When we used rat thoracic aorta we faced some problems such as some rat aorta having a high response to Phenylephrine for contraction while others had slow responses to PE to induced contraction.
We also have similar results with wistar rats in our team, sometimes the results are contradictory, so we performed the test many times, with differents rats (generally 5 or 7) and we consider the tendance that is exhibited by the majority
These variations may occur depending on the presence or absence of endothelium. Endothelium blunts the vasoconstrictor responses to phenylephrine in the aorta of Wistar rats. Mechanical removal of the vascular endothelium is not very easy. To test the presence of the vascular endothelium is necessary to assess the vasodilator response to acetylcholine.
I would say that such variation is normal between animals. We often see different sensitivies to compounds between animals, batches of rats, strains of rats etc etc. Sometimes even different segments of the same artery will behave differently in different organ baths. I imagine the variation is due to a plethora of reasons like quality of the dissection, handling of the arery during mounting, organ bath temperature, oxygen tensions, health of the animal, age of the animal, hweterogeneity in the expression of the various receptors invovled from one animal to another, and from one segment of artery to another...and probably lots more reasons!
Thank you Mireille Kameni
Thank you Cristina Antoniali
Thank you Saoirse Elizabeth O'Sullivan
Thanks for all you gave me more information about my problem
You need control a number of parameters (as indicated Dr. O'Sullivan) including basal tension and cross-sectional area but endothelium functional integrity appears to be more important (as Dr. Antoniali supposed).
I realy care about the endothelium. I never touch the aorta or strech it during the dissection. I touch only the surroinding of fat tissue, lay it on paraffin block with several drop of Krebs on it, divide it new sharp blade.
I think randomization is important:
When I divide the thorasic aorta in to four, the top segment always give more tension. I places each segment in to different bath every time, not in order. (my system has 4 transducers and bath). So every day, for example, the first transducer records different segment.
Additionally, I change the drug incubations too. For example the first channel is with Silymarin (SM), the second INDO+SM, the third LNAME+SM, the forth is control,. The next time I label the baths randomly in different way.
Do not forget to brush the glass chambers properly end of experiment with a lots of distilled water for next day. we have to get rid of remains!
I would agree that there are a plethora of reasons why you may see variability. From experimental set up to the endotheluim.
While inter animal variance is possible I actually see very little if a careful and consistent procedure is used; it is more likely to stem form technical errors made during dissection mounting and during the experiment
The main source in variability of constriction in my lab is from damage to or removal of the endotheliial cell layer. Removal of the endothelium can increase constriction to PE by greater than 10 fold. The reasons for this are complex but most researchers agree that there is a basal release of NO from the endothelium that suppresses constriction, many find that his suppression wears off a little as the the day wears on, i personally don't see this effect.
The other most likely source of variance is stretching the tissue during dissection it is very important to be careful to avoid this overstretched arteries tend to constrict less and are more likely to also have a damaged endothelium.
I also noticed on another thread that you had problems with pH of your solutions when gassing as you do not have the medical oxygen 95% O2 5% CO2 gas mix required for bicarbonate buffers. This is likely to be causing some of your variation, I would strongly suggest to buffering your pH with another buffer such as MOPS if you cannot get the gas mix correct, you could then bubble with a simple air pump.
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models