All methods to remove aortic endothelial cell and which one are the best?
Hi Mudhir,
Actually it depents on your Project. You can remove the endothelium by using mechanical tools, chemical or pharmacological agents. If you are working on aorta segments (like in isolated tissue baths) you can easily use a thin stick to remove the endothelium. Just you need to get some experience to keep the vascular smooth muscle intact. There are some chemical agents (you can find them in several papers). After the application endothelial cells layer removes. The third option is pharmacologically stop endothelial cell activation according to your Project.
Hope this knowledge help you to solve your problem or at least give an idea to solve it.
Good luck.
The mechanical method may be a bit traumatic for underlying smooth muscle layer. Sure, you will not totally destroy it, but it will take quite a time to "calm down" disturbed SMCs. You can just pump air through lumen of aorta for 2-3 min (using a fishtank air pump).
Mudhir,
We find (often inadvertently!) that the gentle rubbing of the lumen of the vessel with the steel wire we use to mount vessels in the myograph is sufficient to denude the endothelium. Smooth muscle function is left intact following this method. Alternatively, one can chemically denude (as others have suggested) with deoxycholate but I have no experience of this/don't see it published much any more. I wouldn't advise a pharmacological route (L-NAME etc.) as one would need to block a whole variety of enzymes (NOS, COX and others...) in order to remove endothelial-derived products that may influence your prep.
Cheers!
Ross
I use the small forceps tip inside the lumen, and spin the aorta gently five time to each direction. Do not forget to make aort segment wet enough with Krebs on your finger tips.
I would concur with much of the above. I think the key point is why you want to remove it. In the past we have denuded the endothelium to remove its influence on smooth muscle repsonses in an organ bath/myograph set-up. To do this gently rubbing the lumen with wire or hair works well. Its effectiveness can then be ascertained by a diminished ACh-induced dilatation.
We have also used collagenase along with some gentle rubbing to isolate the endothelium for primary culture.
Again it depends on why you want to remove the endothelium.
You can rub the lumen gently with a cotton thread 2-3 times in each direction. This way is very simple and effective.
I would agree with Ross and Christopher's answers you have to remove mechanically, for the aorta gentle rubbing with, a swab, cotton, or stainless steel wire are probably the best options human or horse hair (like from a brush or violin bow). In this artery there is quite a thick internal elastic lamina so if you are careful there should be no damage at all to the smooth muscle
I use one of my own hairs!... I do it while the artery is mounted between wires with some tension on and gently rub in a circular motion with very gentle pressue against the artery wall/endothelium in one direction....this decreases Ach responses to less then 10%.
- Badges
- Science method