Hi Mudhir, it could be due to one of several simple reasons. 

Presuming that endothelium is not damaged during mounting of the rings, you can try different protocol for testing endo-dependent relaxation. 1. Use EC50 of phenylephrine instead of EC80. 2. Try Carbachol instead of Ach. I am pretty sure you will see some relaxation. At the end of the experiment, you can use an endo-independent relaxant such as SNP or SNAP if you have, to confirm smooth muscle is responding to dilators, which I presume you are already doing.

The first thing I thought is the temparature. I always use the conventional thermomether (glass stick) together with the automatic electronic set-up. 

Next I would have reprepared Krebs and check the pH... What about carbogen? The environment can brake the endothelium responses.

as you see I only look for the tecnical problems. When we switched on the software system, every time we need to the  calibration of transducers as well.. 

I agree with all the above answers. Also because it depends on the origin of the aortas you are using. The aortas of a model of atherosclerosis will not relax to ACh  and will in fact even contract because the endothelial layer is so damaged and/or there is eNOS uncoupling.

The tlack of response to ACh can result from many causes. I said before, I suggst you to check all technical parameters : mainly temperature and pH in the bath.

Then, I would test endothelium-independent vasorelaxation with sodium nitroprussiate (or other NO donor) or papaverin. 

From which model do you obtain your aortic rings ? Rat ? mouse ? other ? healthy or not ?

I wish you goog luck

Dear François Guerrero ·

I checked Temperature that is 37 oC its Ok

But about pH we have some problem, because we didn't have Carbogen 95% O2 and 5% CO2 ,

we maked it by two way

1. mixing O2 and CO2 in bottle, and checking pH and set it on range 7.30 to 7.55

2. we have two bottle O2 100% and CO2 100% and then connected with organ bath and by regulating pH 7.30 to 7.55. 

About other paragraph test endothelium-independent vasorelaxation with sodium nitroprussiate

As you shown in the attached image , you can see that relaxation occur by SNP pre-contracted with  PE

about last paragraph We used Thoracic aorta in rats and all of the are healthy

please did you have any protocol from using anaesthesia by ketamin and zylacin and heparin until binding aorta and oxygenation 

Best regard

Dear Yagna PR Jarajapu

I used also EC80 for PE, because we didn’t have carbachol I can’t test it is there another materials

I used SNP as shown I attached file

Dear Buket Demirci

Also I check conventional thermomether 37 oC

I have some problem with carbogen

I solved by following

because we didn't have Carbogen 95% O2 and 5% CO2 ,

we maked it by two way

1. mixing O2 and CO2 in bottle, and checking pH and set it on range 7.30 to 7.55

2. we have two bottle O2 100% and CO2 100% and then connected with organ bath and by regulating pH 7.30 to 7.55.

A Calibrate transducers all day before beginning experiment

Best Regard

Dear Joao Carvas

We Used rat aorta its healthy sometimes we used male or female

Dear Cristina Pérez Ternero ·

 I checkedthe tension as well its Ok.

The protocol didnot  work before under the same conditions only for ACh

Best Regard

Thanks for showing more of your work, which is great and you are doing everything right. Did you say EC80 of PE, please try EC50 whatever it is in your studies n use Ach for endo-relaxation. You will see some relaxation. I also suggest you look into other technical aspects such as maintaining tension, looking at your data you seem to have taken care of all that!

As long as you are maintaining pH in the range that you mentioned, you are doing good. I just want to mention that either carbogen (oxygen 95+CO2 5) or medical air (oxygen 20+CO2 5+Nitrogen 75) are extensively used for in vitro vascular studies, if you have access to. I use and prefer medical air.

Dear All 

Also I used the following krebs solution as in attached file

some time I tried to use  Krebs–Henseleit  but aorta didnot work

Sorry for bothering

Best regard

Thanks for showing the recipe for the buffer. I don't think it would really matter that much because you are not doing electrophysiology expt. Anyway here is the recipe of the buffer that I used: NaCl 118mM, KCl 4.7mM, NaHCO3 25mM, MgSO4 heptahydrate 1.18, KH2PO4 1.18mM, CaCl2 2.5mM and glucose 11mM. You can try this one as I do see some differences from yours, again it should not make a lot of difference!!

I also would like to admit that when I started these experiments in 1996 I didn't see relaxation for some number of experiments, yet I continued doing. During the course of time I started seeing relaxation ranging from 20% to 80% and further along I started seeing 80-95% in all four rings that I was putting up. Soon after I have moved on to studying small arterioles though. When I trained students with different backgrounds I did see almost same trend of learning curve. I am not sure how long you have been doing these experiments, and what stage of learning curve you are at. Its just the matter of time and for sure you will see everything going so smooth, no matter how you handle the aorta it will give beautiful responses.

Drop a note whenever you get there...

How did you confirm that the endothelium was intact?  I have found that the endothelium can be easily traumatized while isolating and mounting the vessel.

Hi Mudhir, 

I've had similar things happen with arteries that I have mounted.  Based on what you've said most everything looks alright.  I think the most likely cause is that the endothelium is being damaged in the mounting process.  You can check this by assessing endothlial function with another endothelial dependent dilator like Bradykinin.  If it doesn't dilate to both bradykinin and ACh but does dilate with SNP then it is probably that the endothelium is damaged.  Best of Luck.

Jayson is correct it is incredibly easy to accidentally remove the endothelium in the mounting and dissection processes, any stretching or rubbing of the artery during this process is likely to damage the endothelium. It it constricts to PE and relaxes to SNP I would be 99% sure this is your problem. Excessive PE and basal stretch will reduce but not remove endothelium dependent relaxation. Tips on how to avoid this are to use thinner wires/hooks for mounting (0.25 mm steel wire is more than thick enough) and to take care to minimise handling of the tissue avoiding lateral and longitudinal stretch. I find handling with fine forceps is the best way to do this. Some prefer a dissection microscope to thread the wires through the lumen but it is not necessary if you take care. Also looking at you Krebs solution I would suggest the composition should be more like (mM): NaCl, 118.0; NaCO3, 24; KCl, 3.6;

KH2PO4, 1.2; MgSO4·7H2O, 1.2; glucose, 11.0; CaCl2, 2.5

The above comments pretty much cover everything. Just as a reassurance (Yagna in particular alluded to this) myography is one of those things that for the first few weeks you'll be pulling your hair out trying to get it to work, then one day all of a sudden it'll work, and thereafter it'll work. You'll wonder what you've done differently..... you've simply just got used to the technique and perfected it without realizing it. It just takes practice. Ali McNeish has given some sound technical advice.

A plateau is really vital for good curves, as Sarah states here. I prefer not to add a bolus of PE when contracting out of fear of "burning out" the vessel to the point that Ach wont ever elicit a stable response. I wait 5-10 min in between PE doses from -9 to -6 to see a 50% KCl tension achieved (whichever comes 1st). The area of the aorta matters. Try to avoid abdominal aorta, shoot for the centimeter between the ascending aorta and above the renal artery bifurcation (in a mouse at least, of couse rat aorta is more generous with tissue).

This happens from time time to time as a result of precipitation of salts in buffer. Try to wash the organ bath with 1N HCl, leave for 15 min then wash with deionized water several times.

I also have been experiencing this problem of minimal relaxation of the rat thoracic aorta, despite a profound vasoconstriction to phenylephrine (PE). However I am using the pins instead of the wires (DMT myograph) for mounting the aortic rings. Could the pins be less sensitive than the wires?....This does not make sense since the pins could detect the increase in tension due to PE.

Any advice or thoughts?