth Through Repetitive Ischemia

Summary

This paper presents a method to study postnatal coronary collateral growth induced by repetitive ischemia in mice, including the surgical implantation of a pneumatic occluder on the left anterior descending artery, an automated inflation system for the repetitive ischemia protocol, and potential methods to evaluate collateral growth.

Abstract

Coronary collaterals are a natural bypass in ischemic heart diseases (IHD), and so for many years, coronary collateral growth (CCG) has been a promising therapeutic target for IHD, particularly in patients with type 2 diabetes or metabolic syndrome in which CCG is impaired. However, this process is understudied, partly due to the lack of mouse models of CCG, even though other animal models, such as pigs, dogs, and rats, have been established. A mouse model can take advantage of the many genetic modifications available for the species, including lineage tracing and gene regulation (overexpression or knockout), to elucidate the process and mechanism of CCG, including the pathways and cell types involved.  We, therefore, set out to develop a mouse model of CCG induced by repetitive ischemia (RI) via transient, repetitive occlusion of the left anterior descending artery (LAD). This manuscript provides details of this mouse CCG model, including the RI surgery to implant a pneumatic occluder on the LAD, the automated pressure-based inflation system used for controlling the pressure and timing of inflation, and the sequence of the RI protocol. This method has already generated one publication to elucidate the process of CCG induced by RI, showing that sprouting angiogenesis gives rise to mature coronary arteries in CCG in adult mouse hearts.

Introduction

Ischemic heart disease (IHD) is the leading cause of mortality in the United States, and more than 200,000 coronary artery bypass surgeries are performed annually in an effort to treat the disease¹. Coronary collaterals, anastomoses between branches of the coronary arterial tree, are a natural bypass that can resupply blood to ischemic tissue downstream of a blockage²; however, people exhibit a wide variation in the extent of their native collateral networks^3,4. Patients with IHD who have more extensive coronary collateralization have better outcomes during cardi....

Protocol

The described animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Northeast Ohio Medical University.

1. Surgical preparation

NOTE: For the RI protocol, use C57BL/6 mice of either sex weighing at least 25 g. Use aseptic technique throughout the surgery.

1. Setup
   1. Sterilize all tools in an autoclave or bead sterilizer. Sterilize delicate materials and implants, such as occluder, tether, and PE tubing, with ethylene oxide (EtO).
   2. Clean the surgic....

Results

Out of 136 C57BL/6 mice, including both males and females, the survival rate of the RI surgery was 93.4%, with 80.9% of mice surviving through the entire 17-day RI protocol.

The mouse RI protocol was optimized based on previous animal RI models^12,16, which have short episodes of ischemia without permanent injury to the myocardium. During the surgery, functional assessment of the occluder can be done by observing visible blanching of th.......

Discussion

Coronary collaterals are a natural bypass for IHD patients. After the failed clinical trials targeting angiogenesis¹⁷, promoting coronary collateral development might be a better therapeutic approach for these patients. Different from capillaries derived from angiogenesis, which have only a single layer of endothelial cells, collaterals are mature arteries with the coverage of smooth muscle cells. Collaterals resupply blood flow to the regions of myocardial ischemia caused by obstructive arteries........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#5/45 degree forceps	Fine Science Tools	11251-35
1/4" Closed Brass Electric Solenoid Valve	U.S. Solid	USS2-00054	inflation system
1/4" Open Brass Electric Solenoid Valve	AceCrew		inflation system
1/4" pneumatic tubing	‎China SNS Pneumatic Co.,Ltd	‎APU1/4-32.8ft	push-button device
1/4" push-in connectors	RuoFeng	543Y	push-button device
1/8" brass fittings	Edge Industrial		inflation system
2 Position Pneumatic Electric Solenoid Valve	U.S. Solid	USS- PSV00033	push-button device
20G angiocath	BD	381703
45 degree Castroviejo needle holders	Roboz	RS-6421
6-0 polyglactin sutures	DemeTECH	G176011B13M
6-0 polypropylene sutures	AD Sugical	XS-P618R11
70% Ethanol
8-0 polypropylene sutures	DemeTECH	PM19800, 65G0P
Betadine	Purdue Products	367618150085
Blunt nosed scissors	World Precision Intruments	500366
Carbon fiber arrow shaft			post-surgical cage; cut to 12.5 cm
Cotton swabs (3")	Puritan	872-PC DBL
Curity Gauze Sponges (2x2)	Cardinal Health	2146
Dipsey swivel sinkers	Water Gremlin		post-surgical cage
Electrode cream	Signacreme	17-05
Glycopyrrolate	Westward	0143-9679-01
Hartman hemostats	Fine Science Tools	13003-10
Isoflurane	Covetrus	29404
Ketofen (ketoprofen)	zoetis	10004031
Lidocaine (2%)	Covetrus	14583
MICROFIL (yellow)	Flow Tek	MV-122
Mini Push Button	Interactivia	E-SWC-PBM-PBS-105	push-button device
Miniature Air Pressure Regulator	PneumaticPlus	PPR2-N02BG-4	push-button device
Mini-Colibri spring retractor	Fine Science Tools	17000-01
MiniVent ventilator	Harvard Apparatus	73-0044
Occluder			Custom made
Octagon handled forceps	Fine Science Tools	11041-08
Ohan Rodent Intubation System	BMR Supply	Ohan-201
Paraformaldehyde solution 4% in PBS	Santa Cruz	sc-281692
PE20 tubing
PE50 tubing
Plastic swivel (1 channel)	Instech	375/25PS	post-surgical cage
Premixed PBS Buffer, 10x	Roche	11666789001	Diluted to 1x
Pressure Gauge	PIC Gauges	102D-158D-10/32	push-button device
Programmable Digital Outlet Timer	BN-LINK	‎BND-60/SU105	inflation system
Puralube Vet Opthalmic Ointment	Dechra	17033-211-38
Retractors w/ 18200-07 elastomer	Fine Science Tools	18200-10 and 18200-11
Rodent Surgical Monitor+	Scintica	900-0053-01
Round handled suture tying forceps	Fine Science Tools	18026-10
Snap-lock barrel swivel (size 5)	Eagle Claw	‎01032-005	post-surgical cage
Straight needle holders	Fine Science Tools	12060-01
Tether	Instech	PS62