I'd say at around 60% acetylcholine-induced dilatation, you can be confident that your endothelium is largely intact.

Ross

Thanks, it's what I've read (around 60-70%). But at what percent can I consider that the endothelium is correctly removed?

For the pre-contraction, the concentration of Norepinephrine of 5x10-8 or 10-7M is fine. In rabbit aorta Furchgott and Zawadsky used about these concentrations for precontraction and for endothelium NO liberation (what is a test for the functional integrity of endothelium) acetylcholine of 10-8, 10-7, and 10-6 in a cumulative manner (Nature, 1982). They had almost complete relaxation. I personally think that in the rat aorta 10-6M acetylcholine which would produces at least 60% immediate (!) relaxation, is a proof of the functional integrity of the endothelium.

Yet, I used in rat aorta routinely for the precontraction, at the end of the experiment, Phenylephrin (5x10-7 M) and then, while expecting a relaxation of about 60-70%, Acetylcholin (only two concentrations, 10-7 and 10-6 M). Higher concentration could be less effective due to the muscarinic stimulation on the muscle cells themselves, what may counter the relaxing effect of liberated NO. If after a challenge with 10-6M acetylcholine, the relaxing effect is less than 60%, I would consider that some endothelium was not functional or that was removed.

To consider a preparation as “without endothelium”, you should obtain, after a precontraction with a medium alpha-1 stimulation (NE, Phenylephrine, of not more then 10-7M), absolutely no relaxation with acetylcholine. I would discard the preparations that had an immediate 10% relaxation.

Thank you very much for your precise answer. Based on old publications from our laboratory, to test the integrity of the endothelium, I use 3x10-8M NE or phenylephrine to precontract rat's aorta and 10-4M acetylcholine (Ach) for the relaxation. I generally obtain ≥70% relaxation. It appears that the concentration of 10-4M Ach I use seems high!

After mechanical removal of the endothelium, using the same precontractors and Ach at the same concentrations, I consider the well denuded when Ach-induced relaxation is less than 20%. Perhaps this remains too high?

Dear Hubert,

You must have almost immediate effect of Acetylcholine (well after 1-2 min if mixing is good). Yet, you will always have some NE degradation due to oxidation, in particular if you bubble the organ bath with oxygen.I did number of experiments in almost absolute obscurity or had to add ascorboc acid as an antioxydant and bubble with N2.

Well, this is why you probably see 20% reduction even if endothelium was "removed". If you let NE work for 5 minutes, degradation will be so important that your tension will start diminishing “spontaneously”. Therefore it is better to use slightly higher concentrations of contracting agent, close to its EC50, for NE is probably 5x10-7M.

In fact, you may do relatively fast cumulative dose-response with acetylcholine and when you get 70% relaxation in preparations with endothelium, to consider that the preparation had intact endothelium. Or inverse, and this is better, particularly in the preparations without endothelium, start with stronger concentration of acetylcholine, 10-6M and 5x10-6M and if you do not have any relaxation, consider the endothelium absent. With those concentrations in endothelium present, you will certainly have 70% relaxation even if precontracted with NE 10-7M.

The endothelium removal procedure is important. I probably described it somewhere in the papers. We used to produce cotton wrapped metal sticks, introduced them in each aortal ring and ruled it (between Krebs solution moistened filter paper) as a weal for about 20 sec. Histology showed afterwards that there was practically no endothelium left.

This is a very interesting discussion, at least for me, and I thank you for spending your time for me. Effectively, the effects of Ach are immediate (1-2 min) and the maximal effect obtained about 5 min after administration. NE slowly develops arterial tension, which reached its maximum about 15-20 min after NE injection. Usually, I administered Ach at the maximal effect of NE before its effect began to slowly decrease. My organ bath is at 37.4°C, pH 7.4 and bubbled with a mixture of 95% O2, 5% CO2. I’ll try your proposed concentrations next time.

For the endothelium removal procedure, I’ve read with interest one of your paper downloaded from ResearchGate (tanks for uploading it). The paper is by Ribback et al., Journal of Physiology and Pharmacology 2012, 63 : 119-125. Personally, I introduce a small forceps tip inside the lumen of the vessel and ruled the vessel on the back of my finger. I think  there are many technical procedures for mechanical removal of the endothelium but none is described step by step (as a tutorial)

In all my experiments, the integrity of the endothelium is check before beginning the experiments (generally, dose-response curves). In the above-mentioned paper, it is written that “The integrity of the endothelium in aortas was tested, at the end of the experiment, … At least 40% relaxation was taken to indicate intact endothelium.” My question is: do you still consider 60-70% relaxation as required for considering the integrity of the endothelium?

In the same paper, it is later written: “In the third set of experiments endothelium intact preparations were pre-incubated either with …”

My question is: If the integrity of the endothelium is tested at the end of the experiment, how the endothelium intact vessels are selected?

Thank you again for your help and your time

Yours

With lower pretension (Phenilephrine 10-7M or even 5x10-8M) and Acetylcholine concentration that is high enough (5x10-6M), in principle, yes, 70% must be achieved or even 100%. But all must be performed quite fast to avoid degradation of catecholamine.

However, to speed up the procedure (you may do it also) we pre-contracted with quite LOW concentration of Phenilephrine (10-7M) and when the tension achieved the first fast portion of contraction, we perfused it with quite HIGH concentration - 5x10-6M of Acetylcholine and if the immediately reached relaxation was 40% or more, we considered that endothelium was functional. We seldom waited for maximal contraction - to reach real plateau that happens after 15 min or more (!) and also seldom waited for maximal relaxation. We did number of preliminary experiments (hundreds!) where we found that the relaxation is very profound and even 100%. Later, at the stage of the experiments, we were sure that the relaxation reached would be 70% or more. This is highly reproducible, so that if you would establish your experiment, the test will be poor routine, and even may be not always necessary. However, you must have established that routine! We did not test ALWAYS and in that particular experiment if we did not remove endothelium we assumed that it was present. I never had an aortal ring that we treated for removal of endothelium that relaxed on Acetylcholine and very seldom, may be once or twice, out of hundreds not treated endothelium intact rings, that did not relax on the application of Acetylcholine. This may happen in some special experiments where some aortal rings are incubated for a week for example, or if you use KCl for contracting; and then afterwards the endothelium functionality may not be always perfect.

By the way, endothelium may be functionaly destroyed during the experiment so you should test it after the experiment, I think.

I am in Paris, so...if you need more help...

It will be a pleasure to meet you to discuss this subject. How can i contact you?

% relaxation will be closer to zero the better job you've done removing the endothelium. Don't bother with a concentration response curve for this specific purpose. Just a bolus high concentration... see above.... is fine. Good practice would be to check the smooth muscle has not been damaged during the procedure. To do this, immediately after you're sure there is no response to ACh add a bolus of NO donor, such as MAHMA NONOate or sodium nitroprusside

David

Did you down vote my response above? Please explain why?

I do not understand your answer. To use one dose may be a good routine, but is basically not always correct.

The relaxation is a nitric oxide effect and a test described above (with ACh) is for endothelium functional property. The ACh test after the experiment may be (and sometimes should be) a dose-response experiment also. It would test for the NO producing capacity of the endothelium, and if too high ACh concentrations is used (one good bolus, as you suggest), in an endothelium "present" but in a low NO producing vessel, it may produce even a contraction of the vascular smooth muscle (muscarinic effect). This is why we should start with some lower concentration of ACh and 50% relaxation would suggest functional endothelium. If we are not happy with the response, we should increase the concentration of ACh.

The second part of your comment is either not clear or also false.

If the smooth muscle is injured following removal of endothelium, it will not react well to any contracting agent including ACh – but with a CONTRACTION! not with relaxation (!). This means its contraction is diminished or absent. The "NO-donor" will be useless - it cannot test the contractile property of the smooth muscle!

I have a feeling that you do not understand what I am talking about.

Dragan, if your response was a little more polite I'd probably take the time to give you an answer, or rather where you have misinterpreted my answer. I actually do what I am talking about. 

Hubert, I was simply expanding on what others had said. It wasn't intended to be an alternative to what others had suggested, rather an add on to the discussion, and it was said that the discussion in general was of interest. Adding an NO donor at the end of the experiment is a perfectly logical procedure, whether for an intact vessel or a denuded one. For a denuded vessel it shows the lack of response to ACh is not simply due to a reduced capacity of the smooth muscle to respond to preformed NO (regardless of whether it contracts well or not - that's a separate issue, Dragan!). It's good practice with minimal effort or expense. 

Kind regards, Dave

Dear David

I am terribly sorry. This is a complete misunderstanding. We communicate here pretty directly. Sorry again.

Could you please explain your logic.

Thanks very much indeed.

And incidentally, whether a denuded vessel contracts in response to ACh depends on what vessel you're studying. They don't always contract, some do nothing

Dear David,

Okay. But please explain.

D.

No problem Dragan.

If you have an intact vessel, once you have your Rmax from ACh you can add a bolus of NO donor at a concentration that regularly gives maximal relaxation. From this you can see in your data (your "n"s overall) if there are rogue experiments. For example, if your normal ACh response is 70%, and in one or two experiments you get 50%, an NO donor at the end will tell you whether you've inadvertently damaged the endothelium during dissection, or whether the smooth muscle simply isn't responding to NO. Some researchers even calculate %relaxation as a function of the maximal response to NO donor, rather than simply the % reversal of agonist induced tone.

In a denuded vessel the same practice can be applied. While 0% relaxation to ACh is very unlikely to be due to zero response of the smooth muscle, it is an easy box to tick that gives you even more confidence that your endothelium is fully removed.

Kind regards,

Dave

Dear David

Again, I am terribly sorry for misunderstanding. This is science and we are a priory friendly. I have a long list of publications so you probably did not realize that my main interest is smooth muscle contraction.

We discussed here contractions of rat aorta and appropriate protocols. I described one and I do not think what you proposed is valid. What was of interest was the integrity of endothelium and Ach test is an appropriate test.

The viability of the blood vessel is the other problem and this is tested with a preliminary test contraction. The NO-donor may test relaxation mechanisms, but rat aorta will, if injured, relax spontaneously anyway.

Best wishes.

Hi Dragan & David, I’ve just read your discussion. I’m pleased to see that this question still has some interest. Thanks for your answers. To summarize, if I’ve got well, the discussion now rather concerns the integrity of the smooth muscle – that is also important for its relaxation. I think that you are right as well. Indeed, if the smooth muscle is damaged following removal of the endothelium, the effect of the vasoconstrictor is very weak, even absent and the vasorelaxation impossible to test. Therefore, the use of an NO donor might be useless (Dragan’s answer). I also agree with David when he suggests injecting sodium nitroprussite (SNP) immediately after Ach (during the test of the viability of the endothelium). However, what would be the necessity of doing this if one has a “good contraction” (suggesting the integrity of the smooth muscle)? But what is a “good contraction”? Moreover, what would be the necessity of doing this at the end of the experiments, as he said later, especially when the drug tested induced 100% relaxation? To say all, in my practice, I used to inject SNP (10-4M) at the end of my experiments, but now I wonder whether this kind of practice may add value to my experiments.

Most often the smooth muscle is damaged mechanically or the endothelium is partially destroyed. During the preparation, you should not touch the muscle that you will test in vitro. You should be holding just surrounding tissue. You also should not stretch the muscle at all during preparation. I did not describe that procedure in my book, but I will add a section in the next edition.

If you used some agents that either blocked cGMP production or that blocked myosin dephosphorilation (block phosphatises) or if you use some other agents that increase contraction and you want to test for their effects, NO-donor is a way to do it. Indeed you need a control for this, which may be the same vessel before the experiment, or a control vessel that is from the same animal.

You cannot use too many various combinations of the agents on the same vessel during one long experiment since the residual effects of the agents that were used previously may change the responses. A simple protocol is the most valuable protocol.

The above should be taken to be speculations and the only reliable test is the very in vitro test.

Hubert, if you're getting 100% reversal of tone with ACh then there is no need to add SNP.... It is pointless in that particular case. It does come in handy in vessels where there is not 100% relaxation, though. I have seen instances where maximal responses to SNP are low (as low as 70-80% if I remember rightly), so if you have a relatively craps response to ACh in that preparation you know it's probably just a rogue experiment, and likely not a damaged endothelium. 

What Dragan says about taking care in tissue preparation is very important. A couple of my own tips would be: 1. Hold the vessel by the surrounding fat tissue when dissecting from the animal, 2. When trimming the adipose tissue off and for mounting in organ bath hold the tissue with the very tip of the forceps on the outermost layer of the tissue I.e. the adventitia. Also when cutting the vessel into rings discard the end bits (the first 2mm or so) as these usually become damaged during prep. And as Dragan says avoid stretching, and also linking the vessel. 

Something else we used to do was file the forceps to toughen them up a little. This makes them more grippy... Particularly handy when gripping adipose tissue, or adventitia by the very top of the forceps. 

Sorry Dragan... You did also mention holding the tissue by the outer layer away from the smooth muscle. 

... and try to take the tissue “in block” out of the body of the animal and place it in the BPS in less than 1 minute... For the rat aorta 30 seconds is good time.

The smooth muscle is very robust and is viable after many hours, in a solution for a week or more, but some cells that you have with the smooth muscle (blood cells, endothelium, or some their functions, may deteriorate.

If done as David also indicated, EVERY single preparation will function perfectly well. There will be almost no failed preparations.

During the experimentation, well then, the serious problems arrive. The smooth muscle has its “history”: its stretch history and its contraction history, at least. What responses you will get will depend on its history. The only solution to this problem is to try to perform identical experiments in the control tissues and in fact to work always in parallel with the controls. You must have 4, or preferably 8 or more experimental setups.

Thanks to all 2 of you. I think dissection – removing the vessel from the animal, cleaning the vessel of fat and surrounding tissues, cutting away the 2 ends of the vessel – is now ok in my practice. After a long discussion with Dragan, I’ve ameliorated in particular removing of the vessel from the rat. Now I think that one most important thing is to perform all experiments in similar conditions.

Good point Dragan. We used to remove iliac arteries from rabbits in a block that included abdominal tissue, skeletal muscle from the upper hind leg, the lower abdominal aorta and upper femorals. The iliacs were nicely protected in this block along with, usually, quite a bit of adipose tissue. Time ex vivo can be a real issue for some aspects of endothelial function. Gap junctions have a half life of a few hours or so. For small and microvessel function, epoxygenases are an issue because these decline rapidly when haemodynamic stimuli cease. Some researchers flow-adapt the vessel first if ex vivo for a while. It is, overall, best to get the most info out of the vessel as quickly as possible. Not always feasible, however. 

And paired data, as you say Dragan, is also very important for organ bath and myograph. I think most don't do it though. Use Bonferroni test also, and your data is pretty much bullet proof