בדיקה מכנית של העורקים עכבר הראש: מ יילוד כדי למבוגרים בלבד
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
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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