Glucocorticoid levels in body fluids are frequently used as indicators of stress. Corticosterone is the major glucocortieosteroid secreted in laboratory rodents. Urinary concentrations of eorticosterone were measured as indicators of stress induced in rats by four different anaesthetic regimens. Testostemne levels were also measured, as a potential stress parameter.Three of the four anaesthetic regimens used resulted in increased urine levels of eorticosterone. Elevated levels of testosterone were only observed in one of the treatments.In conclusion, eorticosterone may be a useful indicator of stress associated with anaesthetic regimens in rats. However testosterone does not seem to be as sensitive a parameter as eorticosterone but it may prove to be a useful supplement

Hau, J

O'Brien, Deirdre

Obi, P Tipi

Saibaba, P

Stodulski, G

English

Journals from University of Tartu

Semd. ,1, Lab. Anim Sci N0. 2 . 1993 . V01. 20Comparison of stress induced in rats by four differentanaesthetic regimens as recorded by urinaryconcentrations of corticosterone and testosteroneby Deirdre O’Brien, 1’. Tibz 0171'. G. Stodulski. P. Sazbaba (52 J. HuuLaboratory Animal Science and Welfare, Department ofPathology and Infectious Diseases,Royal Veterinary College, University ofLondorL London, UKIntroductionStress in animals is not always easy to re—cognise or define. [Vraser Pt (11. (1975) de-fined stress as a state that occurs when ananimal encounters adverse physical or emo-tional conditions which cause a disturbanceof its norma1 physiological and mental equi-librium. Glucocorticoids are relased by theadrenal cortex in response to various stres—sors. Corticosterone is the major glucocorti-eosteroid secreted in laboratory rodents(mice and rats) and is frequently used as amarker to measure stress in animals. Themain problem in assessing stress by meansof glucocorticoids is that various factors canaffect the levels of these hormones in bothstressed and unstressed animals. Circulatingtestosterone levels have been suggested as apotential stress parameter in male primates(Sapa/sky 1985) but this finding still re—mains to be confirmed and the potential use-fulness in other species has not been exam-inedAnaesthetic agents provide various leve1s ofanalgesia against pain and discomfort duringa procedure. However, their use is associ—ated with some risk of mortality and un»pleasant effects during induction and reco-very. These drugs frequently affect the car—diovascular, respiratory and thermoregula-tory mechanisms including the central ner-vous system. Wl'zelan & Fleckne/l (1991)defined surgical anaesthesia in laboratoryanimals as the state in which an animal isimmobile, unaware of the procedure beingperformed and has an attenuated stress re—sponse.The objective of this study was to comparefour different anaesthetic regimens in orderto assess which anaesthetic regimen is theleast stressful in rats as measured by the se—cretion of urinary corticosterone and testo-sterone.Materials and methodsA total of 12 male SPF Wistar rats (CharlesRiver, UK) of 5—8 weeks of age were ran-domly divided into groups A, B and C, andallocated in pairs into metabolic cages (24.2cm x 18 cm, area 500 cm2) (Techniplast,Italy). Control animals (group A) were nothandled except during cleaning and weigh-ing procedures. Animals from groups B (2ndcontrol group) and C (experimental group)were handled twice daily between 9.00 amand 4.00 pm. Group C contained four pairsofrats and each pair received one of the fourdifferent anaesthetic regimens on day 14(Table 1). Food (RMI, Special Diet ServiceLtd, Essex) and water were supplied ad lib,except for one hour each day when food waswithheld. This simulated procedures prior toanaesthesia. The temperature range of theanimal room was 20—23’C and re1ative hu—midity in the range of 45—62 %. The lightvariation consisted ofa photoperiod of 12 hlight: 12 h darkness. The rats were grouped14 days prior to anaesthetic induction inorder to recover from transport and adapt totheir new environment. Food and water in-take were measured throughout the experi-ment. Urine samples were collected every 24hours throughout the experiment.113Scand. J. Lab. Anim, Sci. No. 2 . I993 . V01. 20Table 1. Dose and duration 0fthe different anaesthetic agents administered on day 14. Group Anaesthetic Agents Concentration MaintenanceC1 1 part Hypnorm, 2 parts Hypnorm: 2.7 mg/kg HypnormH20 & 1 part Midazolam Midazolam=2 mg/kg 0.1 mg/'kg i.m.mixture injected i.m. after 30 minsC2 Xy1a7ine + Ketamine Xylazine = 3 mg/kg Ketamine(1 :30) injected i.p. Ketamine = 90 mg/kg 25 mg/kg i.m.after 30 minsC3 Mcthoxyflurane : < 4 % 0.4—1 .0 %(volatile)C4 Halothane = 3—4 % 0.5—2 %(volatile) Experimental designThe animal were moved to the metaboliccages on day 0 of the experiment. On day 14the animals in group C were anaesthetisedfor 45 minutes and returned to the meta-bo1ic cages following recovery. The animalswere killed on day 22 by guillotine. Rats ingroup C were weighed and given atropinesulphate (0.04 mg/kg 5.0.) thirty minutesprior to anaesthetic induction. Atropinesulphate reduces salivary and bronchial se—cretions. The first pair (C1) were injectedi.m. with a mixture oi'Hypnorm and Mida-zolam. The second pair (C2) received firstlyXylazine and then Ketamine i.p. The third(C3) and fourth (C4) pairs were anaestheti-sed in an anaesthetic chamber with Me-thoxyfluarane and Halothane respectively.Table 1 summarises the concentrations usedfor induction and maintenance of anaesthe—sia for 45 minutes. Respiration was monito-red every minute by direct clinical observa—tion. Temperatures were recorded using adata logger (LOGIT, Griffin and George,Leicstershire UK). The rats were placed oncotton wool and on a heating pad duringanaesthesia. Room temperature was main—tained at 25°C until the rats were fully re-covered.Measurement ofcortieosterone andtestosteroneThe urine concentrations of corticosteroneand testosterone were measured dailythroughout the study period using commer—cial radioimmunoassay kits (BIOGENESISLIMITED, Bournemouth and IMMUNO-DIAGNOSTIC SYSTEM LIMITED, TyneTable 2. Daily corticosterone (ng/ml) and testosterone (nmolc/ml) in urine beforeand after anaesthesia. 7,, A B C1 C2 C3 C4Corlicosterone 2.41 1.98 1.75 2.43 1.98 2.31Before : 2.25 i 1.9 i 0.77 :- l.1 : (1.71 i 0.78After 1.5 2 4.5"“ 7* 35* 2.5Testosterone 0.47 0.65 0.71 0.72 1.01 0.66Before 10.13 :0.17 :0.18 10.19 :099 :018After 065 0.65 125* 0.9 0.4 0.65* significantly elevated levels.C1 = Hypnorm/Midazolam, C“ = Xylazine/Kctaminc, C3 = Methoxyflurane,C4 = Halothane.114& Wear 11 K respectively). These assays arebased on the competitive double antibodytechnique.Statistica1eva1uationDifferences in food and water intake be-tween groups were evaluated by one wayanalysis of variance. P-Values < 0.05 wereconsidered statistically significant. Whencomparing pre-treatment corticosterone andtestosterone levels with post-treatment, va—lues different from the mean + 2 s. d. wereconsidered significantly different.ResultsThere was no significant variations in foodor water intake in the control and experi-mental groups throughout the study.There was no significant difference in pre-treatment eorticosterone leve1s in urine be-tween handled and non handled animals(Table 2).A significant increase in corticosterone 1e-vels after anaesthesia in groups C1, C2 andC3 was recorded. There was no significantdifference in the corticosterone levels of thecontrol groups and group C4 (Tab1e 2).Testosterone concentrations increased sign—ificantly following anaesthesia in group C1only (Table 2).DiscussionGlueocortieoid levels in body fluids areoften used as indicators of acute stresswhereas they are not useful as measures ofchronic stress and this has been reviewedrecently by Manser (1992). Minor stress cg.blood sampling results in increased plasmalevels of glucocorticoids and in order toavoid this and eliminate the diurnal varia-tion we chose to measure corticosterone inurine collected in metabolic cages. Handlingcan cause significant increases in glucocor—ticoids in animals not habituated to it. Therats from groups B and C were handledtwice dai1y for fourteen days, prior to ana—esthesia in order to reduce stress associatedwith handling.Stand]. Lab.Anim.Sci.No.2 .1993 . Vol.20Three of the four anaesthetics used (Table 2)resulted in increased urine levels of corti-eosterene. Hypnorm/Midazolam and Xyla-zine/Ketarnine are injeetable anaestheticsand resulted in greater increases in corti-costerone synthesis than induction withMethoxyfluarane which is a volatile anae~sthetics. There was no significant increase incorticosterone levels when Halothane (v01a—tile) was used as an anaesthetic. This wasprobably due to the short fast induction ofilalothane. Volatile anaesthetics probablyprovide the safest means (in experiencedhands only) of anaesthetising small inani-mals. The depth of anaesthesia can be ad-justed relatively quickly and easily and nor-mally the animals recover rapidly (Flecknell1991). When anaesthetising small animals,injection of the drug intravenously is oftendifficult and another route must be chosen.The effect of the anaesthetic is slower andthe depth more difficult to ascertain whenusing intraperitoneal or intramuscular rou~tes. Administration of an antagonist canoften be necessary. Some antagonists reducethe analgesia provided by the anaestheticand the animal may experience some dis-comfort.The present results demonstrate that corti-costerone, which is the active glucocorticoidin rodents, may be a useful indicator of shortterm stress associated with anaesthetic regi-mens in rats.Transiently elevated levels of testosteronehave been recorded in baboons followingacute stress (Sapolsky 1985). In the presentstudy elevated levels of testosterone in urinewere only observed after Hypnorrn/Midazo-1am anaesthesia. Although testosterone doesnot seem to be as sensitive a parameter ascorticosterone to measure acute stress inmale rats, it may prove to be a useful sup»plement.AcknowledgementsThis study was kindly supported by SmithKlineBeecham.115Scand. .1. 13h. Anim. Sci. No. 2. 1993 . Vol. 20SummaryGlucocorticoid levels in body fluids are frequentlyused as indicators of stress. Corticosterone is themajor glucocortieosteroid secreted in laboratoryrodents. Urinary concentrations of eorticosteronewere measured as indicators of stress induced inrats by four different anaesthetic regimens. Testo~stemne levels were also measured, as a potentialstress parameter.Three of the four anaesthetic regimens used resul—ted in increased urine levels of eorticosterone.Elevated levels of testosterone were only observedin one of the treatments.In conclusion, eorticosterone may be a useful indi-cator of stress associated with anaesthetic regi—mens in rats. However testosterone does not seemto be as sensitive a parameter as eorticosteronebut it may prove to be a useful supplement.SammendragGlueoeonieoid-niveauer i hlod og/eller urin erhyppigt anvendt som stress-indikatorer. Cortico-steron er det mest betydende glucocorticosteridhos gnavere. Cortieosteron-koncentrationen blevmalt som indicator for stress induceret i rotter iforbindelse med fire forskellige anaestesimetoder.Testostemn niveaucr blcv ligeledes malt som mu~lig stress parameter.Tre af de fire anacstcsimetoder resulterede i egedeniveaucr af eorticosteron i urin. Qgede niveauer afIcstostcron blev kun observeret i forbindelse medcen anaestesimetode.Det konkluderes, at eorticosteron kan were enbrugbar indikator for stress i t'orbindelse medanaestesi af rotter. Imidlertid ser testosteron ikkcud til at were 523 felsom en parameter sem corti-costeron i denne sammenhéeng, men testosteron116kan mz‘iskc bruges sorn et supplement til andrestress-markm‘er.theenveto / K. PUl/mnenKortikosteroidien miiéiriiéi ruumiin nesteiss'zi pide-titan usein stressin osoittajina. Kortikosteroni onlaboratoriojyrsijéiden ensisijaisesti erittfiméi gluko-kortikosteroidi. Tyiiss'a seurattiin rotan virtsankonikosteronipitoisuutta neljan erilaisen nukutu-stavan aiheuttaman stressin osoittajina. Samanai-kaisesti mitattiin myo's tcstosteronin miiéiréia,mahdollisena stressimuuttujana.Knlmessa neljéista tutkitusta nukutustavasta virt-san kortikosteronin maaréi nousi. Testosteroni—mfiiiréi nousi vain yhdessa' nukutustavassa.Pfiateltiin ettéi kortikosteroni sopii rotilla nukutus-stressin osoittajaksi. 'l‘estostcroni ei niiytéi oleyanyhti herkka, multa saattaa osoittautua kfiyttfikel-poiseksi havainnon varmistajaksi.ReferencesFlecknell P.‘ Anaesthesia and post-operative careof small mammals. In Practice, September1991.180—189.Fraser D. JSD Richie & AF Fraser: "Stress” in aVeterinary Context. British Veterinary Jour-nal 1975,131,653—662.Manser CE: The Assessment of Stress in Labora-tory Animals. Published by the RSPC‘A 1992.Sapolrkv RM: Stress-induced suppression 01" testi-eular function in the wild baboon: role ofglucocorticoids. Endocrinology 1985, 116.2273—2278.Whe/an G & PA Flecknell: The Assessment ofdepth of anaesthesia in mammals and man.Laboratory Animals 1991, 26, 153-162.

Comparison of stress induced in rats by four different anaesthetic regimens as recorded by urinary concentrations of corticosterone and testosterone

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Comparison of stress induced in rats by four different anaesthetic regimens as recorded by urinary concentrations of corticosterone and testosterone

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