Our group recently established a protocol to perform endothelial-dependent vasodilation studies using a DMT pressure myograph system. We can obtain vasodilation of 60-100%, but the response to acetylcholine varies quite a bit from one experiment/artery to another. Others in the literature show vasodilation of 80-100% with tighter error bars compared to our results.
I've attached data that we have collected to illustrate this point. Carotid #1 and #2 are two carotids dissected from the same mouse. Attempts 1-3 are vasodilation experiments performed within the same carotid artery. The cumulative average graph is the results of all of our successful vasodilation experiments (n=5). Note the large error bar at 10-7 M acetylcholine.
We are using male C57Bl/6 mouse carotid arteries between the ages of 12-23 weeks old. The HEPES-PSS pH is maintained between 7.38-7.42 at 37C. We gradually increase the pressure to 50mmHg over 20 minutes, followed by an additional 40 minutes of equilibration. We typically obtain KPSS constriction between 10-15%. All vessels are dissected within 10 minutes using ice cold HEPES-PSS. Experiments are peformed either 6-8 hours or ~24 hours after dissection.
Does anyone have advice to minimize vasodilation variability? How long do you wait between vasodilation experiments, and how many vasodilation experiments do you perform on one carotid artery?
Thank you for your response!
1. We're using pressure myography so the vessels are mounted on stainless steel cannulas. I believe they are 400um in diameter. I try to only touch the ends of the carotid, unless I need to remove connective tissue in my region of interest.
2. Yes, we're using a DMT pressure myograph system, Model 114P.
3. The vessels are dissected and mounted on cannulas and not cut into rings.
4. We use KPSS to test vessel viability and phenylephrine to pre-constrict the vessel prior to acetylcholine treatment. The force increases as the vessel constrict. We typically wait 5 minutes until the constriction diameter plateaus.
5. I will try this on our next experiment. I make 1mL aliquots and freeze them in -80C. I dilute the aliquots to appropriate concentrations on the day of the experiment.
6. We have done experiments to show that the myograph chamber pH steadily increases over time, thus we change the PSS every 15 minutes. The PSS solution is gased with carbogen. (5% CO2, 95% O2).
7. I will keep a closer eye on the force readings. We wait 5 minutes after phenylephrine application because we notice the diameter plateaus.
8. Is there a way I can minimize myogenic tone?
9. We use phenylephrine to pre-constrict vessels.
10. I'll continue to do experiments to get an idea of relaxation.
11. I have noticed that at the lower acetylcholine concentrations that the relaxation is transient.
12. We have used nononate and noticed 100% vasodilation (sometimes greater than 100%).
Do you have additional recommendations based on these responses?
Thanks again for your help!
We expose the carotids to 50mmHg based on what others have done in the literature. I've used 100mmHg, but the carotids don't dilate well. We make sure that there are no branches and the carotids are dissected from the same location from all animals (below bifurcation to the top of the clavicle). Both HEPES-PSS and KPSS are gased with carbogen to maintain pH.
I need to keep a closer eye on the force generated between the vessels. I will also try the phenylephrine and nononate dose-response experiments.
How many consecutive dose-response experiments have you successfully performed? How much time do you wait between dose-dependent experiment? I worry that I am moving too fast from one experiment to another.
Thank you for taking the time to help me.
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