Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

Summary

Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.

Abstract

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall¹. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation². In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.

Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental³ and aging studies⁴ can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression⁵ and disease treatment⁶. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process⁷. One drawback of the mouse model, especially for examining young ages, is the size of the arteries.

We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.

Protocol

1. Pressure myograph set-up

1. Summarized from the manufacturer instructions (Danish Myotechnology). A schematic of the complete system with optional accessories is shown in Figure 1.
2. Turn on the air tank and myograph control boxes and start the MyoView software (Danish Myotechnology). In the software, load the appropriate diameter calibration file, inflation protocol file and name the file for saving data for the first protocol. Recommended inflation protocols for each age are given in Table 1.
3. Choose the required cannulae and suture size for the test system depending on the mouse age. Dimensions of the custom-designed stainless steel can....

Discussion

The protocol presented here provides a straightforward and repeatable method for characterizing the passive mechanical behavior of mouse carotid arteries. Although smooth muscle cells and endothelial cells are critical to the function of smaller, muscular arteries and capillaries, they do not contribute significantly to the mechanical behavior of large elastic arteries. Poisoning the cells with KCN has no significant effect on the pressure-diameter behavior of mouse carotid arteries¹⁷. Passive mechanical chara.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of the reagent/equipment	Company	Catalogue number	Comments
Air tank and regulator	Airgas Mid America	UN3156	For pressurizing myograph
Pressure myograph and software	Danish Myotechnology	110P, MyoView	With custom cannulae (Figure 2)
Inverted microscope, 5x lens and camera	Zeiss	Axiovert 40C	For tracking artery diameter
Physiological saline solution (PSS)	Chemicals from Sigma		Recipe and details in Table 2
Surgical tape	Various suppliers		For securing the mouse during dissection
Dissection board	Fisher Scientific	09-002-24A	For securing mouse during dissection
Dissecting microscope with camera	Zeiss	Stemi 2000-C	For arterial dissection and mounting
Dissecting scissors	Fine Science Tools	14058-11	For cutting skin and opening the chest
Fine tweezers (2)	Fine Science Tools	11200-14	For grasping artery ends
Curved forceps	Fine Science Tools	11274-20	For clearing tissue and exposing carotid arteries
Micro-scissors	Fine Science Tools	15005-08	For precise cutting of arteries
7-0 and 10-0 silk suture	Various suppliers		For estimating length and fastening arteries on cannulae
Digital calipers	Fisher Scientific	806-93-111	For measuring suture length and checking artery length
Disposable scalpel	Feather	No. 15	For cutting artery rings
Activated charcoal	Sigma	C4386-500G	For marking cut locations on vessels
18G Needle	Beckton-Dickinson	305136	For applying activated charcoal to vessels, clearing blood and filling myograph tubing
20 mL syringe	Various suppliers		For clearing blood and filling myograph tubing
Petri dish	Fisher Scientific	08-757-13B	For inserting vessels after dissection and testing to take pictures
Microfuge tube	Fisher Scientific	02-682-550	For storing vessels before testing
Fine wire	California Fine Wire Company	100192	For clearing clogged cannula
ImageJ software	National Health Institute	www. rsbweb.nih.gov/ij	Open-source image processing program developed by NIH
Matlab software	Mathworks		Useful for analyzing data and fitting constitutive equations