Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Summary

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Abstract

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Introduction

Heart disease is the leading cause of death for both men and women in the United States, accounting for 600,000 deaths annually¹. Murine models of cardiovascular disease are critically important for investigating pathophysiological mechanisms and for exploring potential therapies. Myocardial delivery of gene therapy vectors, stem cells, modified RNAs, and other therapeutic agents permits investigation of their therapeutic potential for heart disease^2-7. Currently, there are limited options for myocardial delivery of therapeutic agents in mouse models⁶. Intramyocardial injection under direct visualization is commonly used, but requires ....

Protocol

All described steps were performed under protocols approved by the Institutional Animal Care and Use Committee of Boston Children’s Hospital.

1. Preparation

1. Perform baseline cardiac anatomical and functional assessment by echocardiography prior to beginning the injection protocol, as the optimal fixed transducer position for intramyocardial injection may not yield the optimal standard views for delineating anatomy and assessment of function.
2. The injection setup is shown in Figures 1A-1C. Place an empty syringe with a sheathed needle, bevel oriented upwards, in the syringe clamp (see Figur....

Results

Murine Intramyocardial Injection with Blue Dye or Fluorescent Microspheres

Injection of Evan’s blue dye is useful for training purposes. Soon after injection, euthanize the mouse and remove the heart to visualize the location of the injected blue dye. Figure 5 shows an example of a successful injection, with blue dye infiltrating the myocardium at the mid-papillary muscle level (Figure 5A, region enclosed by dotted line). Evans blue dye will wash out of the tissu.......

Discussion

Biologics can be delivered to the myocardium by direct intramyocardial injection, intrapericardial injection, or indirect administration via the bloodstream. Recent cell based therapy trials in myocardial infarction models have described an open thoracotomy approach to the delivery of injectate^12-14. An important factor in the success of a myocardial therapeutic intervention hinges on the choice of delivery route. The highest local dose of biologic is achieved by intramyocardial delivery^15,16. Intra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vevo 2100 ultrasound imaging system	Visualsonics
Vevo Integrated Rail System III	Visualsonics
Microscan MS400 transducer	Visualsonics
Microscan MS550D transducer	Visualsonics
PrecisionGlide needles	BD	305128	30 G x 1 in/2.5 cm
1 cc slip tip syringe	Exel International	26048	or equivalent
Gastight 50 μl glass syringes	Hamilton	1705
Trypan blue stain (0.4%)	Gibco	15250	or equivalent
Isoflurane	Baxter	AHN3640	or equivalent
Aquasonic 100	Parker Laboratories	(01-08)	or equivalent
Polystyrene microspheres (red fluorescent)	Life Technologies	F-8842	or equivalent