An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.
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Method Article
Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography
In This Article
Summary
Abstract
Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 μm. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.
Protocol
1. Isolation of rat mesenteric small artery
- Anesthetize animal with isoflurane in a closed chamber and wipe the abdomen with alcohol.
- Perform a mid-line laparotomy to expose mesenteric bed.
- Using scissors, remove about 85 cm of intestine with feeding vasculature with the superior mesenteric artery. Cut the proximal end of intestinal section close to the pylorus and the distal end near the ileo-coecal junction. Segments of the intestine are isolated from rats that are deeply anesthetized with isoflurane and euthanized by open-chest cardiac puncture.
- Placed excised section in a coated petri dish containing PSS and perform the di....
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Discussion
Hypertension is a leading cause of cardiovascular, cerebral and renal morbidity/mortality. The occurrence of hypertension is high in the population and salt-sensitive hypertension is particularly high in the aging population, and more prevalent in blacks than whites. This is believed to be due to the tendency of blacks to retain sodium in their kidneys9. Salt-sensitivity is a major contributing factor to kidney disease and is associated with endothelial dysfunction, but the mechanism is not fully understood. R.......
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Disclosures
No conflicts of interest declared.
Acknowledgements
The project described was supported by Award Numbers R01 HL064761, R25 HL059868, 1SC1 HL099139 and P20 MD000175 form National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Auto Dual Wire Myograph System-510ADMT-USA, Inc. | Atlanta, GA. | 100151 | |
| PowerLab/4SP Data Acquisition System |  ADInstruments | ML750 | New models with 4-16 input channels are available. |
| Dell Dimension XPS Gen 4 Computer | Dell | ||
| Stemi SV II (Apo) Dissection Microscope with Ocular | Carl Zeiss, Inc. | ||
| Wistar, Dahl salt-sensitive, Dahl salt-resistant rats | Harlan Laboratories | ||
| Rodent chow | Harlan Laboratories | ||
| Phenylephrine | Sigma-Aldrich | ||
| Other chemicals | All chemicals used were of the purest grades available commercially. |
References
- Mulvany, M. J., Halpern, W. Mechanical properties of vascular smooth muscle cells in situ. Nature. 260, 617-619 (1976).
- Bevan, J. A., Osher, J. V. A direct method for recording tension changes in the wall of small blood vessels in vitro. Agents Actions.
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