Hello. I've posted a question before, however, it may have been a bit wordy, so i'll try to be more brief here.
We are working with 2.5mm mouse thoracic aortic rings in a 25mL Radnoti tissue bath @ 37 degrees Celsius . Right now the buffer is Krebs ringer. We are oxygenating with 100% O2 (I've read that may be incorrect?). Passive tension is at 1.5 grams (but we've tried lower). As of yet we have not had a measurable response (we've tried insanely high concentrations of KCL, and L- phenylephrine in a desperate attempt to get any type of signal).
Hello Dennis,
first I have to say that murine aortas have been pretty weird in my experience and actually harder to prepare than small arteries, so having problems with this model is not unusual.
First idea - instead of using passive tension of X grams I would try to perform a separate normalization procedure for every ring. There was a how-to PDF on the website of myograph company DMT, but now I can't find it anymore, so some other resources:
Simple description of the normalization procedure (same procedure as the one used by DMT):
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230209/
More detailed description of the normalization procedure including determination of the normalization factor:
https://www.researchgate.net/publication/49730527_Accurate_normalization_factor_for_wire_myography_of_rat_femoral_artery
Characterization of various murine arteries, including thoracic and abdominal aorta:
http://www.ncbi.nlm.nih.gov/pubmed/24006308
Also:
- If you use cervical dislocation the aorta is probably suffering.
- If you prepare several rings from one aorta write down their positions and compare only rings from the same area.
Article Accurate normalization factor for wire myography of rat femoral artery
In the mean time, could you explain a little of the reasoning behind this normalization? Right now we are just trying to get any sort of response, not conduct a formal experiment (that will come after our response). Is the normalization absolutely required for a response?
Hi Dennis.
I noticed that the debate cites our work. If I understand correctly you interested in the reactivity of the blood vessels. You using standard Radnoti tissue bath (RTB). In these normalization not done routinely. We have the case dealt with several times. But what I noticed. Using mouse blood vessels and preload 1.5 g. My used rat blood vessels and weight 1g. This is just my estimate but I think that 1.5 g is a lot. It is possible that this damage the blood vessels.
So few advice:
1. Try on one vessel, 3x or 4x randomization change the balance weights (0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75), but one of weight make such comparison. Let stabilize the approx 30/45 min. Than vascular contraction (KPSS or Ser or phenylephrine). You will see the maximum contraction in a single administration. Max is good at constant vessel length.
2. The preparations must bathed in modified physiological salt solution (PSS) oxygenated with a mixture of 95% O2 and 5% CO2. Mabye, try 125 mmol / L KPSS (https://www.hindawi.com/journals/bmri/2014/658479/)
3. The answer to the question: In RTB normalization is not required, the length of the vessel segment respects (not just some) and it is determined by agiven grammage.
4. We are working on mulvany myograf ... it isn´t compatible with the RTB. If the vessel is small, it could be measured by a mouse aorta in mulvany myograph. Unfortunately I do not know the dimensions of the mouse vessels.
5. Helen's comments are correct, protocol must be uniform.
If you have any questions about the reactivity of aortic write Doc. MD. Jozef Torok, Csc., or MD., Ing. Angelika Puzserová, PhD And Mgr. Soňa Čačanyiová, PhD. These people work with it every day. e-mail contact can you find at http://www.sav.sk/index.php?lang=sk&doc=ins-org-ins&institute_no=59&action=users
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models