In vivo animal models in preclinical evaluation of anti-inflammatory ...

Available online at www.ijpras.comcarrageenan (1%). After 4 h the animals were killedwith an overdose of ether, the thorax was openedand the pleural cavity was washed with 1.0 ml ofsterile PBS, containing heparin (20 IU per ml).Samples of the pleural lavage were collected fordetermination ofexudation, myeloperoxidase ,adenosine-deaminase activities, and nitric oxidelevels, as well as for determination of total anddifferential leukocyte counts . Total leukocytecounts were performed in a Neubauer chamber.The cytospin preparations of pleural wash werestained with May–Grunwald Giemsa for thedifferential count which was performed under anoil immersion objective. The serum level of the C-reactive protein was also analysed. In another set ofexperiment animals were treated 30 min beforecarrageenan with a solution of Evans blue dye (25mg/kg, i.v.) in order to evaluate the degree ofexudation in the pleural space. A sample (500 µl)of the fluid leakage collected from the pleuralcavity was stored in a freezer (−20 °C) to furtherdetermine the concentration of Evans blue dye. Tothis end, on the day of the experiments, a batch ofsamples was thawed at room temperature. and theamount of dye was estimated by colorimetry usingan Elisa plate reader at 600 nm, by interpolationfrom a standard curve of Evans blue dye in therange of 0.01 to 50 µg/ml.Evaluation: One ml (the added Hank’s solution) issubtracted from the measured volume. The valuesof each experimental group are averaged andcompared with the control group. ED50 values canbe calculated using various doses. [9][2]7. Granuloma pouch technique: With theintroduction of an irritant substance into an s.c. airpocket, granulation tissue begins to proliferate andsoon covers the whole inside of the pouch. Thistissue consists of fibroblasts, endothelial cells andan infiltrate of macrophages andpolymorphonuclear leukocytes. In the GPA thisrapidly growing tissue can be exposed tocarcinogenic and mutagenic sub-stances. One ofthe major advantages of the system is thepossibility of bringing the test compounds intodirect contact with the target cells, by injectingthem into the air pocket. It is also possible toadminister the material by the oral and parenteralroutes. It does not provide quantitative informationon cytotoxicity of the test compounds in vivo.Procedure: Male or female Sprague-Dawley ratswith a body weight between 150 and 200 g areused. Ten animals are taken for controls and fortest groups. The back of the animals is shaved anddisinfected. With a very thin needle apneumoderma is made in the middle of the dorsalskin by injection of 20 ml of air under etheranesthesia. Into the resulting oval airpouch 0.5 mlof a 1% solution of Croton oil in sesame oil isinjected avoiding any leakage of air. Forty-eighthours later the air is withdrawn from the pouch and72 h later any resulting adhesions are broken.Instead of croton oil 1 ml of a 20% suspension ofcarrageenan in sesame oil can be used as irritant.Starting with the formation of the pouch, theanimals are treated every day either orally orsubcutaneously with the test compound or thestandard. For testing local activity, the testcompound is injected directly into the air sac at thesame time as the irritant. On the 4th or the 5th daythe animals are sacrificed under anesthesia. Thepouch is opened and the exudate is collected inglass cylinders. The pouches are washed with 1 mlof saline, exudates are immediately cooled on iceand the volume is recorded. Total no. of leukocytesmigrated into the pouch are evaluated after stainingwith Erythrosine B and remaining exudates iscentrifuged at 3000 rpm for 10 min at 4 degreesand supernatant stored at -20 degrees until use.Evaluation: The average value of the exudate ofthe controls and the test groups is calculated.Comparison is made by statistical means. [10][11][2]Methods to evaluate proliferative phase ofinflammation:1. Cotton wool granuloma: The foreign bodygranulomas were provoked in rats by subcutaneousimplantation of pellets of compressed cotton. Afterseveral days, histologically giant cells andundifferentiated connective tissue can be observedbesides the fluid infiltration. The amount of newlyformed connective tissue can be measured byweighing the dried pellets after removal. Moreintensive granuloma formation has been observedif the cotton pellets have been impregnated withcarrageenin.Procedure: Male rats weighing 180–200 g wereused. Test drugs were administered orally on aonce daily dosage regimen for 7 days, and thecontrol group received vehicle. Two sterilizedpellets of cotton wool were implantedsubcutaneously, one on each side of abdomen ofthe animal, under the light ether anesthesia andsterile technique. The rats were sacrificed on theeighth day. The implanted pellets were dissectedout and recorded for wet weight. Thymuses werealso dissected out. Both pellet and thymus weredried at60 ◦C for 18 h and the dry weight wasrecorded.Evaluation: The weight of the transudate and thegranuloma as well as the percent granulomainhibition of the test drugs were calculated. Thebody weight gain was also recorded. [12][13][2][7]2. Glass rod granuloma: These reflects thechronic proliferative inflammation. Of the newlyformed connective tissue not only wet and dryweight, but also chemical compositionandvmechanical properties can be measured.Procedure: Glass rods with a diameter of 6 mm arecut to a length of 40 mm and the ends rounded offby flame melting. Male Sprague-Dawley rats withan initial weight of 130 g are anaesthetized with4

Available online at www.ijpras.comether, the back skin shaved and disinfected. Froman incision in the caudal region a subcutaneoustunnel is formed in cranial direction with a closedblunted forceps. One glass rod isintroduced intothis tunnel finally lying on the back of the animal.The incision wound is closed by sutures. Theanimals are kept in separate cages. The rods remainin situ for 20 or 40 days. Treatment with drugs iseither during the whole period or only during thelast 10 or 2 days. At the end the animals aresacrificed under CO 2 anesthesia. The glass rods areprepared together with the surrounding connectivetissue which forms a tube around the glass rod. Byincision at one end the glass rod is extracted andthe granuloma sac inverted forming a plain piece ofpure connective tissue. Wet weight of thegranuloma tissue is recorded. Finally, thegranuloma tissue is dried and the dry weight isrecorded. In addition, biochemical analyses, suchas determination of collagen andglycosaminoglycans can be performed.Evaluation:Several parameters can be determinedby this method. Granuloma weight was reduced bytest compound is compared with that of standard. [2]“Cite this article”Mitul Patel, Murugananthan, Shivalinge GowdaK,P “In Vivo Animal Models in PreclinicalEvaluation of Anti-Inflammatory Activity-AReview” Int. J. of Pharm. Res. & All. Sci 2012;Volume 1, Issue 2, 01-05Bibliography:1. Harsh M. Text book of Pathophysiology. 5 th ed.New Delhi: Jaypee publication. 2005. p. 126-34.2. Gerhard vogel H, Wolfganga HV, BernwardAS, Jurgen S, Gunter M, Wolfganga FV. Drugdiscovery and evaluation pharmacologicalassays.2 nd ed , Berlin, Germany : Spinger;2002. p. 725-71.3. Snyder. DS. Effect of topical indomethacin onuvr-induced redness and prostaglandin e levelsin sunburned guinea pig skin. Prostaglandins.1976;11(4):631-43.4. Yashraj.Y, Mohanty.P.K, Kasture.S.B. Antiinflammatoryactivity of hydroalcoholicextract of Quisqualis indica Linn. flower inrats. International Journal of Pharmacy & Lifesciences. 2011;2(8):977-81.5. Tadafumi.T, Toru.A, Kenji.O, Haruhiko.M.The effects of olopatadine hydrochloride onthe number of scratching induced by repeatedapplication of oxazolone in mice. Eur JPharmacol. 2005;524:149-54.6. Rogerio.A.S, Mariana.K.A.Araruna,Romagna.C.Oliveira, Kleber.D.P.Menezes,Gerlania.O.Leite, Marta.R.Kerntopf, et al.Topical anti-inflammatory effect of Caryocarcoriaceum Wittm. (Caryocaraceae) fruit pulpfixed oil on mice ear edema induced bydifferent irritant agents. J Ethnopharmacol.2011;136:504- 10.7. Gupta.SK. Drug screening methods. 2 ed. NewDelhi: Jaypee; 2009.8. Penna.S.C, Medeiros.M.V, Aimbire.F.S.C,Faria-Neto.H.C.C, Sertie.J.A.A, Lopes-Martins.R.A.B. Anti-inflammatory effect ofthe hydralcoholic extract of Zingiber officinalerhizomes on rat paw and skin edema.Phytomedicine. 2003;10:381–5.9. Vargas.A.J, Geremias.D.S, Provensi.G,Fornari.P.E, Reginatto.F.H, Gosmann.G, et al.Passiflora alata and Passiflora edulis spraydriedaqueous extracts inhibit inflammation inmouse model of pleurisy. Fitoterapia.2007;78:112-9.10. Maier.P, Manser.P, Zbinden.G. Granulomapouch assay i. induction of ouabain resistancein vivo. Mutation Research. 1978;54:159--65.11. Martin.S.W, Stevens.A.J, Brennan.B.S,Davies.D, Rowland.M, Houston.J.B. The sixday-oldrat air pouch model of Inflammation:Characterization of the inflammatory responseto carrageenan. JPM. 1994;32(3):139-47.12. Intahphuak.S, Panthong.A, Kanjanapothi.D,Taesotikul.T, Krachangchaeng.C, Reutrakul.V.Anti-inflammatory and analgesic activities ofMallotus spodocarpus Airy Shaw. JEthnopharmacol. 2004;90:69-72.13. Smita.S, Shwetha.K, Prabhu.K, Maradi.R,Bairy.KL, Shanbhag.T. Evaluation ofantiinflammatory activity of Tephrosiapurpurea in rats. Asian Pac J Trop Med.2010:193-5.5