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.
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.
1. Pressure myograph set-up
2. Carotid dissection
3. Mounting an artery in the test system
4. Testing protocols
5. Unloaded dimensions and opening angles
6. Data analysis
7. Clean up
8. Representative Results
All results are shown for C57BL6J 3-day-old mouse carotid arteries. Example closed and open rings are shown in Figure 7 for calculating unloaded dimensions and opening angles12. Example raw pressure, diameter and force data versus time are shown in Figure 8. Single loading cycles with no artifacts are isolated from this data for each protocol (Figure 9), which can then be used for further calculations, analyses and modeling to determine differences in the mechanical behavior between arteries3,11,15,16.
Approximate age (days) | Max pressure (mmHg) | Wait time (sec) | Pressure steps (mmHg) | Deflation time (sec) | Suture length (mm) |
3 | 90 | 8 | 9 | 90 | 3 |
7 | 120 | 9 | 12 | 90 | 3.5 |
14 | 140 | 10 | 14 | 90 | 4.5 |
21 | 160 | 11 | 16 | 90 | 5 |
30 and above | 180 | 12 | 18 | 90 | 6 |
Table 1. Recommended automatic myograph inflation protocols and suture lengths for different age specimens. The maximum pressure avoids damage to the artery from overinflation, while capturing the nonlinear mechanical behavior, and ranges from 1.5 to 3 times the systolic pressure for each age3. The wait time provides enough time for the system to stabilize at each pressure and allows operator intervention if necessary to correct tracking problems. The pressure steps provide ten steps for each inflation cycle and an overall rate of 1 – 2 mmHg/sec from a starting pressure of 0 mmHg. This is considerably slower than the physiologic loading rate in an adult mouse artery (about 330 mmHg/sec for a 40 mmHg pulse pressure at 600 bpm), but soft biologic tissues are generally insensitive to loading rates over about three orders of magnitude10. Preliminary data showed no differences between mechanical behavior of mouse arteries when pressurized at the maximum rate of the system (approximately 7 mmHg/sec) and the rates listed here11. The deflation time allows the artery to fully return to the starting dimensions, while minimizing total cycle time. The suture length is approximated from the available carotid artery length at each age.
Chemical | Conc (mM) | For 1 Liter (g) |
NaCl | 130 | 7.6 |
NaHCO3 | 15 | 1.25 |
Dextrose | 5.5 | 1 |
KCl | 4.7 | .35 |
MgSO4-7H2O | 1.2 | .29 |
KH2PO4 | 1.2 | .16 |
EDTA | .026 | .01 |
CaCl2 solution, pH 7.2 | 1.6 | 1.6 mL of 1M solution |
Table 2. Recipe for physiological saline solution (PSS). All chemicals from Sigma. PSS can be stored in the refrigerator for up to 3 days.
Figure 1. Schematic of the mechanical test system with optional accessories. Used with permission from Danish Myotechnology.
Figure 2. Dimensions of the custom-made cannulae for different aged specimens. All parts are made from 316 stainless steel hypodermic tubing (Small Parts). 7-0 suture should be used to secure the arteries to the large cannulae and 10-0 suture should be used for the small cannulae.
Figure 3. Diagram of the carotid artery dissection. The heart, aorta, right and left common carotid arteries and cut locations for the arteries are shown.
Figure 4. Images of a 3-day-old right common carotid artery in vivo (A) and ex vivo (B). The reference length is determined with a suture and the cut locations are marked with carbon particles. Note the decrease in length upon dissection. Scale bar = 0.25 mm.
Figure 5. 3-day-old mouse carotid artery mounted on the cannulae in the myograph bath at its unstretched, ex vivo length. Scale bar = 0.2 mm.
Figure 6. Screen shot of the MyoView software showing outer diameter tracking of a 3-day-old carotid artery stretched to 1.2 times the in vivo length and pressurized to 0 mmHg (A) and 90 mmHg (B).
Figure 7. Example closed (A) and open (B) rings of a 3-day-old carotid artery cut to measure unloaded dimensions and opening angle. Scale bar = 0.1 mm.
Figure 8. Example raw pressure, diameter and force data versus time for a single inflation protocol for a 3-day-old carotid artery.
Figure 9.Example isolated loading cycles showing the recorded pressure, diameter, force and calculated axial stretch for all six mechanical test protocols for a 3-day-old carotid artery. The left panels show the inflation protocols at constant axial stretch (λz) with respect to the in vivo length and the right panels show the axial stretch protocols at constant pressure (P, mmHg). The manual axial stretch protocols are performed at a faster rate than the automated inflation protocols, so less data points are recorded.
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 characterization is critical for determining the effects of changes in the matrix composition of large elastic arteries due to development, aging, disease or injury and the subsequent effects on cardiac and cardiovascular function. Mechanical characterization can be used to better understand the developmental process and recreate tissue-engineered arteries with appropriate mechanical properties. It can also be used to test disease treatments aimed at reversing mechanical changes that compromise human health.
Commercial myograph systems have been used to obtain pressure-diameter relationships for large, elastic arteries8 and to investigate contractility and smooth muscle cell function in arterioles18 from adult mice. More complete mechanical characterization of these vessels has been prevented by the use of traditional glass cannulae that do not allow substantial axial stretch of the vessel. Axial stretch is important for duplicating the in vivo configuration of the arteries and some large elastic arteries, such as the abdominal aorta, have in vivo stretch ratios of up to 1.711. We have designed custom stainless-steel cannulae that can be used for mechanical testing of carotid arteries from mice aged 3 days to adult3. The cannulae allow substantial axial stretch and are less prone to breakage than glass. The smallest size can get clogged by residual salts or vessel pieces, but these can be removed by insertion of a fine wire into the cannula tip and mostly prevented by thorough cleaning after each experiment. Limitations of the Danish Myotechnology myograph system used in this protocol include the lack of automated axial stretch and that only step changes in pressure can be programmed into the software.
The data gathered in this protocol, namely in vivo axial stretch, unloaded dimensions, opening angle and pressure, diameter, force and axial stretch for multiple inflation and stretch protocols, provides the required information to rigorously characterize the passive arterial mechanical behavior. The protocol takes more time and effort than a single pressure-diameter or contractility protocol, but provides enough information to compare the behavior for any loading condition and may highlight subtle differences in mechanical behavior that are not apparent in traditional pressure myograph experiments. Microstructurally-based constitutive equations can be used to correlate the mechanical behavior with measured differences in matrix protein amounts or organization13-16.
The protocol characterizes the in vitro passive mechanical behavior of carotid arteries, hence care must be taken to extrapolate any results to the in vivo condition. In vivo, the loading rate will be higher, there will be blood flowing through the artery, the artery will be tethered by surrounding tissue, and the artery will be exposed to a variety of chemical stimuli. The protocol is applied to large, elastic arteries, but can be modified to any vessel type suitable for the cannulae. Different buffers and bath additives may be used to isolate passive and active behavior for the carotid artery and for more muscular, contractile vessels. We have used the protocol for vessels ranging in inner diameter from 150 – 1000 μm and 1.5 – 8 mm in length. As the vessels decrease in length, end effects due to constraints at the suture ties will affect the results. We have performed a finite element study with estimated nonlinear mechanical properties to show that the stresses are homogeneous over at least 60% of the length when the length:diameter ratio is at least 2.519.
The authors have nothing to disclose.
This work was funded, in part, by NIH grants HL087653 and HL105314. Some of the methods described in this work were developed in the laboratory of Dr. Robert Mecham at the Washington University School of Medicine.
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 |