During organ bath, I am using aorta as a model, how I know that the function of endothelium detoriated beside using acetylcholine?
Could you provide more information about your experimental setting and research question? Do you plan to isolate ECs from aorta for in vitro experiments or do you plan to do experiments with intact aorta as an ex vivo model? Are you interested to detect changes in vasoconstriction/relaxation or vascular permeability in response to some chemical induction or alterations in EC metabolic systems e.g. apoptosis, proliferation, and chemokine or ECM secretion?
Thanks. Usually we are using organ bath study with intact aorta, but I am searching for a substance or a method to induce endothelial dysfunction, then how I can confirm that this dysfunction is occurred?
Easy way to induce endo-dysfunction is to expose the aortic rings in organ bath to high glucose, 25mM or even higher. You can look into the extensive literature available online in regards to organ bath/intact artery prep studies in high glucose. You can test the dysfunction by checking the relaxation of aortic rings pre-constricted with phenylephrine by using carbachol or acetylcholine. I recommend using pre constricting agent in a concentration EC50 or no higher than EC75 and test the relaxation by taking a concentration response curve. For high glucose, I recommend a control experiment using L-glucose, and beware of the dearly price of this.
You are very welcome. Good luck with your research my dear friend.
You can also remove the endothelial layer mechanically with e.g. a hair by rubbing the lumen gently. Afterwards you can check the vasoconstrictor capacity with high concentration potassium (e.g. KPSS 60 mM) and the endothelium dependent relaxation with acetylcholine/carbachol after pre-constriction with e.g. 30 mM KPSS. Good luck
In vitro, the endothelium can be removed either mechanically or chemically. For the mechanical removal, the blood vessels most commonly are placed on paper towels soaked with saline. The tip of a wooden stick (or a small forceps) is inserted into the lumen and the endothelial layer removed by gentle rubbing of the preparations back and forth over the paper towel -' This procedure removes endothelial cells, while preserving the ability of the smooth muscle to contract and to relax." ='"'"' Alternatively, blood vessels can be perfused with col lagenase.' saponin,2' or 31 (3 -cholamidopropyl)dimethyl amonio 11 -propanesul fonate (CHAPS).The latter techniques are particularly useful in small arteries to avoid mechani-cal damage of the vascular smooth muscle. Morphological studies using conventional histologi-cal or electron microscopic (Figure 3) techniques demonstrate the effectiveness of these interventions to remove endothelial cells.` In vivo, the endothelium of large conduit arteries can be removed by means of a balloon catheter without damaging the underlying vascular smooth muscle . In the cerebral microcirculation (pial arterioles), responses to endo-thelium-dependent vasodilators can be prevented (while preserving those to direct inhibitors of vascular smooth muscle) by exposing the blood vessels in situ to mercury light in the presence of sodium fluorescein dye.'•" The injury leads only to minimal morphological changes of the endothelium (endothelial vacuolation. blebs, and lucencies). A modification of this tech-nique uses a helium-neon laser in the presence of intravascular Evans blue dye which sensitizes the blood vessels to the laser.unc"" After injury. dilatations to acetylcholine and bradykinin recover within 1 h except at the very center of the impact zone of the laser." Air embolism also has been proposed as a means to damage the endothelium of pial arteries in vivo
http://books.google.iq/books?id=5IL07d_YhRYC&pg=PA7&lpg=PA7&dq=endothelium+removed+by&source=bl&ots=Y8dxI2w0F2&sig=wXzBwZhhtsWSw9vaoeVt15GYF2A&hl=en&sa=X&ei=f-MJVLKKFNKw7AaD_4CgCQ&ved=0CEYQ6AEwBA#v=onepage&q&f=false
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