Alterations in pulmonary structure by elastase administration in a model of emphysema in mice is associated with functional disturbances.

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www.revportpneumol.org

ORIGINAL ARTICLE

Alterations in pulmonary structure by elastase administration in a model of emphysema in mice is associated with functional disturbances

D. Vidal

^a^,^b

, G. Fortunato

^a^,^c

, W. Klein

^d^,^e^,^f

, L. Cortizo

^a

, J. Vasconcelos

^a^,^b

, R. Ribeiro-dos-Santos

^a^,^b

, M. Soares

^a^,^b

, S. Macambira

^a^,^b^,^g^,∗

aLaboratóriodeEngenhariatecidualeImunofarmacologia,CentrodePesquisasGonc¸aloMoniz,Fundac¸ãoOswaldoCruz, Salvador,BA,Brazil

bCentrodeBiotecnologiaeTerapiaCelular,HospitalSãoRafael,Salvador,BA,Brazil

cProgramadePós-Graduac¸ãoemBiotecnologia,UniversidadeEstadualdeFeiradeSantana,FeiradeSantana,BA,Brazil

dFaculdadedeBiologia,InstitutodeBiologia,UniversidadeFederaldaBahia,Salvador,BA,Brazil

eInstitutodeBiociências,InstitutoNacionaldeCiênciaeTecnologiaemFisiologiaComparada,UNESP,RioClaro,SP,Brazil

fDepartamentodeBiologia,FaculdadedeFilosoﬁa,CiênciaseLetrasdeRibeirãoPreto,UniversidadedeSãoPaulo,SP,Brazil

gLaboratóriodeEletroﬁsiologiaCardíaca‘‘ProfAntônioPaesdeCarvalho’’,InstitutodeBiofísicaCarlosChagasFilho, UniversidadeFederaldoRiodeJaneiro,RJ,Brazil

Received6July2011;accepted23December2011 Availableonline17March2012

KEYWORDS Pulmonary emphysema;

Ergometrictest;

Porcineelastase;

C57Bl/6

Abstract Severalexperimentalstudiesofpulmonaryemphysemausinganimalmodelshave beendescribedintheliterature.However, onlyafewofthesestudieshavefocusedonthe assessmentofergometricfunction asanon-invasivetechniquetovalidate themethodology usedforinductionofexperimentalemphysema.Additionally,functionalassessmentsofemphy- sema arerarely correlated with morphological pulmonary abnormalitiescaused by induced emphysema. The present study aimed to evaluate the effectsof elastase administered by trachealpunctureonpulmonaryparenchymaandtheircorrespondingfunctionalimpairment.

This wasevaluated by measuring exercisecapacity inC57Bl/6 mice inordertoestablish a reproducibleandsafemethodologyofinducingexperimentalemphysema.Thirtysixmiceunder- went ergometric testsbefore and28 daysafter elastaseadministration. Pancreaticporcine elastase solution was administered by tracheal puncture, which resulted in a signiﬁcantly decreased exercise capacity, shown by ashorter distance run(−30.5%) anda lower mean velocity(−15%),aswellasinfailuretoincreasetheeliminationofcarbondioxide.Themeanlin- earinterceptincreasedsigniﬁcantlyby50%intrachealelastaseadministration.Inconclusion, applicationofelastaseby trachealfunctioninC57Bl/6inducesemphysema,asvalidatedby

∗Correspondingauthor.

E-mailaddress:simone@bahia.ﬁocruz.br(S.Macambira).

doi:10.1016/j.rppneu.2011.12.007

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morphometricanalyses,andresultedinasigniﬁcantlylowerexercisecapacity,whileresulting inalowmortalityrate.

PALAVRAS-CHAVE Enﬁsemapulmonar;

ensaioergométrico;

Elastasesuína;

C57Bl/6

Alteraçõesnaestruturapulmonardevidoàadministraçãodeelastasenummodelo deenfisemaemratosencontram-seassociadasadistúrbiosfuncionais

Resumo Váriosestudosexperimentaisdeenfisemapulmonaremmodelosanimaistêm sido descritosnaliteraturacientífica.Noentanto,apenasalgunsdestesestudostêmsidoconcentra- dosnaavaliaçãodafunçãoergométricacomotécnicanão-invasivaparavalidarametodologia utilizada para ainduçãodo enfisemaexperimental.Alémdisso, asavaliaçõesfuncionaisde enfisema raramentese encontram correlacionadascom anomaliasmorfológicas pulmonares causadasporenfisemainduzido.Opresenteestudotevecomoobjetivoavaliarosefeitosda elastase administrada porpunçãotraquealno parênquima pulmonareasuadisfunçãofun- cionalcorrespondente. Estafoi avaliadaatravésdamediçãodacapacidadedeexercícioem ratosC57Bl/6, deformaaestabelecerumametodologia reprodutível esegura deinduzir o enfisemaexperimental. Trinta e seisratosforamsubmetidos atestes ergométricos antese 28diasapósaadministraçãodeelastase.Asoluçãodeelastasepancreáticasuínafoiadmin- istradaporpunçãotraqueal,oqueresultounumadiminuiçãosignificativamentedacapacidade de exercício, demonstradapela diminuiçãoda distânciapercorrida (menosde 30,5%)e por umavelocidademédiainferior(menosde15%),assimcomopelaincapacidadedeaumentara eliminaçãodedióxidodecarbono.Aintersecçãolinearmédiaaumentousignificativamenteem 50%naadministraçãotraquealdaelastase.Emconclusão,aaplicaçãodeelastaseporpunção traquealemratosC57Bl/6induzenfisema,conformefoivalidadoporanálisesmorfométricas,e resultounumacapacidadedeexercíciosignificativamentemaisbaixa,emborasetenhaobtido umabaixataxademortalidade.

Introduction

Emphysema is a chronic obstructive pulmonary disease (COPD), limiting air flow during expiration, and is associ- atedwithanabnormalinflammatoryresponseofthelungsto noxiousparticlesorgases(mainlycigarettesmoke).Thisdis- easeischaracterizedbyapermanentabnormaldilatationof alveolarspaces.Thedestructionofpulmonaryparenchyma impairs alveolar gas exchange, compromising thephysical capacityofapatientsinceitisassociatedwithairflowlim- itationsthatarenotfullyreversible.

No therapycapable of reversingemphysematous tissue lesions is available to date and, in severe chronic stages the only treatment that remains is lung transplantation, representingaprocedurewithhighlevelsofmorbidityand mortality.Emphysema,togetherwithothertypesofCOPD, areresponsibleformorethan2.5milliondeathseveryyear, representing the ﬁfth leading cause of mortality in the world.

The severityof itspathology togetherwiththelack of anyeffectivetreatmenttransformsemphysemaintoagreat medical challenge.There is a need for studies aiming to understandthepathogeniccellularmechanismscausingtis- sue destruction in order to elucidate the disease and to open newtherapeutic avenues. Another aggravator is the factthataconsiderablevariationexistsinthecourseofthe diseaseamongdifferentsmokers.Onlyabout15%ofsmokers developCOPD.^1,2

Animal modelsrepresent a fundamental instrument to correlate pre-clinical research with clinical studies. The

experimentalmodelallowsthedetailedinvestigationofdif- ferentfactorsinﬂuencingemphysema.Thesefactorsinclude inﬂammatorycellrecruitment,geneticbackground,abnor- mal matrix repair, lung cell apoptosis, and misbalance betweenapoptosisandreplenishmentofstructuralcellsin thelung.Additionalfactorsareresearchofpotentialthera- peuticagentsandstrategies,suchasadministrationofstem cellsordifferentgrowthfactors.³

UntilnowtherearemanyexperimentalmodelsofCOPD, eachof themwithadvantages anddisadvantages.In real- ity,anumberofapproacheshavebeentriedbecauseuntil nownoneofthemconstitutesamodelthatexactlyrepro- duces all phases of development and clinical features of emphysemaor any other abnormalities that make upthis clinicalentity,theCOPD.Otherfactorthataffectstherepro- ducibilityofdataisthedifferencesinanatomicalstructure withrespecttodevelopment,maturation,structuralorga- nizationofrespiratorybranches,andconstituentcellsand vascularization.⁴Ontheotherside,somecommoncharac- teristicscanbeobservedbetweenanimalmodelandhuman disease.Forexample,rodentsdevelopthephenomenonof metaplasiainduced by reactionto injury,⁵ which is a fre- quentlyobserved responsetoan insult indifferentorgans inhumans,suchasinrespiratorytract,urinarybladder,and esophagus.

The ﬁrst animal model of emphysema was devel- oped more than 40 years ago⁶ and it was evoked in rats by intratracheal instillation of the plant proteinase papain.The experimental modelsof emphysema arecon- tinuously improving and became a research tool for

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several groups of scientists. These include enzyme air- way administration model,^7,8 tobacco smoking model,^9,10 apoptosisinduced emphysemaby intratrachealinstillation ofactivatedcaspase-3,¹¹ AIAT deﬁcient animals,¹² natural mutants,¹³knockoutmice,¹⁴andtransgenicmice.¹⁵Despite themodelchosenallofthempresentrestrictions.^16---22

Taking theseinformations into account,in the present studywechosetoreproducehumanpulmonaryemphysema in mousemodel usingintratracheal instillationof porcine pancreaticelastasebecauseitis usedformorethanthree decadesandhasbeenwellcharacterized.²⁹ Untilnowitis consideredtobethemostconsistentandimpressivemodel ofairspace enlargement.³⁰ This is due totheexposure of elastinﬁberstoporcinepancreaticelastaseleadingtoacute lunginﬂammationandpulmonaryparenchymadestruction.

This modelis very appropriatetoinvestigate the relation betweenstructureandfunctionabnormalitiescharacteris- ticsofemphysemaprogression,aswellaselucidatedfactors associatedto thesedisturbances and collagen remodeling failure related to this pathology.³¹ This model was used toinvestigate and validate a new methodology to evalu- atethedynamiclungmechanicalfunctioninexperimental model ofemphysema.³² The pathogenesis for emphysema includesmanyeventssuchasinﬂammation,elastase,matrix metalloproteaseimbalance,apoptosis,andoxidativestress, whicharereproducedbyelastaseadministrationinanimal models.³³WechosetodevelopthemodelinfemaleC57Bl/6 micebasedonclinicalevidencesthatpointoutwomenare moresusceptible toemphysema developmentthan men³⁴ andonpre-clinicalassaysthatdemonstratedthehighersus- ceptibilityoffemaleA/Jmicetoemphysema.³⁵ Theuseof anappropriatemodelofemphysemaisessentialtoinvesti- gaterelevantquestionsinvolvedinthepathophysiologyof the disease and totest the therapeutic potential of new drugs. Another essential point is the use of methods to evaluatethephysiologicalparametersofrespiratoryinthe emphysemamodel.

Evaluation of pulmonary function and measurement of physiologicalparametersarecrucialtostudiesthat inves- tigatenewtherapeuticstrategies,pathogenesisofdisease and toxicology. There are invasive and noninvasive pulmonary function tests available which are sensitive in detecting abnormalities in mechanical respiratory. The invasive methodology evolution allowed the evaluation of relevant parameters transpulmonary pressure, expiratory and inspiratory ﬂows, such as pulmonary resistance and compliance. Although these procedures give precise data, they involve invasive surgical procedures²³ (oro- tracheal intubated animal) and anesthesia that impose non-physiological conditionsto experimentthat may gen- erate signiﬁcant artifacts, such as decreased respiratory frequency,²⁴ besides the risk of death of animals due to the procedure which implies the use of a larger number of animals per experimental group. The non-invasive methodologystartedintheendof70s²⁵andtherearebasi- cally two types of plethysmograph: double-chamber and single-chamber whole-body instrument. This noninvasive serial measurements reduced the number of animal per study,butlong-termexaminationisnotallowedduetothe stressimposedbyoftheneckcollarrestrained.Noninvasive techniquestoevaluaterespiratoryparametersinconscious mice are easy, convenient, repetitive and reproducible,

appropriate for quick and repeatable screening of respiratory function in a large number of conscious animals,²⁶ however arenotsoaccurate astheinvasive method,and someimportantdataaboutpulmonaryfunctionmaybemis- leading.

This kind of evaluation is essential to reveal informa- tion about pulmonary functionbut does not provide data onthedegreeofimpairmentofphysicalcapacityoftheani- mal. Lüthjeetal.²⁷ demonstratedexercise intoleranceby treadmill asaconsequence of pulmonary emphysemaand its systemic consequence in elastase-induced-emphysema modelintransgenicmice.

Followingthisrational,wesoughttoevaluateadifferent proceduretoestablishexperimentalpulmonaryemphysema in C57Bl/6 usingelastase via tracheal puncture. To prove theefﬁcacy ofemphysemainduction, weinvestigatedthe degreeofimpairmentofphysicalcapacitybytheevaluation ofrespiratoryfunctionduringexercisetestingontreadmill, acquiring data about distance run,exercise time, oxygen consumption, and carbon dioxide production. To evaluate the structure alterations we measured LM, an objective parameter that representsthe average distance between alveolar walls. The increase of Lm with age is relatively smallanditisindependentofbodysize.Thismeasurement is easy tomake andis independentof the observer. This measurementisconsideredtobesuitablefordetailedstud- iesthataimtocorrelatestructureandfunctiondisturbances inlungs.²⁸

By the investigation of functional disturbances and structural alterations associated with elastase-induced emphysemainmice,weaimedtoestablishanexperimen- tal model withhigh reproducibilityandlow mortality. We analyzedsafety,feasibilityandreproducibilityofourmodel applyingintratracheallyanintermediarydoseofelastaseto producethe enlargementof alveolar spaceand itsconse- quencesonrespiratoryfunction.

Materials and methods

Animals

Two-month old female C57Bl/6 mice (n=36), weighing around 20g, raised and maintained at the animal facili- tiesattheGonc¸aloMonizResearchCenter,FIOCRUZ,were used in the experiments, and were provided with rodent dietandwateradlibitum.Allanimalswereeuthanizedina CO2chamber,andhandledaccordingtheNIHguidelinesfor ethicaluseoflaboratoryanimals.

Experimentalgroups

The animalswererandomlydivided intothreegroups: (A) control:non-manipulatedanimals(n=12);(B)intratracheal elastase: 100␮l (n=12); and (C) intratracheal PBS100␮l (n=12).

Emphysemainduction

For elastase instillation, mice were anaesthetized with ketamine (Vetbrands) and atropine (Allergan), intubated

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orotracheally,and ventilatedusinga ventilator/respirator (Mini-VentSmallAnimalVentilators,KentScientiﬁc).Exper- imental animals received 2U/100g body weight porcine pancreaticelastase(Sigma,Aldrich,Taufkirchen,Germany) dissolvedin100␮lphosphate-bufferedsaline(PBS)solution or100␮lPBSalone.Controlanimalswerenotmanipulated.

Therespectivesolutionwasadministeredintratracheally via tubing followed by 200␮l of air for an even distribu- tionoftheliquidthroughoutbothlungs.Theanimalswere submittedtoanteriorcervicalincisioninordertovisualize thetracheaandmakethetrachealpuncturetoadminister oneofthesolutions.Afterwards,miceweresutured, kept on a warm plate (30^◦C) until restoration of spontaneous breathingandthenwereextubated.

Treadmill

Amotor-driventreadmillchamberforoneanimal(LE8700, Panlab,Barcelona,Spain)wasusedtoexercisetheanimals.

Thespeedofthetreadmillandtheintensityinmilliampsof theelectricshock appliedtoan stainlesssteel gridat the rearendofthetreadmillwerecontrolledbyapotentiometer (LE8700treadmillcontrol,Panlab).Roomairwaspumped into the chamber at a controlled flow rate (700ml/min) by a chamber air supplier (OXYLET LE 400, Panlab). Out- flowwasdirectedtoanoxygenandcarbondioxideanalyzer (Oxylet00;Panlab)tomeasureconsumptionofoxygenand productionofcarbondioxide.Themeanroomtemperature wasmaintainedat21±1^◦C.Afteranadaptationperiodof 40min in the treadmill chamber themice wereexercised atdifferentvelocities,startingat7.2m/minandincreasing thevelocity7.2m/minevery10min.Theinclinationofthe treadmillwasmaintainedatanuphillangleof10^◦.Veloci- tieswereincreaseduntiltheanimalcouldnolongersustain agivenspeedandremainedformorethantensecondson theelectrified grid, which providedan electrical stimulus (1 milliamp) tokeep the mice running.Total running dis- tanceandrunningtimewererecorded.Treadmilltestswere carriedoutonallmicebeforetheinductionofemphysema and28daysafterexperimentalprocedures.

Histopathologicalanalysis

Twenty-eight days after application of elastase or PBS, the mice were euthanized using CO2 and the lungs were exposed. The trachea was canullated with gelco number 18andthelungswereperfusedwithbuffered4%formalin applyingaconstanttranspulmonarypressureof20cmH2O for2h.Afterthisprocedurethetracheawassuturedinorder toholdtheintrapulmonarypressureat20cmH2Oandthe entirecardiopulmonarytissueblockwasremovedandﬁxed informalin(4%).

Morphologic examinations were performed following Thurlbeck²⁸ method modified. Briefly, the mean linear intercept (Lm) of each lung was determined using light microscopyon20randomlyselectedfields,originatingfrom randomlyselectedtissuesamplescoveringthe entirelung andcontainingapical aswellasbasal areasof theorgan.

Fivepictures weretaken of each lung. The Lmasindica- torofairspacesizewascalculatedfromcountinglinesof deﬁnedlengththatwererandomlyplacedoneachofthe20

Table 1 Percentage of mortality related to different methodology to induce experimental pulmonary emphysema. (A) Control:non-manipulatedanimals; (B)intratracheal 100␮l of elastase administration; (C) intratracheal 100␮lofsalineadministration.

Group Numberofmice Mortality %

A 12 0 0

B 12 2 16.7

C 12 2 16.7

Total 36 4 11.1

lungsectionsof5␮mandthenumberofinterceptscrossing thelinescounted.TheLmwascalculatedfromthelength ofthelinesmultipliedbythenumberofthelinesdividedby thesumofallcountedintercepts.

Statisticalanalysis

Numericvalueswereexpressedasmean±SDunlessstated otherwise. Treadmill performance was analysed using a pairedt-testtocomparegroupsbeforeandaftertreatment andmorphometricdatawerecomparedapplyingaOne-way ANOVAfollowedbyaTukeytesttoidentifygroupsthatare signiﬁcantly different.A value of P<0.05 was considered statisticallysigniﬁcant.

Results

Safetyoftheprocedure---mortality

Fouroutof 36mice(11.1%) diedduring theexperimental procedure.The groupsmanipulated byintratrachealfunc- tion showed a similar mortality (two death per group), independentofthekindofsolutionadministrated(Table1).

Functionalanalysis--- ergometry

Maximum velocity and running distance reached by each groupbefore (pre) andafter (post) administration of PBS orelastasearesummarizedinTable2.

Before the manipulation, all mice were capable of exercising on the treadmill. After manipulation, intratra- chealelastaseadministration(groupB)showedasigniﬁcant decreaseinthemaximumexercisevelocitybeingabletosus- taintreadmillvelocitiesupto15.1m/min(Table2).These differencesresulted in a signiﬁcant reductionin distance coveredbytrachealelastase-treatedmicewhencompared totrachealsaline-treatedmice,reducingrunningdistance by30.5%intheformergroup(Table2).

Concerningoxygenconsumptionandcarbondioxidepro- duction, all mice treated with elastase increased oxygen consumption after induction when comparedat the same speedbeforeandafteradministrationofelastase(Fig.1A).

Inrelationtotheproductionofcarbon dioxide,nosigniﬁ- cantdifferencewasobservedatthesamespeedbeforeand afteradministrationofelastase(Fig.1B).

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Table2 Miceperformanceduringexerciseonamotorizedtreadmillevaluatedbymaximumvelocityreachedandmaximum distancecoveredbefore(pre)andaftertreatment(post)andtherespectivepercentagechange(%).GroupsasinTable1.

Group Velocity(m/min) Distance(m)

Pre Post Pre Post

A 19.8±3.0 19.2±3.2 900±124 810±173

B 19.2±2.7 16.2±3.1^* 810±161 570±45^*

C 18.0±3.0 18.0±3.6 710±157 720±201

* Indicatesavaluesigniﬁcantlyloweraftertreatment(P<0.05).

Morphometricanalysis---meanlinearinterseptum measurement

The administration of 100␮l of elastase by the tra- chealroutesigniﬁcantlyincreasedLm(99.6±2.9␮m)when compared to intratracheal saline-treated (33.3±0.6␮m) and non-manipulated mice (25.6±0.1␮m). A slight, but

Rest ing

7.2 m/

min 14.4 m

/min 21.6 m/min

28.8 m/min 36 m

/min Exhaustion Rec

overy 0

2000 4000 6000 8000 10000

Pre-induction Pos-induction

** *

* ***

VO2(mL/Kg-1/h-1)VCO2(mL/Kg-1/h-1)

Resting 7.2 m

/min 14.4 m

/min 21.6 m

/min 28.8 m

/min 36 m/min

Exhaustion Recovery 0

2000 4000 6000 8000

A

B

Pre-induction Pos-induction

Figure1 Comparisonofrespiratoryfunctionduringphysical effort in mice before and after the administration of elastase.Oxygenconsumption(A)andcarbondioxiderelease(B), duringresting conditions, exercisingat 5different velocities (7.2,14.4,21.6,28.8and36m/min)onamotorizedtreadmill, immediately(Exhaustion)and25minafterexercise(recovery).

Dataaremeans±SD;10mice/group.Notethatmiceelastase- treatedwereunabletorunatavelocityof36m/min;therefore, nodataaregiven.*p<0.05;**p<0.01;***p<0.001.

150

120

90

*

60

30

µm

0

A B C

Figure2 Comparisonofthemeanlinearinterceptlengthin threegroupsofC57Bl/6micetreatedin(A)non-manipulated animals(n=12),(B)animalsreceivingtrachealelastase(n=12), or (C) receiving tracheal saline (n=12). Data are presented as means±SD. * Indicates a signiﬁcant difference (P<0.05) betweengoups.

signiﬁcant, increase in Lm was found comparing traqueal applicationofsalinewithnon-manipulatedanimals(Fig.2).

Discussion and conclusion

Inthisworkweinducedemphysemabyintratrachealinstil- lation of porcinepancreatic elastase, which resulted in a meanlinearinterceptof99.6␮m.Ishizawaetal.²⁹applied thesameconcentrationofelastasebyintranasalinstillation toinducetheexperimentalemphysemainmiceand,after threeweeks,theyobservedpulmonaryabnormalitiesanda meanlinearinterceptof 80␮m.Thesamegrouprepeated the same procedure inorder totest the effectsof Hepa- tocyteGrowth Factorininjuredlungsbyelastase,butthe meanlinearinterceptwasslightlysmaller:65␮m.³⁰Another study,³¹usingbothC57BL/6miceandSP-C/TNF-atransgenic mice,usingsimilardosageofelastasefoundameanlinear interceptrangingbetween60and85␮m.Comparingthese morphometricdatasuggeststhatintratrachealadministra- tionofelastaseseemsmoreefﬁcienttoinduceemphysema, coupledwitharelatively lowmortality. The experimental proceduresusedinourstudytoinduceemphysemacanbe consideredtolerablesincethemortalityindexinallexper- imentalgroupsremainedlow(Table1).Ishizawaetal.^29,30 didnotmentionthemortalityrateobservedintheirstudies.

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Recently,²⁷ emphysematous disturbances were induced in a transgenic strain of mice (NMRI), which possesses a higherendogenousanti-proteaseactivity.Twoexperimental groupswereusedtoadministerporcinepancreaticelastase by intratracheal instillation: 5U/100g body weight, sin- gleadministrationand3.3U/100gbodyweight,repetitive administration.Inbothtreatmentstheabsolutemortalityof animalswasmuchhigherthanthatinourwork(sixandﬁve mice,respectively).However,the authorsdonotmention thetotalnumber ofanimalsusedper group,butit seems clearthatthehigherconcentrationsofelastaseusedintheir studyresultedinagreatermortalityrateamongexperimen- tal animals whencompared tothe present study.²⁷ These resultsindicatethattheexperimentalprotocolusedinour studyismoreapplicabletoinduceemphysemainmice.

Comparing the morphological parameters of our study withtheonesmeasuredbyLüthjeetal.,²⁷weobservedthat theLmwasgreaterinthelatterstudy(260.7␮m)whencom- paredtoourdata(99.6␮m).Thegreaterdestructionoflung parenchymainduced inthemicebyLüthjeetal.²⁷ canbe attributedtothegreaterconcentrationofelastaseused,as wellastothemousestrainused,whichismoresusceptible tothedevelopmentofpulmonarystructureabnormalities.

The physical activity is significantly impaired in many patients with COPD, significantly altering their qualityof life. The pathogenesis of physical incapacity is complex andinvolveslossofrespiratorymusclestrength,changesin gasexchange,andabnormalitiesinlung mechanics,which reflectsinexerciseintolerancethatisadirectconsequence ofimpairmentinrespiratorymechanics.However,themea- surementof parametersrelated to respiratory mechanics involved invasive or noninvasive methodology, which it is nottotallysecure,requiringalargenumberofanimalstobe usedoraloss ofaccuracyof measurementsof respiratory parameters,sinceitdoesnotreflectthephysiologicalreal- ity of an undisturbed animal. The invasive measurements requirethatanimalsareanesthetizedandventilated,²³orotracheally or endotracheally intubated, tracheotomized³² or underspontaneous breathing.The advantages of these methods includeno stressfor theanimal and theevalua- tion of gold standard parameters, such as resistance and compliance.However,theanesthesiacouldaltersomephys- iological parameters and depending on methodology the measurement cannot be repeated.³³ Hantos etal.³⁴ tried to establish a link between the mechanical properties of the respiratory system and absolute lung volumes in elastase-induced emphysema in order to investigate how these physiological findings are related to the patho- physiological changes in lung volumes and the associated alterations in respiratory resistance and elastance in patients. Thus, the tissue damage did not correlate with airwaydysfunctioninthismousemodelofemphysema.³⁴

On the other side, noninvasive techniques are easy to perform,allowspontaneouslybreathing,andcanbedonein largenumbersofconsciousanimalduringthesameperiod.

As previously mentioned, there aretwo types of plethysmograph:double-chamberandsingle-chamberwhole-body instruments. In the ﬁrst one the animal is restrained at the neck. The animals are placed in the body plethysmograph, while thehead protrudedthrougha neck collar into a ventilated head exposure chamber.^35,36 This appa- ratus which is attached toan ampliﬁer is connectedto a

pneumotachographand adifferentialpressure transducer, which allows airﬂow measurement, tidal volume, minute volumeandrespiratoryrate.³⁷Thesenoninvasiveserialmea- surements reduced the number of animal per study, but long-term examination is not allowed due to the stress imposedbytheneckcollarrestrained.Thereisalsoaunre- strained double-chamber instrument where the animal’s head and body are separated in a chamber by a rubber collar aroundthe neck, which creates two environments:

nasal chamber and thoracic chamber. In single-chamber whole-bodyplethysmograph,norestraintisrequiredandthe animalshavefreeaccesstowaterandfood.Theserecord- ingsmaybedonealldaylongbutphysiologicalmeasurement is indirect since it is done based on pressure changes in single-chamberwhole-body plethysmograph.³⁸ The results arereproducible despite of the chamber plethysmograph employed.The single-chamber is appropriate tomeasure airwayresistancereactivitytodrugadministration,butnot toevaluatetidalvolume.Onotherside,thedouble-chamber istoprovideaccurateresultsabouttidalvolumeandrespi- ratoryrate.³⁷Whenthistechniqueisperformedinadouble chamberwithrestrainedanimalrecordsofinspiratoryand expiratory ﬂows,tidal volume, minute volume and respi- ratoryrate canbe done directly,but it is not possible to measuretranspulmonarypressureorairwayresistance.The noninvasive measurements with unrestrained animal in a singlechamber allowphysiological measurementbasedon pressurealterations inside thechamber, thus the inspira- toryandexpiratoryﬂowsareevaluatedindirectly,andthe transpulmonarypressurecannotbeanalyzed.Thelimitation istheshortageofaccuracyontheevaluationofphysiological parameters,suchasrespiratoryrateandtidalvolume.³⁹

Inadditiontothereasonsdescribedabove,thatjustify theuseoftreadmillexercisetoassessthefunctionallimita- tionsoftherespiratorysysteminducedbyemphysema,^29---31 thereisthe factthat acutephysical exercise isknown to markedly increase the blood ﬂow and oxygen uptake in thelung.Theseverityofpulmonaryparenchymalesionson exerciseperformancecanbeeasilyevaluatedbyergomet- rictests, and in experimental models of emphysema this factmaybeevaluatedbytestingexercisecapacityintread- mill,asperformedbyLüthjeetal.,²⁷whichdemonstrateda reductioninrelativerundistanceinemphysematousmice.

Here, not only the distance run and exercise time were measuredbuttheconsumptionofoxygenandcarbondiox- ide were evaluated. The results obtained demonstrated thatemphysemainductioncommittedtheexercisecapac- ityprobablyduetoanimpairmentingasexchange(Fig.1 andTable2).

Inourwork,withasingleintratrachealadministrationof elastaseandasmalldegreeofinclinationinthetreadmill, weobservedareductioninrunningdistanceof30.5%when comparedtopre-treatmentexercisecapacityofemphyse- matousmice. The greatestdecreasein maximum velocity wasalsoseen inthegrouptreatedwithelastasebyintra- trachealroute (Table2).Areductioninmaximum velocity duringtreadmillexercisewasalsoobservedinaratmodelof emphysemaafterintratrachealadministrationofelastase.³⁴ Thetreadmill performanceofmiceinthe studybyLüthje and co-workers revealed that there wasno difference in runningdistance between controlandsingleintratracheal administrationofelastasegroups,whereasthereductionin

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runningdistance inthegroupwithrepetitiveintratracheal administrationof elastasewas29.7%.Therefore,although the pulmonary tissue destructionwas more severe in the studybyLüthjeandco-workers,asshownbylinerintercept length,probablyduetostrainsusceptibilityandthenum- berofelastaseadministration,ourexperimentalprocedure resulted in the same level of serious functional impair- ment,asrevealedby theexerciseperformance. Different fromthepreviousworks,we analysedthedynamicsofgas exchangeduringexercise.Theaugmentinoxygenconsump- tion(Fig.1A)couldreﬂecttheabilityofthehearttoincrease cardiacoutput in ordertoattend themetabolic demands of animal during physical effort, trying tohold the blood oxygenpartialpressureinthefaceofbloodcarbondioxide partialpressurealtered.The maintenanceofcarbondiox- ideproduction(Fig.1B)couldmeantheimpairmentofgas exchange. These respiratory dysfunction is reﬂected in a lowerexercisecapacityandcouldbeconsequence of pul- monaryparenchymalesion.

Uptodate,allofthestudiesinducingemphysemainani- mal models are insufficiently capable of reproducing the slow and progressive inflammatory process that leads to chronicpulmonaryinjuryinhumans.However,theyhelped to elucidate the pathogenic mechanisms and the development of new clinical therapeutic protocols. Different animals such as hamsters, rats, and mice were used to establishmodelsofemphysema,whicharebasedongenetic manipulations (in mice), exposure to cigarette smoke, or administrationofproteinases intotheairway. Knockoutor transgenicmicemodelsforemphysemarevealeddifferent genesthatcouldbeinvolvedinthepathogenesisofemphy- sema,suchasplateletderivedgrowthfactorA,⁴⁰fibroblast growth factor receptors 3 and 4,⁴¹ metalloproteinase- 1 transgenic mice,¹⁵ platelet derived growth factor B transgenic mice,⁴² macrophage elastase,⁴³ and neutrophil elastase,⁴⁴ thelastonesgivenresistancetosmoke-induced emphysema. However, it is noteworthy that these gene- targetedmicemayhavealterationsinlungstructure,suchas alveolarenlargement,possiblyassociatedtoalterationsin lungdevelopmentinsteadofemphysemadevelopmentdue todestructioninpulmonaryparenchyma,sincethesegenes areexpressedthroughoutthetissuedevelopment,growth, and maturation.⁴⁵ The smoke-induced animal models of COPDincludenose-onlyexposureor wholebodyexposure.

Thismodel maybedeveloped indifferentanimal species, such as rat,⁴⁶ guinea-pig,⁴⁷ or mouse.⁴⁸ The guinea-pig reproducesthemostappropriatemodelofemphysema,but therearemanyproblems relatedtothe cost,shortageof tools to investigate molecular mechanisms, and reduced numberofreagent speciﬁctothisanimal species.^4,49 Rats are very resistant and if the time and volume of smoke exposureareextrapolatedtheseanimalsdevelopedinspe- ciﬁc abnormalities.⁵⁰ With regards to mice, in addition to the advantages described above, functional parameters such as compliance, can be accurately measured.⁴ However,the smoke-induced-emphysema model is not so reproductible,^51,52 the time for developing the disease is long (about sixmonths), produceonly a mild diseaseand afterthecessationof smokethelesionsdonot appearto progress.⁴ Currently, mice are considered the gold standard toreproduce and study many human diseases, since themousegenome shows agreat similaritytothe human

genome, having about 300 genes uniquely present in one species or the other.⁵³ Furthermore, more than 10,000 geneticmarkers havebeen mappedin themouse, alarge numberofinbredstrainsandtransgenicmiceareavailable, anddataontheanimals’anatomy,biology,immunology,and physiologyareavailable.

Themodeltestedinourstudyprovidessomeadvantages, includingthe use of a lowerelastase concentration when comparedtootherstudies.Nonetheless,itinduced severe structuralabnormalities thatmayberesponsiblefor functional disturbancescharacterizing pulmonary emphysema.

Furthermore,theemphysemacouldbeinducedwithavery low mortality rate and a high degree of reproducibility, which could befunctional analyzed by non-invasive tech- nique that reﬂects the physical impairmentassociated to structuralabnormalitiescharacteristicofpulmonaryemphy- sema.

Conﬂicts of interest

Theauthorshavenoconﬂictsofinteresttodeclare.

Acknowledgements

Thiswork wassupportedbygrantsfromtheBrazilianMin- istryofScienceandTechnology,CNPq,FINEP,FIOCRUZ,and FAPESB.

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