Carrageenan-Induced Paw Edema 115

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From: Methods in Molecular Biology, vol. 225: Inflammation ProtocolsEdited by: P. G. Winyard and D. A. Willoughby © Humana Press Inc., Totowa, NJ

1. IntroductionCarrageenin, from the Irish word “carraigin” meaning Irish moss, refers not

only to a species of red alga Chondrus crispus found along rocky areas of theAtlantic coast of the British Isles, Europe, and North America, but also refersto its mucopolysaccharide extract, discovered by the British pharmacistStanford in 1862. The name was later changed to carrageenan so as to complywith the “-an” suffix for polysaccharides. Structurally, the carrageenans are acomplex group of polysaccharides made up of repeating galactose-relatedmonomers and are of three main types; lambda, kappa, and iota (see Chapter33, Note 1). Each has their own gel characteristics which are all thermally re-versible. The lambda form does not gel strongly at room temperature and is inject-able to induce an inflammatory response. Inflammation induced by carrageenan,originally described by Winter (1), is acute, nonimmune, well-researched, andhighly reproducible. Cardinal signs of inflammation—edema, hyperalgesia,and erythema—develop immediately following subcutaneous injection, result-ing from action of proinflammatory agents—bradykinin, histamine, tachy-kinins, complement and reactive oxygen, and nitrogen species. Such agentscan be generated in situ at the site of insult or by infiltrating cells. Neutrophilsreadily migrate to sites of inflammation and can generate proinflammatoryreactive oxygen and other species. The inflammatory response is usually quan-tified by increase in paw size (edema) which is maximal around 5 hpostcarrageenan injection (see Fig. 1) and is modulated by inhibitors of spe-cific molecules within the inflammatory cascade. The nonsteroidal antiinflam-matory drug (NSAID) indomethacin is a clinically useful example (see Fig. 1).The model, therefore, has had, and will continue to have, a vital role in noveldrug development.

Carrageenan-Induced Paw Edemain the Rat and Mouse

Christopher J. Morris

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2. Materials1. Animals

Species: Rat MouseStrain: Wistar, Albino SwissSex: Male MaleWeight: 165–220 g 25–35 gHealthy animals were kept in approved plastic cages, with metal mesh lids andbottoms, at a temperature of 20 ± 2°C and were exposed to a 12-h light dark cycle.Food and water was supplied ad libitum throughout the duration of the study.

2. Carrageenan (lambda form, FMC Marine Colloids Division, NJ, or type IV, Sigma-Aldrich, Poole, UK) was prepared as a 1% W/V solution in 0.9% saline, no morethan 24 h before use. Carrageenan powder becomes extremely sticky on contactwith water and may form lumps that are difficult to dissolve. Complete solutionof solid material is vital to prevent blockage of the hypodermic needle bore andpotential injury to the investigator by pressurized ejection of the needle from thesyringe or breakage of the syringe barrel in the hand (see Note 1).

3. 25-gauge hypodermic needles five-eighths-in long. Becton Dickinson, Oxford,UK. www.bd.com

4. 1-mL disposable plastic syringes. Becton Dickinson, Oxford, UK. www.bd.com5. 100-μL gastight syringe. Hamilton Co., 1700 series, Cat. No. 81001

www.hamiltomcompany.com6. Plethysmometer, Cat. No. 7150 www.ugobasile.com7. Digital calipers with computer link. Digimatic 500, www.mitutoyo.com

Fig. 1. Inflammatory response post-carrageenan injection.

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3. Methods

3.1. Induction of Inflammation1. Animals are weighed, randomized into groups (n=6), and kept for 1 wk to accli-

matize to the laboratory conditions. This helps to keep stress levels low, which isimportant for the development of a good inflammatory response.

2. All animals were marked on the tail with an indelible pen for identification.3. If required, test compounds are administered to animals at an appropriate time-

point before carrageenan injection. The timing will depend on the pharmacologi-cal profile of the compound or may be determined by time-course studies. Effectsof unknown compounds are usually compared to reference compounds whosepharmacology and action in this model are known. NSAIDs [such as indometha-cin (5 mg/kg per-orally)] are good examples (see Subheading 3.5.).This model is unsuitable for comparative studies where individual drugs cause asignificant fall in blood pressure. This compromises the oedematous response.

4. Volume of preinjection paw/paws measured immediately prior to carrageenaninjection. (For more details, see Subheading 3.2.).

5. 100 μL (rat) or 25 μL (mouse) of a 1% solution of lambda carrageenan in 0.9%saline is injected subcutaneously into the plantar region of the left hind paw.Injections are performed in lightly anesthetized (ketamine hydrochloride, 20 mg/kg, intraperitoneal) animals using a five-eighths-in, 25-gauge needle insertedinto the pad region of the glabrous skin on the underside of the paw. The injec-tion site lies close to the center of the plantar region, an important point in rela-tion to the mouse which does not have a well-defined pad region on the undersideof the hindpaw (see Note 2).

6. Carrageenan injected and control paw volumes are measured hourly as requiredfrom 1–6 h and again at 24 h (For more details, see Subheading 3.2.)

7. Animals are killed after the final assessment and paws removed by cutting at thetibio-tarsal level. Paws may then be used for a wide variety of assays linked tothe inflammatory response, though detailed protocols are beyond the scope ofthis chapter. Paw tissue may be examined immunohistochemically or by in situhybridization for cellular localization and quantification of specific proteins, i.e.,growth factors, cytokines, tachykinins, or homogenized and extracted for themeasurement of, for example, prostaglandin E2 and elastase activity (2) andcyclooxygenases (3). Edema fluid may also be removed by centrifugation ofwhole paws and direct measurement of specific molecules made (4).

3.2. Quantification of Paw SwellingIn the case of the rat paw, there are three commonly used methods, the one

selected being dependent on equipment available and personal preference.Results from the various methods correlate well (5).

1. The most basic method (which is not suitable for mice), requiring minimal equip-ment but much practice, is measurement of paw circumference (PC) by a length ofcotton thread looped round the paw at the metatarsal level and gently tightened.

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The loop is then carefully opened out and its length measured to the nearestmillimetre with a ruler. Accurate measurement is dependent on near immobilityof the paw and to this end rats are placed in a plastic cylinder (20 × 6 cm), and thetail and paws left free. The preinjection PC of the left hind paw is measured fol-lowed by hourly measurements of the same paw as detailed in Subheading 3.1.6.

2. Edema may also be assessed by measurement of paw thickness in the dorsal-plantar axis at the metatarsal level by caliper. The point of measurement shouldbe premarked on the top of the foot with an indelible pen for reference at subse-quent measurements. If modern electronic digital calipers are used, multiple mea-surements may be made at a given time-point and the results fed directly into acomputer. An index of paw thickness is calculated as mean difference of pawthickness ( paw thickness/thickness of the contralateral paw).

3. The most convenient, rapid, and accurate assessment technique and the one mostapplicable to the mouse paw is plethysmometry. A plethysmometer directly mea-sures changes in paw volume by water displacement and consists of two verticalwater-filled interconnecting perspex tubes (see Fig. 2A,B). The larger one (A -18-mm diameter) is used to measure fluid displaced by the paw, a volume change thatis precisely mirrored in the smaller tube containing a transducer. The transducer islinked to a decoder capable of digitally displaying volumes and/or feeding datadirectly to a computer.The paw is immersed in vessel A until a fixed and visible anatomical point on theankle and the chamber water meniscus coincide. At this point, several recordingsare made. The most visible point at the ankle is the lateral malleolus and a lineshould be marked on the skin just above this protrusion with an indelible pen toallow easy and accurate repeated measurement. Accuracy is also helped by keep-ing the foot steady during the measurement process.The volume of both the contralateral and injected paws can be measured at eachtime-point and a volume increase calculated by subtraction. A far simpler andequally effective protocol is to measure the left paw volume for each animal priorto carrageenan injection and then at hourly intervals (see Note 3).

3.3. Results and Statistics

Values are expressed as mean ± standard error of the mean (SEM). Datawere analyzed by analysis of variance (ANOVA) followed by post hoc analysiswith a one-tailed Dunnett’s t-test for multiple comparisons. Comparisons aremade with preinjection values or with the contralateral paw as appropriate tothe protocol adopted.

3.4. Applications

Inhibition of carrageenan-induced inflammation has been shown to be highlypredictive of antiinflammatory drug activity in human inflammatory diseaseand doses of NSAIDs in this model correlate well with effective dose in patients(6). Using antagonists of various mediators of inflammation, Di Rosa et al. (7)showed that the inflammatory response to carrageenan consisted of three

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phases. The primary phase mediated by both histamine and 5-hydrox-ytryptamine, is followed by a secondary kinin-mediated phase notably theendogenous nonapeptide bradykinin produced by kallikrein. Novel histamineH3-receptor agonists (8) bradykinin agonists (9) and bradykinin receptor antag-onists (10) have been assessed in this model. The final phase is attributed tolocal production of prostaglandins (PG), especially those of the E series. Theprecursor of both PGs and thromboxanes is PGH2, derived from arachodonicacid by the action of cyclooxygenase (COX) enzymes. Inhibition of theseenzymes is the basis of action of the NSAIDs of major clinical importance inthe treatment of pain and inflammation (11). Carrageenan paw edema has,therefore, been a vital tool in the development of NSAIDs and recently devel-oped novel COX inhibitors. A role for neutrophil derived reactive oxygen spe-cies, nitric oxide, and peroxynitrite in carrageenan-induced inflammation hasalso been identified and a number of specific inhibitors have been identified(2,12) which have potential clinical use.

Recently, the paw edema model has become popular as a model of localizedinflammatory pain. Several behavioural and electrophysiological studies haveshown that, after injection of carrageenan, initial edema development is fol-lowed by a period of allodynia, which peaks around 4 h after inoculation andlasts 24–96 h (13).

The literature on pharmacology and specific modulation is extensive.

Fig. 2. A water displacement plethysmometer.

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4. Notes1. The lumpy carrageenan problem may largely be avoided by adding the powder

to saline at 4°C and immediately stirring rapidly to facilitate even distribution.Any remaining lumps are very difficult to dissolve and should be dispersed byhomogenisation. Warming to 50°C with stirring speeds up the solubilizationprocess.

2. At injection, the needle should be directed along the center line of the paw for adistance of 6 mm in the direction of the toes and at an angle of about 5° to theplantar surface. The needle point should be bevel downward and a new needleshould be used for each injection. Slow injection of the viscous carrageenan,followed by brief gentle massage of the plantar region with a finger, gives gooddistribution in the tissue.

3. Some authors measure the baseline foot volume immediately after carrageenaninjection to negate the effect of the injected fluid volume. Experiments in thislaboratory show that this is not necessary.

References1. 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. 111, 544–547.

2. Rioja, I., Ubeda, A., Terencio, M., Guillen, I., Riguera, R., Quintela, J. M., et al.(2000) An inflammatory ditriazine inhibiting leukocyte functions and expressionof inducible nitric oxide synthase and cyclo-oxygenase-2. Eur. J. Pharmacol. 397,207–217.

3. Nantel, F., Denis, D., Gordon, R., Northey, A., Cirino, M., Metters, K. M., et al.(1999). Distribution and regulation of cyclooxygenase-2 in carrageenan-inducedinflammation. Br. J. Pharmacol. 128, 853–859.

4. Smith, C. J., Zhang, Y., Koboldt, C. M., Muhammad, J., Zweifel, B. S., Shaffer,A., et al. (1998) Pharmacological analysis of cyclo-oxygenase 1 in inflammation.Proc. Natl. Acad. Sci. USA 95, 13,313–13,318.

5. Eschalier, A., Kayser, V., and Guilbaud, G. (1989). Influence of a specific 5-HT3antagonist on carrageenan-induced hyperalgesia in rats. Pain 36, 249–255.

6. Otterness, I. G., Wiseman, E. H., and Gans, D. (1979). A comparison of the carra-geenan edema test and the ultraviolet light-induced erythema test as predictors ofthe clinical dose in rheumatoid arthritis. Agents Actions 9, 177–183

7. Di Rosa, M., Giroud, J. P., and Willoughby, D. A. (1971). Studies of the media-tors of the acute inflammatory response induced in rats in different sites by carra-geenan and turpentine. J. Pathol. 104, 15–29.

8. Rouleau, A., Stark, H., Schunack, W., and Schwartz, J-C. (2000). Anti-inflamma-tory and anti-nociceptive properties of BP 2-94, a histamine H3-receptor agonistprodrug. J. Pharmacol Expt. Ther. 295, 219–225.

9. Wirth, K., Hock F. J., Albus, U., Linz, W., Alpermann, H. G., Anagnostopoulos,H., et al. (1991). Hoe 140 a new potent and long-acting bradykinin antagonist. Br.J. Pharmacol. 102, 774–777.

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10. Asano, M., Hatori, C., Inamura, N., Sawai, H., Hirosumi, J., Fujiwara, T., et al.(1997). Effects of a nonpeptide bradykinin B2 receptor antagonist FR167344,on different in-vivo animal models of inflammation. Br. J. Pharmacol. 122,1436–1440.

11. Vane, J.R. and Botting, R. M. (1995). New insights into the mode of action ofanti-inflammatory drugs. Inflamm. Res. 44, 1–10.

12. Jadot.G., Michelson, A.M., and Puget K. (1986) Anti-inflammatory activity ofsuperoxide-dismutases: inhibition of carrageenan induced edema in rats. Free.Rad. Res. Commun. 1, 395–403.

13. Fletcher, D., Kayser, V., and Gilbaud, G. (1996). Influence of timing on the anal-gesic effect of bipuvicaine and epinephrine infiltration in carrageenan injectedrats. Anaesthesiology 84, 1020–1026.