A new model for standardising and treating thermal injury in the rat

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Method Article

A new model for standardising and treating thermal injury in the rat

Lisa Davenport*, Geoffrey Dobson, Hayley Letson

Heart,Trauma&SepsisResearchLaboratory,CollegeofMedicineandDentistry,JamesCookUniversity, Townsville,Queensland,4811,Australia

ABSTRACT

Thermalburninjurymethodologiesareinconsistentlydescribedwithinthecurrentliterature.Topermitthe advancementofnewtreatmentsthereisanurgentneedforthedevelopmentandstandardisationofanacuterat model. We describe a rat thermal burn model that involves: anaesthesia, chronic catheterisation, skin preparation,baselinehemodynamicandphysiologicalmonitoring,andaquantifiablemethodtoreproducea severefull-thicknessburnsinjuryaffecting30%percentofthetotalbodysurfacearea(%TBSA).Followinga 15minpost-burnperiod,treatmentcommenceswithanacutemonitoringphaselastingupto8h,whichcanbe modifiedaccordingtoindividualprotocols.Thismodelreflectstheclinicalcontinuum-of-carefrompoint-of- injury,a15minambulanceresponsetime,a60minprehospitalphaseandhospitaltreatmentmonitoringphase.

Themodelis validatedwithhistological evidenceof full-thicknessinjury, evidenceof thehypermetabolic response(K+,BaseExcess,lactate)andchangesincompletebloodcounts.

Ît^has^been⁵⁰^years^since^Walkerând^Mason^published^their^widely^popular^“Â^StandardÂnimal^Burn^Model^”^. ^The^model,^however,^lacksquantifiablemethodologyfortheassessmentofburnthickness,surfaceareaburnt

andphysiologicalstatus.

^We^present^a^newstandardisedmethodforevaluationofdrugandinterventionaltherapiesthatmimicthe clinicalscenarioincludingambulanceresponse,pre-hospitalandhospitalphasesafterburn.

creativecommons.org/licenses/by/4.0/).

ARTICLE INFO

Methodname:Severethermalinjuryintheratforevaluationofacuteresuscitation Keywords:Burns,Ratmodel,Thermalinjury,Prehospital

Articlehistory:Received9August2019;Accepted10September2019;Availableonline12September2019

*Correspondingauthor.

E-mailaddress:[email protected](L.Davenport).

https://doi.org/10.1016/j.mex.2019.09.006

creativecommons.org/licenses/by/4.0/).

ContentslistsavailableatScienceDirect

MethodsX

journal homepage:www.elsevier.com/locate/mex

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SpeciﬁcationTable

SubjectArea: MedicineandDentistry

Morespeciﬁcsubjectarea: Laboratory Preclinical

Methodname: SevereThermalInjuryintheRatforEvaluationofAcuteResuscitation Nameandreferenceoforiginalmethod: WalkerHL,MasonAD(1968)Astandardanimalburn.JTrauma8(6):1049-51

Resourceavailability: NA

Methoddetails

Methodologybackground

Thegoalofresuscitationintheacutephaseaftersevereburnsistopreserveorganfunctionwhile avoiding complications of ‘fluidcreep’. The over resuscitation that leads tofluid creepand the associatedcomplications suchas compartment syndromeand pulmonary oedema has not been combatedbyanycurrenttreatment[1].Existingresearchintoimprovingtheformulasandfluidtype haveonlyresultedinminoradvancementsincetheoriginalParklandformulaforfluidresuscitation wasdevelopedover50yearsago[2–4].Astandardisedpreclinicalsevereburnsmodelisneededto allowfornewnoveltherapiesandresuscitationprotocolstobeaccuratelyevaluated.

Burnmethodologyusedinpreclinicalstudiesoftenlacksdetailthatprohibitseasyreplication.A systematicreviewevaluating116studiesconcludedthat moststudieswerepoorlydescribedand lackedreproducibility[5].Thestandardisationofthesevereburnwillallowforbettercomparison betweenstudies.Akeyconsiderationinstandardisingaburnsmodelistheaccuratecalculationofthe percentageofbodysurfaceareainjuredasitdirectlycorrelateswiththedegreeofmetabolicchanges demonstratedonthebloodgasandfullbloodcount[6].Afullthicknessburnterminatesthesensation nerve terminals,reducing pain and discomfort associated with theburns wound, an important considerationforanimalwelfare.However,currentfullthicknessburnstudieswidelyvaryonthe reportedexposuretemperatureandtime;from10scutaneouscontactwith95Cwater[7]to96Cfor 25s[8].Longerexposuresresultingreaterdamagetounderlyingstructuresbelowthedermallayers, making the comparison of organ damage difﬁcult between studies. Standardising the thermal techniqueandexposuretimewillensurethatthedesiredburnsthicknessisachievedwithoutdirectly impactingvisceralorgans.

Burninjuriesoccurinthecommunity,delayingthetimebeforeaninterventioncanbereceived.To replicatethisenvironment,preclinicalstudiesofburninjurymust,therefore,considerthedelayin providinganintervention.Thefirstresponders’arrivaltimevariesaroundtheworldandisinfluenced bygeographicalisolation.Countrieswithadevelopedambulanceservicereportaresponsetimeofless than 15min [9–11]. Time from ambulance response tohospital admission is also an important considerationwhen tryingtoreplicatea real-world scenario.Astandardisedmethodshouldalso incorporatethewidelyacceptedfirstaidofsevereburns,includingcoolingtheburnfor20minand coveringthewound,whicharestandardaroundtheworld[12].Asevereburnsmodelthatreflectsthe clinicalenvironmentimprovesthetranslationalpotentialofpreclinicaldiscoveries.

Animalwelfaredeclaration

All procedures were reviewed and approved by the James Cook University Animal Ethics Committee(ApprovalNumber:A2479).

Animalpreparationandsurgicalinstrumentation

10 Sprague-Dawleyadult male rats(320–340g) wereobtained fromJames Cook University’s BreedingColony,Townsville,Australia.Allanimalswerehousedina14h–10hlight–darkcyclewith freeaccesstofoodandwateradlibitum.Animalswereanaesthetisedwith5%isoﬂuraneinoxygenvia

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noseconeandwerebreathingspontaneously.Tomaintainanaesthesiacontinuous0.5–1.5%isoﬂurane inoxygenwasdelivered,whilemonitoringfortheabsenceofthewithdrawalreﬂexandchangein heart rate. Animals received 0.05mg/kg Buprenorphine Hydrochloride (Temgesic Injection) subcutaneouslybetweentheshoulderblades(abovetheburnarea)forpainrelief.

Underanaesthesia,thehairwasremovedonthedorsaland lateralsurfacesincludingtheleft inguinal area with clippers and Veet depilatory cream. The shaved area was disinfected with povidone-iodine solution and cleaned with 70% ethyl alcohol. All animals were placed on a homeothermicblanket(Physitemp,ADInstruments,BellaVista,NewSouthWales,Australia)andcore bodytemperaturewasmonitoredandrecordedthroughoutthedurationoftheexperimentusinga rectalprobe(T-typePod,ADInstruments)tomaintainacoretemperatureof35.5–37.5C.

Sterilechroniccatheters(AccessTechnologies,Skokie,IL,USA)wereinsertedintheleftfemoral arteryandveinandattachedtoanInstechdualaccessvascularportfittedwithajacket(Walker Scientific,Perth,WesternAustralia).Venousaccessallowedforfluidinfusionandarterialaccess allowedforbloodsamplingandbloodpressuremonitoring.Tofitthechroniccathetersa12mm transverseincisionwasmadeonthedorsalsurfaceof theneckabovethelevelofthe shoulder blades,allowingforthecreationofasubcutaneouspocketthelengthofthebacktothelevelofthe hipsviabluntdissection.Afurtherincisionattheleftinguinalareawasmadetoallowfortheblunt dissection in the subcutaneousspace with long straight forceps, permitting placementof the catheters.Onceinsertedandsecurethecatheterswereflushedwithheparinisedsalinetoensure patencybeforeclosingtheneckandinguinalincisionswith4-0braidedsilksuture.Thistechnique for femoral vascular access has previously been described [13–15]. The arterial catheter is connected to a pressure transducer and Bridge Amplifier/PowerLab (AD Instruments) for continuous blood pressure monitoring. Subcutaneouselectrocardiogram (ECG) leads were also attachedtothefore-andhind-limbpaws forheartratemonitoring.After instrumentation,the animalsremainedanaesthetisedfora30-minstabilisationperiodpriortorecordingtheirbaseline measurements.

Fullthicknessburninjury Surfaceareacalculation

Thetotalbodysurfacearea(TBSA)andtotalsurfaceareaburntwascalculatedusingtheMeeh formula:TBSA=kW^2/3,wherek=9.83,andW=weightinkg[16].

Thermalinjuryapparatus

A cradle witha prefabricated aperture of 68cm² (5cm13.6cm) was created based onthe selectedratweightrangeof320–340g(Fig.1).Thecradlewasconstructedfromcommonlyavailable PolyVinylChloride(PVC)100mmpipewitha3mmwallthicknesstoallowrubbersealtobesecured aroundtheaperture.Theanimalisplacedinthecradleleftlateraldorsalsidedownfirstfollowedby therightlateraldorsalsidetoachieve28.4–29.6%fullthicknessburn(Fig.2).Theholddevicefitsina scaffold,whichensuresthedevice,andthereforetheanimalcanonlybeloweredtojustbelowthe surfaceofthewaterbathandnodeeper,formaximumprotection(Fig.3).Thewaterbath(Thermoline ScientificLaboratory)temperatureissetto96C.

Fig.1.CustomCradlemadefromPolyVinyChloride(PVC)100mmpipewitha3mmwallthickness.

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Afterbaselinerecording,theanaesthetisedanimalisdisconnectedfromtheBridgeAMPandECG leadsaretemporarilyremoved.Thisallowstheanimaltobeplacedinthecradlewiththeleftlateral dorsalsidedownwhiletheisoﬂuranenoseconeremainsattached.Immediatelypriortoperforming thethermalinjury,twoconsecutivetoe-pinchtestsontwodifferentlimbsareperformedtoconﬁrm theappropriatedepthofanaesthesia.Thecradleisthenimmersedinto96Cwaterfor8s,recordedon anelectronictimer.Immediatelyafter,therightlateraldorsalsideisthenexposedfor8s,avoidingthe paws,tailandhead.

Followingburninduction,theanaesthetisedanimalisimmediatelyliftedoutofthecradleandthe dorsalsurfacedriedgentlywithcleanpapertowel.Theanimalisthenplacedproneonaclean,dry sterileunderpadoverahomeothermicblanketsetto37Ctomaintainnormothermia.Continuous hemodynamic monitoring is reconnected including arterial blood pressure, ECG, and rectal temperatureprobe.

Ambulanceresponsephase

Asimulationofthetimeforanambulancetorespondisobservedfor15minwithnointervention.

Onlymonitoringoccurs,andanimalsremainanaesthetisedonamaintenancedoseofisoﬂurane(0.5– 1.5%).

Prehospitalphase

Atthecommencementofthe60-minprehospitalphase,theanaesthetisedanimalshavewater appliedfor20mintotheburnedarea,aspercurrentstandard-of-caretreatmentforburnspatients.To simulatecontinuousrunningwateraroomtemperaturewaterspraybottleisusedcontinuouslyfor 20min,whiletheanimalisproneonthehomeostaticblankettomaintainnormothermia.Theburn siteisthencoveredwitha3MTegadermFilm(Medshop,Australia).Atthistime,itwouldbeclinically appropriatetoadministeraninterventionorresuscitationthroughthevenousaccesstosimulatea paramedicorﬁrstrespondertreatingtheburninjurypriortohospitaladmission.

Hospitalphase

Atthecommencementofthehospitalphase,theisoﬂuranewasceased,andtherectalprobeand ECGleadswereremovedfromtheanimal.Theconsciousanimalsintherecoverycagehadaccessto water,softenedfoodpellets,andDietGelRecovery(ClearH2O).Ahomeothermicblanketwasplaced

Fig.2. Leftlateralsurfacewithschematicrepresentationofsurfacearea.

Fig.3.CradleandWaterBath.

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underthecagetomaintainwarmth(37C)fortheentiremonitoringperiod.Duringthe7-hhospital phase, animals were securedtothe InstechVascular AccessHarness, swivel and tether system, allowingfor continuousarterial monitoring.Thetemperaturewas recorded hourly using a non- invasive Infrared Digital Thermometer. During this phase, an intervention or ﬂuid resuscitation therapymaybeadministeredthroughthevenousaccesstosimulatemedicaltherapyreceivedduring ahospitalstay.Thechroniccatheterscanremaininplaceforupto40daysandthehospitalphasecan beshortenedorextendeddependentonindividualstudyrequirements.

Methodvalidation

Full-thicknessburnwasconfirmedusinghistologicalanalysis.Formalin-fixedskinsampleswere processedusingtheLeicaHistocorePearltissueprocessor,paraffin-embeddedusingtheHistocore Arcadiaembeddingcenter,andsectionedusingtheLeicaRM2255automatedrotarymicrotome.Slides werestainedwithhematoxylinandeosin (H&E)and examinedusinglight microscopy(Olympus CKX41Nikon DigitalSight). Comparedtonon-burned skin,skin samplesfromanimalsreceiving thermalinjuryasdescribedshowedevidenceofinjuryacrossalllayers,includingcompleteepidermal destructionandthermalcoagulativedamagetothefullthicknessofthedermiswithinvolvementof

Fig.4.DeepthicknessburninjuryhistologyinSprague-Dawleyrats8hand15minafterinjury:A)Uninjuredepidermisand dermis,40.B)Burntepidermisanddermis,40.C)Uninjuredhairfolliclewithinthedermallayer,40.D)Burnedhair folliclewithinthedermallayerwithcellulardamageincludingcellswelling,40.

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thesubcutaneousfatand skeletalmuscle (Fig.4b).Burnedhairfolliclesshowed distinct cellular damageandcytoplasmicswelling(Fig.4d).

Thehypermetabolicresponseassociatedwithsevereburnswasvalidatedforthethermalinjury method.Arterialblood gasesand electrolytes(pH,pO2,pCO2,Cl ,Na⁺,K⁺,Ca²⁺,sO2,BaseExcess, HCO3 )andlactateweremeasuredusingtheRadiometerABL800bloodgasanalyser(Radiometer Pacific, Mt Waverley, Victoria), while complete blood counts were obtained using the VetScan HM5Hematologyanalyser(REMSystems,MacquariePark,NewSouthWales).Lactateandpotassium levelswere both significantly increased420min after burn, while baseexcess fell from1.58 to -11.98mM(p<0.001) (Table 1).Totalwhite blood cell countincreasedsignificantly indicating a systemicimmuneresponse(Table2).Whiletotallymphocytesdidnotchange,therewasasignificant decreaseinthelymphocytepercentage,accompaniedbya4.4-foldincreaseinneutrophils(p<0.001).

Platelets remained unchanged, however red cells, hemoglobin and hematocrit all signiﬁcantly increasedasplasmaleaksfromdamagedcapillaries(Table2).

Statistics

SPSSStatisticalPackage24wasusedforstatisticalanalysis(IBM,StLeonards,NewSouthWales).All valuesare expressedas meanSEM. Data normalitywas assessed usingShapiro-Wilk testsand normallydistributeddatawasanalysedusinganindependent-samplest-test.Statisticalsigniﬁcance wassetasp<0.05.

Acknowledgements

SupportforthisstudywasprovidedbyTheKJMcPhersonEducationandResearchFoundation, Queensland Ambulance Service, as well as the College of Medicine and Dentistry, James Cook University.

Table1

BloodChemistryParametersBeforeandAfterBurn.

Parameter Baseline 420min p-valve

Lactate(mmol/L) 0.670.05 1.690.23 <0.001

BaseExcess(mmol/L) 1.581.16 11.981.43 <0.001

Potassium(mmol/L) 4.450.23 6.540.29 <0.001

Table2

HematologyParametersBeforeandAfterBurn.

Parameter Baseline 420min p-value

WhiteBloodCells(10⁹/L) 8.611.07 15.160.94 <0.001

Lymphocytes(10⁹/L) 6.190.74 5.380.38 0.340

Monocytes(10⁹/L) 0.410.10 0.980.14 0.040

Neutrophils(10⁹/L) 2.010.38 8.780.63 <0.001

Lymphocytes(%) 71.902.84 35.751.74 <0.001

Monocytes(%) 5.041.02 6.300.58 0.298

Neutrophils(%) 23.072.67 57.941.64 <0.001

RedBloodCells(10¹²/L) 6.970.23 9.150.17 <0.001

Hemoglobin(g/L) 12.810.41 16.400.29 <0.001

Hematocrit(%) 38.481.33 47.920.89 <0.001

Platelets(10⁹/L) 22146 29619 0.151

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