Buffalo Bulletin (June 2009) Vol.28 No.2

EFFECT OF PIROXICAM ON THE CELLULAR RESPONSEIN BUFFALO CALF SKIN

Neelu Gupta1, A.K. Katiyar2 and Madhu Swamy2

1Department of Veterinary Public Health, College of Veterinary Science & A.H. Anjora, Durg, (Chhattisgarh)India, E-mail: gupta.neelu8@gmail.com2Department of Pathology, College of Veterinary Science and A.H. JABALPUR (M.P.) India

ABSTRACT

The present work was conducted inapparently healthy buffalo calves 3-6 months in age.Calves were pretreated with piroxicamintramuscularly 30 minutes prior to intradermalinjection of Steph. epidermidis suspension andturpentine. Lesions of different time intervals wereobtained for the sequential study of cellularresponses. Maximal suppression of leukocytesoccurred at 3 h in both types of inflammation. In allthe cases, neutrophils were more extensivelysuppressed as compared to other cells.

Keywords: Staphylococcus epidermidis,turpentine, piroxicam, neutrophils, monocytes,lymphocytes, basophils

INTRODUCTION

Specific antagonistic drugs have been usedearlier in buffalo calves (Gupta et.al., 2007, 2008a,2008b) to study the chemical mediation of acuteinflammation in this species. Piroxicam is a cyclo-oxygenase inhibitor and specifically blocks thesynthesis of prostaglandins. However, to ourknowledge, no such studies have been conducted inbuffaloes. Thus, in the present study, the possiblesuppression of the cellular response in theinflammation induced by turpentine and Staph.epidermidis was studied in the buffalo calvespretreated with piroxicam.

MATERIALS AND METHODS

Twelve healthy male buffalo calves, aged 3to 6 months, were divided into two groups: controland experimental, for the study of cellular responsein the buffalo calf skin. The calves were maintainedunder hygienic conditions and fed standard feed.

Substances-Normal saline: (WockhardtLtd. Aurangabad ) 0.9 % w/v, sterile pyrogen free,isotonic solution was used for the preparation of thebacterial suspension.

Staphylococcus epidermidis: (MTCC-35)the culture was obtained from the Institute ofMicrobial Technology, Chandigarh. A bacterialsuspension was prepared, and 0.1 ml was injectedintradermaley at each site. The concentration of thebacteria per ml of the suspension was determinesas 2.3 x 103 million.

Turpentine- (SAM KAM, INDORE) thecommercially available turpentine was usedintradermally for the induction of inflammation. Ateach site 0.05 ml turpentine was injected.

Piroxicam- (Pfizer Limited Batch No.020-04065 Mumbai) of 20 mg /ml strength was used asa prostaglandin antagonist.

Preparation of skin- The site of thecutaneous reaction was prepared according to themethod described in horses (Zarrilli and Calhoun,1970) with suitable modifications. Briefly, one daybefore induction of the inflammation, hair from thelateral thoraco-abdominal region of the buffalocalves was removed by close shaving. The skin wascleaned with a soft cloth moistened with the steriledistilled water. On the following day, cleaning of theskin was repeated.

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Induction of inflammation- Inflammationwas induced in the buffalo calf skin as described inthe following groups.

Control Group I- Six calves of the controlgroup were again divided into two subgroups, i.e.,Subgroup I A and Subgroup I B. Each subgroup hadthree calves.

Subgroup IA- Each calf received twointradermal injections of the Steph. epidermidis(MTCC-35) suspension (0.1 ml) in the normal saline(Wockhardt Ltd. Aurangabad) for each time interval(0-2 minutes, 30 minutes, 1 h, 3 h, 6 h, 12 h, 24 h and48 h) at both sides of the thoraco-abdominal region.At the appropriate time (immediately after the 0-2time interval. The calves were euthanized with asaturated solution of magnesium sulphate givenintravenously. The skin specimens were collectedand fixed in the Cornoy’s fluid for the histo-pathological studies as described for the chicken byShrivastava et al., 1997. Sections were cut to 4-5μm thickness.

Skin sections were stained with thehaematoxylin and eosin and with 0.05 percentsolution of the toluidine blue in acetate buffer (pH3.8) for basophils as described for the chickens byDhodapkar et al. (1987).

Subgroup IB- Each calf received two i/dinjection of the turpentine (SAM KAM, INDORE)@ dose rate of 0.05ml for each time interval (0-2minutes, 30 minutes, 1 h, 3 h, 6 h, 12 h, 24 h and48 h). The rest of the procedure was the same asthat for Subgroup IA.

Experimental Group II- Six calves of theexperimental group were again divided into twosubgroups i.e., Subgroup II A and Subgroup II B.Each subgroup had three calves.

Subgroup II A- Each calf was pretreatedwith piroxicam (Pfizer Limited Batch No. 020-04065, Mumbai) i/m, at the dose rate of 0.3 mg/kgbody weight 30 minutes prior to the i/d Steph.epidermidis and this treatment was repeated every12 h. The rest of the procedure was the same asthat for control group IA.

Subgroup II B- Each calf was pretreatedwith piroxicam i/m, at the dose rate of 0.3 mg/kgbody weight 30 minutes prior to i/d injection of theturpentine (0.05 ml), and this treatment was repeatedevery 12 h. The rest of the procedure was the sameas that for control group IA.

RESULT AND DISCUSSION

Control Group ISubgroup IA- The inflammatory reaction

in the calf was induced by giving intradermalinjections of Steph. epidermidis, and lesions of 0-2minutes, 30 minutes, 1 h, 3 h, 6 h, 12 h, 24 h and 48h were collected for the histopathology. The vascularand cellular changes were not noticeable at the initialintervals (0-2 minutes and 30 minutes). However, afew neutrophils were found at 0-2minutes and 30minutes. At 6 h, hyperaemia of the blood vesselsand oedema of the dermis were well marked. From30 minutes onwards, leukocytes infiltration hadsignificantly increased, and the maximal number ofleukocytes were observed at 12 h. The maximumnumbers of neutrophils, monocytes, lymphocytes andbasophils were recorded at 6, 12, 24 and 1 h,respectively. Infiltration of eosionophils was notnoticed at any time interval (Table 1)

Subgroup IB- The inflammatory reactionin the calf was induced by giving intradermalinoculations of turpentine (0.5 ml), and lesions ofdifferent time intervals were collected forhistopathology. The vascular and cellular changeswere noticed from 30 minutes onward to 48 h. Afew leukocytes were seen adhering to endothelium.From 30 minutes onwards cellular infiltration hadsignificantly increased, and the maximal number ofleukocytes was observed at 12 h. The number ofneutrophils increased gradually up to 6 h, after whichthe number gradually decreased, but there weremore than in the initial stages. The maximal numberof neutrophils was observed at 6 h. The number ofmonocytes increased gradually up to 12 h, afterwhich it decreased. Marked infiltration of monocyteswere observed at 12 h. From 1 h onwards infiltrationof lymphocytes increased, and the maximal numberof lymphocytes was recorded at 48 h. Toluidine bluesections showed infiltration of basophils. Infiltrationof basophils were observed from 30 minutes andgradually increased up to 3 h. Infiltration ofeosionophils was not noticed at any time interval(Table 2).Experimental group II-

Subgroup II A- Calves were pretreatedwith Piroxicam intramuscularly 30 minutes prior tointradermal injection of the Steph. epidermidissuspension, and lesions of different ages were

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Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 1. Section of buffalo calf skin 30 minutes after intradermal injection of Staph. epidermidis suspension. Note - emigration of leukocytes. H &E x 400.Figure 2. Section of buffalo calf skin 30 minutes after intradermal injection of turpentine. Note - emigration of leukocytes. H &E x 400.Figure 3. Section of buffalo calf skin 12 h after intradermal injection of Staph. epidermidis suspension. Note- intense infiltration of leukocytes. H&E x 400.Figure 4. Section of buffalo calf skin 12 h after intradermal injection of turpentine. Note- maximal number of leukocytes. H&E x 400.Figure 5. Section of piroxicam preatreated buffalo calf skin 3 h after intradermal injection of Satph. epidermidis suspension. Note-maximal suppression of leukocytes. H&E x 400.Figure 6. Section of piroxicam preatreated buffalo calf skin 3 h after intradermal injection of turpentine. Note-maximal suppression of leukocytes. H&E x 400.Figure 7. Section of buffalo calf skin 48 h after intradermal injection turpentine. Note- completely disintegrated neutrophils. H&E x 400.Figure 8. Section of buffalo calf skin 12 h after intradermal injection turpentine. Note- the presence of giant cells in the interstitium. H&E x 400.Figure 9. Section of piroxicam preatreated buffalo calf skin 12 h after intradermal injection of Turpentine. Note- the presence of giant cells in the interstitium. H&E x 400.

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Buffalo Bulletin (June 2009) Vol.28 No.2

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Buffalo Bulletin (June 2009) Vol.28 No.2

obtained and processed for histopathlogicalexamination. The vascular and cellular changes werenoticed from 30 minutes onward to 48 h. Themaximal number of leukocytes was observed at 12h, but there were fewer than in the control group.However, maximal suppression of leukocytes wasobserved at 3 h. Infiltration of neutrophils was moremarked at 6 h. While maximal suppression ofneutrophils was recorded at 3 h. The maximalnumber of the monocytes was recorded at 12 h,while maximal suppression of monocytes at 6 h.Marked infiltration of lymphocytes was observed at24 h. However, maximal suppression of lymphocyteswas seen at 3 h. Maximal basophil suppression wasobserved at 1 h. Infiltration of eosionophils was notnoticed at any time interval (Table 1).

Subgropup II B- Thirty minutes beforeintradermal injection of turpentine, the calves werepretreated with the piroxicam intramuscularly, andthe lesions as per the non-pretreated group wereobtained and processed for histopathologicalexamination. The vascular and cellular changes werenoticed from 30 minutes onward to 48 h. Themaximal number of leukocytes were observed at24 h. However, maximal suppression of leukocyteswas observed at 3 h. Neutrophils were more markedat 6 h. While maximal suppression of neutrophilswas recorded at 3 h. Monocytes were noted from30 minutes onwards up to 24 h. The maximal numberof monocytes was recorded at 48 h while maximalsuppression of monocytes was at 6 h. Multinucleatedgiant cells were observed at 12 and 24 h.Lymphocytes were noted at 1 h to 48 h. Markedinfiltration of lymphocytes was observed at 48 h.However, maximal suppression of lymphocytes wasseen at 3 h. Few basophils were noted at 30 minutesonward to 24 h. The maximal number of thebasophils was observed at 3 h, and suppression ofthe basophils was noted at 3 h. Infiltration ofeosionophils was not noticed at any time interval(Table 2). Pretreatment with the prostaglandinantagonist piroxicam caused suppression of the totalleukocyte infiltration in both turpentine and Staph.Epidermidis-induced buffalo inflammation. At 3 h,maximal suppression of leukocytes occurred in bothtypes of injury. However, in the turpentine inducedreaction, piroxicam caused maximal suppression ofthe neutrophils, lymphocytes and basophils at 3 h,

and of monocytes at 6 h. Whereas, in the Staph.Epidermidis-injury it resulted in maximalsuppression of neutrophils and lymphocytes at 3 h,monocytes at 6 h, and basophils at 1 h. Issekutz andMovat (1982) studied the effect of prostaglandin onpolymorphonuclear leukocyte infiltration. Theyconcluded that prostaglandins enhance chemotactic-factor-mediated poly-morphonuclear infiltration.Since, piroxicam is a cyclooxygenase pathwayinhibitor through which the prostaglandins areformed, pretreatment with the drug may also causesuppression of neutrophil infiltration. Gupta et al.(2008) reported that intradermal injection of S.epidermidids suspension and turpentine resulted inan inflammatory reaction in buffalo calf skin. Theinflammation-induced vascular permeability wassignificantly suppressed by injection of theprostaglandin antagonist drug piroxicam, indicatingthat the prostaglandins might be responsible forincreased vascular permeability in inflammation. Inthe present study, the greater suppression ofneutrophils indirectly indicates the suppression ofprostaglandin synthesis due to piroxicampretreatment. Taken together our results indicate thatthe prostaglandins may play a role in the mediationin the cellular response in buffalo inflammation.

REFERENCES

Dhodapkar, B.S., J.L. Vegad, R.G. Dhawedkar andG.N. Kolte. 1984. Pathology of reversedpassive arthus reaction in the chicken. AvianPathol., 13: 93-108.

Gupta, N., A.K. Katiyar and M. Swamy. 2007. Roleof histamine in turpentine induced cellularinflammatory response of buffalo calves.Indian Vet. J., 4: 363-364.

Gupta, N., A.K. Katiyar and M. Swamy. 2008a.Effect of promethazine hydrochloride on thevascular permeability and cellular response ofbuffalo calves. Indian Vet. J., 11: 1152-1154.

Gupta, N., A.K. Katiyar and M. Swamy. 2008b.Effect of piroxicam on the vascularpermeability in buffalo calf . Indian Vet. J.,11: 1354-1355.

*Continued on page 72

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ACKNOWLEDGEMENTS

Authors are grateful to the Professor andHead, Department of Animal Reproduction,Gynaecology and Obstetrics, Veterinary College andResearch Institute, Namakkal, Tamilnadu, forproviding ultrasonography instrument for this study.We also thank Dr. K. Krishnakumar, AssociateProfessor, VCRI, Namakkal, for his help in the initialdemonstration of ultrasonography.

REFERENCES

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Kumar, S., F.A.Ahmed and M.S. Bhadwal. 2004.Biometry of female genitalia of Murrah buffalo(Bubalus bubalis). Indian J. Anim. Reprod.,25(2): 143-145.

Madan, M.L. 1990. Factors limiting super-ovulationresponse in embryo transfer programmeamong buffaloes. Theriogenology, 33: 280.

Madan, M.L., S.K. Das and P. Palta. 1996.Application of reproductive technology tobuffalo. Anim. Reprod. Sci., 42: 299-306.

Misra, A.K., M.C. Yadav, and K.T. Motwani. 1988.Successful embryo transfer in a buffalo(Bubalus bubalis).In Proc. of the SecondWorld Buffalo Congress, New Delhi, India,I: 56.

Honparkhe, M., V.K. Gandotra, A.S. Nanda and S.Prabhakar. 2003. A comparison of rectalpalpation and ultrasonography for the detectionof follicle(s) and corpus luteum in pluriparousbuffaloes. Indian J. Anim. Reprod., 24(2):149-151.

Rohilla, N., U. Singh, R.K. Sharma and I. Singh.2005. Ultrasonic ovarian status in summeranestrus postpartum Murrah buffaloes. IndianJ. Anim. Reprod.Indian J. Anim. Reprod.,26(2): 95-98.

Totey, S.M., G. Singh, M. Taneja and G.P. Talwar.1991. In vitro maturation and fertilization offollicular oocytes from buffalo.Theriogenology, 35: 284.

*Continued from page 66

Issekutz, A.A. and H.Z.C. Movat. 1982. The effectof vasodilator prostaglndins onpolymorphonuclear leukocyte infiltration andvascular injury. Am. J. Path., 107: 300-309.

Shrivastava, A.B., J.L. Vegad and A.K. Katiyar.1997. Inflammatory response of skinautograftin in the chicken. Ind. J. Anim. Sci.,67: 387-391.

Spector, T.S. 1989. An Introduction to GeneralPathology, 3rd ed. Churchill Livingstone,Edinburg.

Vegad J.L. 1979 . The acute inflammatory responsein the sheep skin. Vety. Bull. Weybridge., 49:555-561.

Vegad, J.L. and A.K. Katiyar. 1995. The acuteinflammatory response in the chicken. Vet.Bull., 65: 399-409.

Zarrilli, L.W. and M.L. Calhoun. 1970. Cellularresponse to equine encephalomyelitis vaccinein skin window of horses. Amer. J. Vet. Res.,31: 97-102.

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