You have not provided much detail, but we used an air puch model in rats to collect cytokines after injecting the pouch with LPS [Miller AJ, et al. American Journal of Physiology (Regulatory Integrative and Comp Physiol). 1997;272:R857-R61.] We used only 1ml saline to lavage the pouch. It could be tricky to remove and was done only after the animals were sacrificed. We then disected out and opened the pouch to remove with a pipette. Trying to remove through the skin is pretty nigh impossible, given the volume differences and the fact the fluid sits in a thin film.

Thank you so much for your detailed answer Dr. Hopkins, what we exactly did was the use of a rat air pouch model in order to evaluate the effects a herbal extract on inflammatory response and angiogenesis. Inflammation was induced by the injection of carrageenan into the pouch. after the granulation tissue is formed we have to lavage the exudate for the evaluation of exudate volume and leukocytes total count, etc. The problem is that after we injected 3 ml of PBS, all the PBS was disappeared, even after we opened the pouch! We couldn`t collect the fluid back even with needle and pipette! Is it possible that the fluid may be leaked into the tissue surrounding the pouch?

Our Exp Path group worked using this model from the 1950's headed by Prof Willoughby. We do need a bit more detail - rat air pouch does give a decent volume of exudate, more so in the acute phase, so timing is important. Also the quality of carrageenan (use lambda carrageenan) to get a decent response. Note the PBS needs to be 0.315% citrated or EDTA or heparinised to stop cell activation and coagulation. Another aspect, for longer time periods the pouch will deflate, so can need re-inflating. If it deflates the tissue may consolidate and exudate recovery is reduced or the injection goes into the tissue. Re angiogenesis, have a look at our paper in J Pharmacol Exp Ther. 1995 Sep;274(3):1463-72 . As Dr Hopkins says, we do need a little more experimental detail.

Re: Michael Seed's respnse: we in fact developed our model partly based on some of the work published by the Willoghby group. The pouch is a really nice model to study cell and tissue fluid responses in situ. The points about carageenan are well taken if you need a spontaneous exudate, but we used only LPS, which produces little or no exudate or granulation tissue. However, we were still able to recover small volumes of injected washout fluid. If the pouch starts to deflate it should be reinflatable. I guess the fluid could leak out if the pouch, if it is damaged, but they are usually quite robust and, if you are really careful the pouch can even be disected out, and looks like a vascular balloon, with fluid in if pre-injected. It works well in mice too, with smaller volumes of air.

How much air do you inject and do you re-inflate after 3 days? The pouch should be the size and shape of an egg and after the initial injection and the re-inflation be quite firm and not disappear into the surrounding subcutaneous tissue. Carrageenan in my experience produces a very robust exudate in rats, much better than that seen in mice. There should be exudate in the pouch without a washout and this just allows most of the cells and exudate to be recovered without coagulation after the addition of EDTA or heparin (as already mentioned by others). I agree good quality lambda carrageenan is the key. The pouch should also contain just one single cavity. I have occasionally had to "rescue" pouches with multiple cavities, caused by inexperienced co-workers not keeping the needle in the same place throughout the injection, although this can be more of an issue in mice. Prof Willoughby examined my Ph.D., so I know Michael Seed can give you good advice.

First of all I have to thank all of you for your kind answers.

Repaly (Dr. Seed):

we actually did not add any anticoagulant agent to the PBS., that might be a reason. I was wondering if the position of the rat while collecting the fluid may influence the work. I mean a ventral recumbency or any other ways.. Moreover, our plan was to collect the initial amounts of the exudate using a needle, and then to open the pouch from its margin and collect the rest with a pipette. whats your suggestion in this regard? Another problem that we faced was the restraint of the rats with an air pouch on their back, specially when we had to gavage the animals daily! I think the manipulation of the pouch during restraining caused the pouch to be perforated in a way that it couldn`t maintain the injected PBS anymore! Is that possible? Is there any spacial way of restraining these animals that are less annoying?

Repaly (Dr. Dawson):

we injected 8-10 ml of sterile air into the pouch and we also re-inflated it to maintain the volume.

Dear Hilad, now we are getting somewhere. I would be very interested to hear what others do, lets see if there is a better way, but here is what we do. Take care killing the rat. Do not stretch the neck, as this will stretch and damage the pouch, with loss of luid and bleeding into the fluid. Avoid any method of killing that will damage the pouch. So anaesthetic overdose, CO2 suffocation (with care, rising tide - welfare issue here), or other humane method. see UK Home Office Guidelines for Schedule 1 killing. Lay the rat face down on the bench. Wet the fur with 70% ethanol or isopropanol (to keep fur etc out of your sample). Using rat toothed forceps, pick up the top of the pouch, perhaps slightly forward of the centre, and cut a small hole with scissors, and let air in so fluid settles. Inject your 3ml, or 1ml as you see fit of PBS with an anticoagulant (citrate or EDTA), through the hole to stop coagulation of the exudate and cell activation & aggregation etc. Insert a disposable plastic bulb pipette into the hole (whilst still holding the pouch up with the forceps to stop the fluid coming out of the hole), and gently suck fluid in and out of the bulb to mix the contents. You can gently agitate the pouch at the sides to facilitate mixing, Don't be rough. You can put the pipette into the corners to suck in and out, but not so that the tip touches the limits of the pouch. You then suck out all the fluid. You can lift the rat a bit to let the fluid drain into a corner to get the last drops out. Remember when measuring the fluid exudate volume (or weight), subtract the volume you have added. The mouse is much less fluidic in nature. In the pleurisy for example, one can wash out and count the cells, but to get an index of exudation one needs to inject i.v. a tracer (eg Evans Blue) that will colour your washout which you can then assay. Not sure about mouse air pouch though. I hope this helps... tell us how you get on.

Dear Dr.Seed, I really appreciate your care:

I`m going to try all you mentioned. About the i.v. tracer that you

mentioned we have used carmine red as an an indicator of exudation. As

I said we tried to collect the fluid with a needle initially, which I

think is one of the weak parts of our work because it makes it more

difficult to collect all the exudate. I`m going to try what you said

and I`ll open the pouch and then lavage the pouch. Hope I`ll get a

good result.. and I`ll inform you of the process.

Can I advise caution with carmine red. This is particulate unless solubilised in alakali. In alkali it bleaches. The angiogenesis carmine vascular density assay incorporates carmine suspension in warm gelatine, which on iv injection to the warmed anaesthetised animal circulates, cools and forms a vascular cast. Whilst we have shown it does exude (JPET paper), this remains particulate and does not bind albumin. I would suggest Evans Blue as this is soluble and binds albumin, thus making a much more accurate determination of exudation into the pouch fluid. The particulate nature of carmine will confound your exudation assay. In the rat, as we have all said, there should be pkenty of exudate so no need to use a tracer. Looking forward to seeingy our results!

Thank you so much Dr. Seed for your great information and experience sharing. You mentioned a delicate point, I will definitely take it into consideration..

By the way Dr. Seed, we want to inject the dye before sacrificing the animal through the jugular vein (placing a canula), so there should be a standard concentration for Evance Blue solution in order to be injectable. In the pilot study we used a 5% carmine solution in 5% gelatin. What should be the concentration for Evance Blue solution? Is the solvent distilled water?

Many thanks to dear Dr. Seed, Dr. Hopkins, and all others who kindly guided me to overcome the problem.. I used all of your worthy hints and could finally collect plenty amounts of exudate in our study. The volume of exudate reached 13 ml in some of the groups!

It was a great pleasure and a really nice experience to have you beside me through this study..

13mL, sounds a bit over the top, you might be able to reduce the stimulus a bit...

Great to see it work! Best, Michael S

Hi,

I am about to try the same thing and will be using heparin and PBS as my buffer. It needs to be used at 50 U ml-1, but where can I get it from? They sell the heparin sodium salt, is this the same thing?

Thanks

Lucy

Yes, it's the sodium salt of heparin. There are lots of suppliers (e.g. Sigma H3393 or H9399) and it should/will state how many units there are per mg, so you can calculate the 50U per ml concentration.

We use heparin at 10U/ml for the carrageenan pleurisy, as far as I can see we have not used heparin in the air pouch. Depending on your outcomes (if you need functional cells), heparin is not recommended as a blood anti-coagulant for DNA/PCR work. Looking online, heparin sodium appears to preserve white cells better than the lithium salt. It seems the lithium salt should be avoided.

We also used sodium salt of heparin heparin at 10 U/ml too (Sigma).