Models of Inflammation: UNIT 5.

4
Carrageenan-Induced Paw Edema in the Rat
A solution of carrageenan in saline injected into the hind footpad of rats induces an acute BASIC
swelling of the paw that becomes maximal ∼3 hr after the injection. This model has long PROTOCOL
been used to assess the anti-inflammatory properties of agents such as nonsteroidal
anti-inflammatory drugs (NSAIDs) that inhibit prostaglandin production. This protocol
is a method to elicit and measure carrageenan-induced foot pad edema.

NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals.
Materials
Female Crl:CD(SD)BR rats weighing 45 to 50 g upon arrival (Charles River Labs)
0.5% (w/v) carrageenan solution (see recipe)
0.9% (w/v) saline, sterile (e.g., Irrigation USP, McGaw)
Test compound (new chemical entity, NCE)
CO2 supply
Scales, accurate to 0.1 and 0.0001 g
Repeating pipettor (e.g., Brinkmann)
27-G, 1.25-in Monoject needles (Sherwood Medical)
2.5-ml combitips for repeating pipettor (Brinkmann)
Animal restrainer (e.g., Broome restraint, Plas Labs)
Razor blade mounted in a safety scraper (e.g., Proedge)
Weigh boats
1. Acclimate female Crl:CD(SD)BR rats for 1 week in solid-bottom cages with wood
shavings. Weigh the rats using a scale accurate to 0.1 g. Divide rats weighing 100 to
110 g after 1 week into five groups of eight animals each. Withhold food and water
during the experiment.
Animals are acclimated under standard lighting and temperature conditions with food and
water ad libitum to eliminate the effects of stress.
There should be one group of eight animals for each of the following: carrageenan and
vehicle, carrageenan plus NCE at each of three different doses, and possible control such
as indomethacin (Sigma) at 5 mg/kg administered 1 hr before carrageenan injection.
Male rats may also be used. Animals are selected by weight for reproducibility, handling,
and NCE requirements.
2. Place rat in a temporary animal restraint.
3. Load a 0.5% carrageenan solution into a repeating pipettor and attach a 27-G, 1.25-in
needle to the combitip. Insert the needle, bevel down, through the callus, at an angle
nearly parallel with the footpad and inject 50 µl of solution. Repeat for all animals
in the group using a new needle for each animal.
The needle is inserted to a depth of ∼1 mm into the callus to deliver an accurate and uniform
amount of carrageenan into the subplantar site. The uninjected left hind paw serves as the
control.
4. Return the rat to its cage after injection. Repeat the injection for each animal in the
experimental group.
Animal Models of
Desease

Contributed by Phyllis E. Whiteley and Stacie A. Dalrymple 5.4.1

Current Protocols in Pharmacology (1998) 5.4.1-5.4.3
Copyright © 1998 by John Wiley & Sons, Inc.
 5. Repeat steps 2 through 4 for each experimental group of animals.
Experimental design for testing as NCE is determined based on the chemistry of the
compound and known in vitro and pharmacokinetic data. Saline or another appropriate
solution (e.g., see UNIT 10.2, Reagents and Solutions) should be used as the vehicle. Typically
three to four doses of the NCE are tested, enough doses to produce a linear response curve.
The NCE is administered at a time consistent with the available pharmacokinetic data, but
all experiments are terminated 3 hr after administration of carrageenan.
6. Three hours after carrageenan injection, euthanize the rat in a CO2 atmosphere.
7. Using a razor blade mounted on a safety scraper, remove the hind paws by severing
them at the tarsotibial joint and place them into individual pretared weigh boats.
It is important to remove the paws in a consistent manner in order to produce useful
comparison data. There is typically a <10% difference between the weights of two hind
paws removed from injected animals.
8. Weigh the left and right paws immediately using a scale accurate to 0.0001 g.
9. Calculate the difference in weight between the right (injected) and left (uninjected)
paws for each animal and the mean difference for each group.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

0.5% carrageenan solution

Pour 1 liter sterile 0.9% saline (Irrigation USP; McGaw) into a 2-liter beaker and
add a stir bar. Slowly add 5 g carrageenan powder (Type IV Lambda, Sigma) to the
beaker. Heat the beaker to 90°C with stirring, but do not allow the solution to boil.
When all of the powder is dissolved, pour the solution into a clean 1-liter Pyrex
bottle equipped with a cap and sterilize by autoclaving. Cool the solution to room
temperature and aliquot into sterile, 100-ml capped bottles. This solution is stable
when stored at 4°C.
Heating the solution helps dissolve the carrageenan; it takes ∼1 hr to completely dissolve
the powder.

COMMENTARY
Background Information
Paw swelling, or footpad edema, is a con-
venient method for assessing inflammatory re-
sponses to antigenic challenges and irritants
(Winter et al., 1962; Otterness and Moore,
1988). The model described in this unit uses
carrageenan as the irritant to induce paw edema.
Typically, test materials are assessed for acute
anti-inflammatory activity by examining their
ability to reduce or prevent the development of
carrageenan-induced paw swelling. Nonsteroi-
dal anti-inflammatory drugs (NSAIDs), such
as indomethacin, reduce paw swelling in a
Models of dose-dependent manner to a maximum of
Inflammation: ∼60%.
Carrageenan-
Induced Paw
Edema in the Rat
Critical Parameters
Rats must be acclimated at least 1 week prior
to initiation of the experiment to eliminate the
effects of stress.
The amount of swelling depends on the
carrageenan preparation. When a new lot of
carrageenan is used, it should be titrated to
define the most consistent induction of edema
formation and to compare the effectiveness
with that of the previous lot(s). Although higher
concentrations of carrageenan can produce in-
creased swelling, the maximal edema response
may prove to be too difficult to inhibit. There-
fore, it is useful to determine the best therapeu-
tic window of edema and inhibition with an
appropriate dose of NSAID.

5.4.2
Current Protocols in Pharmacology
 Table 5.4.1 The Inhibitory Effect of Orally Administered Indomethacin on
Carageenan-Induced Paw Edema in the Rat

Increase in hind paw weight Mean %

Test compound Dose (mg/kg)
(mg) mean ± SEMa,b inhibition

Vehicle controlc — 579.3 ± 37.9 —

Indomethacind 1.0 425.0 ± 41.7 26.6
Indomethacind 2.5 339.6 ± 21.3 41.4
Indomethacind,e 5.0 265.3 ± 14.9 54.2
aThe increase in hind paw weight is defined as the difference in weight between the right (injected) paw
and the left (uninjected) paw. The mean increase is the sum of the increases for all animals in a group
divided by the number (n) of animals in the group (n = 8).
bAbbreviation: SEM, standard error of the mean.
cSee UNIT 10.2, Reagents and Solutions for a recipe for a suitable vehicle.
dIndomethacin is administered 1 hr before carrageenan injection.
eIndomethacin at 5 mg/kg can be used as a positive control for experiments with a new chemical entity
(NCE).

To minimize variation, all paws must be Time Considerations

excised at precisely the same site and must be
weighed within 1 hr of excision to prevent
weight variation due to the loss of water.
Troubleshooting
A large variation in paw weights can often
be due to a lack of precision in the paw excision.
Consistency in this regard is critical for obtain-
ing useful results, so it is recommended that the
investigator practice the dissection on untreated
animals until he or she can reproducibly obtain
paw weights for hind paws from the same
animal that differ by <10% of the mean paw
weight.
Anticipated Results
The increase in weight of the right paw over
the left paw is obtained for each treated animal,
and the mean increases in paw weight are cal-
culated for each experimental group. Typically,
the uninjected control paw weighs between 850
and 1050 mg. The paw injected with car-
rageenan generally shows a 400 to 500 mg
weight increase. NSAIDs, such as in-
domethacin, inhibit 40% to 60% of the increase
in paw weight with a visible reduction in red-
ness and swelling. Table 5.4.1 shows the results
of an experiment using indomethacin at three
different doses.
The carrageenan solution is generally made
in advance and stored. The animal procedure
requires up to an entire day, depending upon
the number of animals being studied. The in
vivo incubation time is 3 hr. The paws must be
weighed within 1 hr following their removal.
A typical experiment with five groups of ani-
mals takes an experienced investigator 1⁄2 day
(∼1 day for a beginner working alone).
Literature Cited
Otterness, I.G., and Moore, P.F. 1988. Carrageenan
foot edema test. Methods Enzymol. 162:320-
327.
Winter, C.A., Risley, E.A., and Nuss, G.W. 1962.
Carrageenan-induced edema in hind paw of the
rat as an assay for anti-inflammatory drugs.
Proc. Soc. Exp. Biol. Med. 111:544-547.
Key Reference
Otterness and Moore, 1988. See above.
A comprehensive review of the long-standing car-
rageenan paw edema assay, including alternative
species and measurements.
Contributed by Phyllis E. Whiteley
and Stacie A. Dalrymple
Roche Bioscience
Palo Alto, California

Animal Models of
Desease

5.4.3
Current Protocols in Pharmacology