I have used following protocol,

Since it's a lot of work to mount the arteries it's also wise to do an endothelial curve with ACH and subsequently look and the myogenic tone.

Experimental protocol.

Experiments were performed under no-flow conditions. Vascular reactivity was tested with norepinephrine (NE) (10−5 M). Endothelial function was pretested with ACh (10−4 M). Afterward, the function of arteriolar smooth muscle and endothelium was assessed with cumulative doses of ACh (10−9-10−4 M) and sodium nitroprusside (SNP) (10−9-10−4 M) known to be endothelium- dependent and -independent dilatory agents, respectively. ACh and SNP were added after preconstriction with NE 10−5 M. Only preparations with >50% NE-induced vasoconstriction were used. Intravascular pressure was decreased to 3 mmHg after a wash-out period of 30 min, allowing the vessels to equilibrate. For assessment of passive mechanical properties, vessels were deactivated of myogenic tone by perfusion with Ca2+-free Krebs solution containing EDTA 10 mmol/l for 30 min. Then intravascular pressure was increased to 10, 20, 30, and 40 mmHg and afterward in 20-mmHg steps until 140 mmHg. The pressure was maintained at each pressure step until stable conditions were reached to allow the vessel to reach a steady-state diameter. The changes in internal diameter as well as media thickness of vessels in response to each increase in intravascular pressure were measured at three points along the vessel with use of a calibrated video system (Danish Myo Technology).

Regarding the oxygenation question: Oxygenation is important, depending on the gas you use to oxygenate your Krebs solution, your vascular response will be different. Usually people use carbogen (5% CO2 and 95% O2), which is not a gas composition you will find under physiological conditions. Using a mixture of pressurized air and 5% CO2 is more physiological, but still hyperoxic regarding the gas concentrations in your blood. The advantage of carbogen, in my experience, is that the contractions of your vessels will be stronger. :-)

And there is a nice manual from Mulvany and Halpern, explaining the measurement of myogenic tone.

You may wish to look at:

Saul PJ, Rea RF, Eckberg DL, Berger RD, Cohen RJ. Heart rate and muscle sympathetic nerve variability during reflex changes of autonomic activity. Am J Physiol 1990;258:H713-721.

They were unable to directly measure vascular sympathetic tone. However, they looked at power spectral measures of heart rate variability comparing them to two measures of sympathetic outflow, peroneal nerve sympathetic activity and antecubital vein plasma norepinephrine concentrations. From the comparisons I believe they were able to extrapolate peripheral vasomotor activity.

I'm not sure if this is of any help, But it seems to provide an indirect method of evaluating myogenic vascular tone.

It will be so useful if you can give me a detailed protocol from isolating till diameter measurement.

And if the artery is not oxygenated, can we detect the active myogenic tone?

Thanks for your help.

It really depends on the scientific question you are asking. It is my understanding that the protocol that Sebastian has explained will measure pure myogenic tone, which is the active tone as a percentage of the passive tone. these values change at different intraluminal pressures and flow rates. These experiments can be performed ex vivo using a pressure myograph. The protocol listed by Koen does not look at pure myogenic tone. Experiments which use constrictors and dilators are more leaning toward the investigation of vascular reactivity to these agents, and these experiments can be done on both a wire myograph or pressure myograph system ex vivo . Not having oxygenation is not ideal, not only is it an ozygen supply for the tissue, it also maintains the pH of your solution- very important in vascular studies, and to prevent the precipitation of calcium. However, the use of HEPES, can help if you can't get a gas supply to your set-up/ organ bath, but you will need to change your solution at least every 15 minutes!

Hope this helps

I would go to the microcirculatory literature - myogenic tone is a mainstay - and the rigorous laboratories work with pO2 in the vicinity of 'normal' for tissues at rest in vivo - otherwise you are hyperoxic. Many will actually test the ability of the vessels, in vivo and ex vivo to contract to 10 or 15% O2 as in index of viability; maximal relaxation to SNP or 10-4 M ADO is used to assess capacity. Authors to look up are Michael Davis, Brian Duling, Ingrid Sarelius, Bruce Klitzman, Robert Gore, Steve Segal -( to mention a few & yes, there is a bias to the ones I know personally ). Many of these guys have worked with resistance vessels in vivo and in vitro depending on the questions being asked and they have published lots of controls.

Modar - the way you would know whether you could observe tone under conditions of hypoxia would be to use a pharmacologic vasodilator and see if you observe an increase in diameter - but the condition is not physiologic. Some people have used anoxia (no oxygen) to determine the maximal diameter (instead of SNP or ADO or high K+)

Myogenic tone may be assessed in exoerimental animal if one measures perfusion pressure (P) and a regional flow (F). The ratio P/F is the resistance of that vascular bed. If one increases suddenly the perfusion pressure mechanically (e.g.with the occlusion of the aorta downstream the studied artery), the flow immediately increases because of the distension of the artery by the transmural pressure while P/F decreases. After at least 10 s the flow is back to the control and P/S is now increased. This difference in resistance is due to the myogenic contractle response.

If after 10-15 s from the beginning of the occlusion, one releases the aorta, the pressure decreases below the control because the studied vascular bed is under an elevated myogenic tone so that there is a further increase of P/F. About 10-15 s after the realese of the aortic occlusion a transient fall of P/F occurs with a parallel increase in flow. This change in resistance is a measurement of the vasodilator myogenic mechanism. The experiments should be repeated in the presence of chemical denervation of the vascular bed as well as after inhibition of eNOS, or, better, denudation of the same bed.

What factors actually effect the viability and myogenic tone of vessels in no flow/pressure myograph experiment.