I am interested in doing retro orbital bleeding of mice/rats for doing FACS analysis of the whole blood. I am looking at a rare population. Right now I am using chloroform to do the same but I am not sure whether chloroform affects the cell numbers. Please tell me if chloroform influences the cell counts or any other parameter in the rodents and also suggest alternate and more reliable anesthetics.
Isoflurane works great as mice would recover quickly from it....
I have performed many analyses on whole blood by flow cytometry. Here in the US, I have either used isoflurane or ketamine/xylazine. I have not detected any abnormalities in cell populations with either of these, and they are strongly promoted by our animal care facilities.
Additionally, you may consider looking into another blood acquisition method, as retro-orbital bleeding does not seem necessary for what you are doing. Consider saphenous or submandibular vein bleeding as these often require NO anesthesia and allow for adequate amounts of blood collection.
Good luck,
~Adam
We use the following recipe for 10 ml:
PBS 8.1 ml
Rompun (Bayer) 0.3 ml
Imalgene 500 (Merial) 1,6 ml
Dosis 10-11 microl/gr of animal
Good luck
While ketamine/xylazine anesthesia is another alternative to isolfurane, its effects are typically much longer and your animals will be down for extended periods unecessarily... Another thing not necessarily relevant to your specific questio: being longer under anesthesia will reduce insulin release and glycemia if you are ever interested in that.... Something to keep in mind.
Isoflurane works well in rats. Mice we have always done without anesthesia.
you can do Occular bleeding without using anaesthesia, The animal reacts to the anasthesia which may alter your results, Be careful with the Capillary tube. QED.
All answers are good, I just wanted to precise that you shouldn't use anesthesia when you want to dose cytokines - it decreases your values a lot! For analysis of cells you will realize that you recover more cells when you draw blood without aneshesia (it induces marginalization)
Isoflorane, although if you are experienced, you can eventually do it without anesthesia. This is preferable if you are looking at corticosterone.
Http://vsc.stanford.edu/policies/anesthesia.html
useful site click on animal type for info
another good resource
http://web.jhu.edu/animalcare/procedures/retro-orbital.html
I've bled mice serially across 6 months without using anaesthesia. The trick is to use heparinized or EDTA treated microcapillary tubes, so the edges are smooth. I'd routinely get 300-500 microliters per bleed.
Please remember to check with your institution's animal care and use committee before you perform this procedure without anesthesia. I know the IACUC at Northwestern University wouldn't stand for it.
Subjecting to mild anesthesia using Isoflurane is the best. however with practice one can do orbital plexus bleeding without anesthesia as well subjected it is allowed within the institutional rules and regulations.Goodluck
For rats I don't know but if you are experienced for mice you don't need the anesthesia at all. Of course if your institution allows to perform this procedure without anesthesia.
With practise you will be able to without anesthesia quickly. Rodents are required to be anesthetized incase corticosterone levels are to be estimated and in such case use either Halothan / Isoflurane. In any case, do not use chloroform or ether.
Retro orbital bleeding can be done from mice without given anesthesia by which cells count and morphology remain constant. Another route of bleeding is tail vein.
You might also consider other blood-sampling methods that do not require anaesthesia: (http://www.nc3rs.org.uk/bloodsamplingmicrosite/page.asp?id=419). For example, punction of mandibular vein: http://www.medipoint.com/html/directions_for_use1.html (see the video) .
Retro-orbital bleeding from mice and rats has been banned by the Animal Ethical Committee of the world. In the present situation one could bleed mice and rats from tail vein. In mice and rats bleeding is performed by venisection.
In mice it is done by just making an incision one of the lateral tail veins. Before making an incision the vein should be engorged by submerging the tail in warm water followed by drying in hot air by an hair dryer.
In rat as the tail skin is very thick the skin flap over one of the lateral veins has to be surgically lifted and the vein has to be exposed. After exposure of the vein blood can be drawn from vein with the help of a needle and syringe.
I am happy if i know how to get 200microliters of blood from tailvein per time.
Without anesthetisation it is very tough to handle rats during orbital blood collection. Most of the anesthethetising agent will induce stress and causes hyperglycemia which alter basal levels so i think tailvein method is better than orbital method and less pain for rats also
Compare Ethyl ether, chcl3 and thiopentone. U can chose the best for your requirement.
What volume do u desire?
You can use either ketamine-HCl or Chloroform as anesthetic and hope you will be get result better ................
Isoflurane anesthetic at 3% to induce and 1.5% to maintain allows procedures like this to be much more manageable. Chloroform is very stressful on the animals and would be considered a last resort method.
Nagaraju Raju: use a restrainer when ur collecting blood from tail vein. Its easier.
For drawing out blood from mice, I would not advise it as the blood volume is little and its hard to do a time-span study. Use rats instead
Not sure about rats, though tail vein is probably ok. As for mic, sub-mandibular bleeding works well without anesthesia (but requires practice, so spend time with animal subjects that are not critical for the experiment first). See 2nd link under the first answer to you question. If you want to use anesthesia, then isoflurane delivered by a vaporizer is generally the safest way. Lastly, a fast-asting injectable agent like methohexital or thiopental would also be suitable.
If you have access to an isofluorane inhalation system, that would be superior. There is less risk of dying from the anesthesia and they wake up really quick.
It depends on route of exposure for anesthesia. If through inhalation, isoflurane or sevoflurane or halothane are recommended. If via IP injection, ketamine+xylazine mixture is recommended.
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