Rev. Bras. Anestesiol. vol.67 número2

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REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

PublicaçãoOficialdaSociedadeBrasileiradeAnestesiologia

www.sba.com.br

SCIENTIFIC

ARTICLE

Effect

of

dexmedetomidine

on

acute

lung

injury

in

experimental

ischemia---reperfusion

model

Ömer

Burak

Küc

¸ükebe

a

,

Deniz

Özzeybek

b

,

Ruslan

Abdullayev

a,∗

_,

_Adil

_Ustao˘

_glu

c

_,

Is

¸ıl

Tekmen

d

_,

_Tuncay

_Küme

e

a_Adıyaman_University_Research_and_Educational_Hospital,_Department_of_{Anesthesiology}_and_Reanimation,_Adıyaman,_Turkey b_Dokuz_Eylül_University_Medical_Faculty,_Department_of_{Anesthesiology}_and_{Reanimation, ˙Izmir,}_Turkey

c_Zonguldak_Atatürk_Hospital,_Department_of_{Anesthesiology}_and_Reanimation,_Zonguldak,_Turkey d_Dokuz_Eylül_University_Medical_Faculty,_Department_of_Histology_and_{Embryology, ˙Izmir,}_Turkey

e_Dokuz_Eylül_University_Medical_Faculty,_Department_of_Medical_{Biochemistry, ˙Izmir,}_Turkey

Received1March2015;accepted17August2015 Availableonline21March2016

KEYWORDS

Experimental; Dexmedetomidine; Reperfusioninjury; Acutelunginjury

Abstract

Purpose: Ischemia---reperfusioninjuryisoneoftheconsequencesoftourniquetapplicationfor

extremitysurgery.Theaimofthestudywastoestablishtheeffectofdexmedetomidineonthe acutelunginjuryfollowinglowerextremityexperimentalischemia---reperfusionmodelinrats.

Methods:Twenty-eight Wistar-Albino breed Rats were recruited after Ethics Committee

approval and allocatedinto 4 groups,each with 7subjects. Group 1(SHAM) received only anesthesia. Group 2 (IR) hadexperienced 3h ofischemia and3h ofreperfusion using left lowerextremitytourniquet afteranesthesia application.Groups3(D-50)and4(D-100)had undergonethesameproceduresasintheGroup2,exceptforreceiving50and100mg·kg−1_,

respectively,dexmedetomidineintraperitoneally1hbeforethetourniquetrelease.Blood sam-pleswereobtainedfortheanalysisoftumornecrosingfactor-␣andinterleukin-6.Pulmonary tissuesampleswereobtainedforhistologicalanalysis.

Results:Nosignificantdifferenceregardingbloodtumornecrosingfactor-␣andinterleukin-6

valueswasfoundamongthegroups,whereaspulmonarytissueinjuryscoresrevealedsignificant difference.HistologicalscoresobtainedfromtheGroup2weresignificantlyhigherfromthose intheGroups1, 3and4withp-values 0.001foreachcomparison.Moreover, Group1scores werefoundtobesignificantlylowerthanthoseintheGroups3and4withp-values0.001and 0.011,respectively.NosignificantdifferencewasobservedbetweentheGroups3and4.

Conclusion: Dexmedetomidine is effective in reduction of the experimental

ischemia---reperfusion inducedpulmonary tissueinjury in rats, formedby extremity tourniquet appli-cation.

∗_{Corresponding}_author.

E-mail:ruslanjnr@hotmail.com(R.Abdullayev). http://dx.doi.org/10.1016/j.bjane.2015.08.002

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PALAVRAS-CHAVE

Experimental; Dexmedetomidina; Lesãodereperfusão; Lesãopulmonar aguda

Efeitodedexmedetomidinasobrelesãopulmonaragudaemmodeloexperimental

deisquemia-reperfusão

Resumo

Objetivo:Alesãodeisquemia-reperfusãoéumadasconsequênciasdaaplicac¸ãodotorniquete

emcirurgiasdeextremidades.Oobjetivodoestudofoideterminaroefeitodedexmedetomidina emlesãopulmonaragudaapósmodeloexperimentaldeisquemia-reperfusãoemextremidade inferiorderatos.

Métodos: Vintee oito ratos albinosWistar foram recrutadosapós aprovac¸ão do Comitêde

Éticaealocadosemquatrogrupos,cadaumcomseteindivíduos.OGrupo1(Sham)recebeu apenasanestesia.OGrupo2(IR)foisubmetidoa3horasdeisquemiae3horasdereperfusão comousodetorniquete em extremidadeinferiorapósaaplicac¸ãodaanestesia.Osgrupos 3(D-50) e4 (D-100)foramsubmetidos aosmesmos procedimentos doGrupo 2, exceto por receberem50e100mg·kg−1_de_{dexmedetomidina,}_{respectivamente,}_por_via_{intraperitoneal}₁

horaantesdaliberac¸ãodotorniquete.Amostrasdesangueforamcoletadasparaanálisede TNF-␣eInterleucina-6(IL-6).Amostrasdetecidopulmonarforamcoletadasparaanálisehistológica.

Resultados: Nãohouvediferenc¸asignificativaquanto aosvalores sanguíneosdeTNF-␣e

IL-6entreosgrupos,enquantoosescoresdelesãoemtecidospulmonaresrevelaramdiferença significativa.OsescoreshistológicosobtidosnoGrupo2foramsignificativamentemaioresque osdosgrupos1,3e4,comvalores-pde0,001paracadacomparação.Alémdisso,osescores doGrupo1foramsignificativamentemenoresqueosdosgrupos3e4,comvalores-pde0,001 e0,011,respectivamente.Nãohouvediferençasignificativaentreosgrupos3e4.

Conclusão:Dexmedetomidina mostrou eficácia na reduc¸ão de lesão em tecido pulmonar

induzidaporisquemia-reperfusãoexperimentalemratos,ocasionadaporaplicac¸ãode torni-queteemextremidade.

Introduction

Cessation of the blood flow, namely tissue ischemia,and

reperfusion after a period of time are the main

patho-physiological mechanisms responsible for organ damages

following some clinical conditions like organ

transplan-tation, cerebral ischemia, stroke, myocardial infarction,

thromboemboli,cardiopulmonarybypassand vascular

sur-geries, major trauma and hemorrhagic shock, as well as

surgicalprocedureswithtourniquetuse.1,2_Ischemic_damage ismainlytheresultofthenecrosisofthecellsdueto

oxy-gencessation;oxygensupplyduringischemiaisnotenough

tomeet the metabolic needs of the cells and this

condi-tionresultsintheactivationoftheanaerobicmetabolism.3

When the tissue blood flow is provided again, i.e.

reper-fusion starts, it paradoxically increases the local tissue

damage,togetherwithaninflammatoryresponseresulting

indistantorgandamage.2,3_Many_inflammatory_agents_have

been shown to be responsible for this damage, namely

ischemia---reperfusioninjury(IRI).2

Cytokines have been blamedfor distant organdamage

of IRI.4 _Blood, _kidney _and _lung _tumor _necrosing

factor-␣ (TNF-␣), interleukin (IL) IL-1 andIL-6 levelshave been

found to be high after lower extremity ischemia.5 _The

mediatorsreleasedafterischemia---reperfusion(IR)resultin

localand distantorganinjuries through freeoxygen

radi-calsandleukocytes.6,7 _Negative_{immunoregulatory}_effects

of noradrenaline have been demonstrated in the

forma-tion of monocyte/macrophage origin mediators (TNF-␣,

IL-1, IL-6) andthis effectwaslinkedtoitsboth ␤ and␣2

adrenoceptors.8

Dexmedetomidineisastrongandselective␣2-adrenergic

agonist that is an active d-isomer of medetomidine

and metabolized in the liver. It has been approved to

be used for sedation in intensive care unit and

pos-sessessedativeandanalgesiccharacteristics.9,10_There_are

few studies in the literature regarding anti-inflammatory

effects of dexmedetomidine.11---16 _Yagmurdur _et _al.14 _have

demonstrateddexmedetomidine todecrease thelevels of

malondialdehydeandhypoxanthine(thatareformed after

IR), following tourniquet application for upper extremity

surgery. Although it has been shown that

dexmedetomi-dine has some protective effects for IRI locally, we have

notencountered astudy regardingits effectsonsystemic

inflammatoryresponseanddistantorgandamage.

We aimed to establish the effects of two different

doses of dexmedetomidine on the acute lung injury

fol-lowing lower extremity experimental IR model in rats.

The hypothesis was based on possible protective effects

of dexmedetomidine taking into consideration its

anti-inflammatoryeffects.

Methods

This studywascarriedoutin DokuzEylulUniversity

Medi-cal Faculty Multidisciplinary Animal Laboratory between

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approval of Dokuz Eylul University Experimental Animal

Studies Ethics Committee, ˙Izmir, Turkey (24/11/2008, n◦

134, Chairperson Prof. M. Olguner). Twenty-eight adult

femaleWistar-Albinobreedratsweighting250---340gwere

usedforthestudy.Theanimalswereprovidedwithstandard

pellet foodand waterandwere keptin 12-hourcyclesof

lightanddarkforadaptationbeforethecommencementof

the study.The temperature and humiditywere also

stan-dardized.

The rats were fasted for 12h before the surgical

pro-cedure and were allowed to drink water only. Each rat

was weighted before the experiment and the doses of

dexmedetomidine were calculated. They were randomly

allocated into one of the 4 groups using closed envelope

method. Group 1 (SHAM, n=7) received only anesthesia

with blood sample obtainment at the 1st hour of

reper-fusionandbloodandtissuesampleobtainmentat theend

ofthereperfusion.Group2(IR,n=7)hadexperienced3h

ofischemiaand3hofreperfusionusingleftlowerextremity

tourniquetafteranesthesiaapplication.Bloodsamplewas

obtainedat the1st hourof reperfusionandat theendof

the reperfusion blood and tissue samples were obtained.

Group3(D-50,n=7)hadundergonethesameproceduresas

intheGroup 2,exceptforreceiving 50␮g/kg

dexmedeto-midine(Precedex,AbbottLaboratories Ltd.NorthChicago

ABD,100␮/mL)intraperitoneally1hourbeforethe

tourni-quetrelease.Group4(D-100,n=7)hadundergonethesame

proceduresasintheGroup2,exceptforreceiving100␮g/kg

dexmedetomidine intraperitoneally1h before the

tourni-quetrelease.Againthebloodsampleswereobtainedatthe

1sthoursofthereperfusionwithblood andtissuesamples

attheendofthereperfusionfortheGroups3and4.

Anesthesia was performed using 50mg·kg−1 _ketamine

(Pfizer Pharma GMBH, Germany) and 10mg·kg−1 _xylazine

hydrochloride (Alfazine, 2%, Egevet) mixture applied

intraperitoneally.Ischemiawasperformedusingelastic

ban-dage(1cmwidthand30cmlength)appliedtotheinguinal

region of the left lower extremity. The tourniquet was

deflated to establish reperfusion after the certain period

oftime.Theenvironmenttemperaturewaskeptstableand

the operation table was heated to protectthe rats from

hypothermiaandthebodytemperatureswerekeptbetween

36.8◦_C_and_37.5◦_C._The_rats_were_hydrated_with₃_mL/kg/h

isotonicsodiumchloride subcutaneouslythroughouttheIR

procedure toavoid dehydration. The subjects were

sacri-ficedaftertheobtainment ofsamplesofbloodandtissue.

Theproceduredurationswerekeptequalinallthegroups.

Attheendof the1sthourof3-hourreperfusionperiod

1.5mLbloodwasdrawnfromthetailveinoftheratsforthe

analysisofTNF-␣andIL-6, wereputintosteriletubewith

ethylenediaminetetraaceticacid(EDTA)andcentrifugedfor

10min with1200rpm speed.Plasma ofthe blood samples

wereseparatedputintoEppendorftubes,freezedandkept

at 70◦_C _until _the_analysis _day. _The _rats _received _isotonic

sodiumchlorideintraperitoneallyequaltotheamountlost

for sampling. At the end of the reperfusion thorax was

opened by subdiaphragmatic approach. Blood sample for

TNF-␣andIL-6analysiswasobtainedin2mLvolumedirectly

fromtheheartandwaskeptusingthesamemethodas

men-tionedabove.Attheendoftheprocedure5mLof10%formol

wasslowlyappliedthroughthecardiacapexvia20Gneedle

andheartandlungbedwereexcisedafterthesacrification.

Asectionfromthemiddlezoneoftherightlungandtheleft

lungtotallywereputinto10%formolandkeptfixedforat

least24h.

Analysisofinflammatorymediators

The analysis of inflammatory mediators (TNF-␣ vs. IL-6)

was performed via Enzyme-Linked Immunosorbent Assay

(ELISA).Absorbentvalueswerereadat450nmintheELISA

device(Organon TechnicaMicrowell SystemReader 230S).

Theresultswereexpressedinpg/mL.

Histopathologicalanalysis

Pulmonarytissuesobtainedfromthesubjectswerestained

using hematoxylin-eosin method and examined by light

microscope(OlympusBH-2) under200×magnification.Six

randomregions wereselected fromeach sample and 0---4

point scoring were made taking into account

histopatho-logical findings like alveolar and interstitial hemorrhage,

alveolarandinterstitialneutrophileinfiltration,edemaand

atelectasis.Scoringwasmadeaccordingtotheinjuredarea

proportionas0:noinjury,1:<25%injury,2:25---50%injury, 3:50---75%injury,4:diffuseinjury.

Statisticalanalysis

ThedataobtainedwasevaluatedusingSPSS11.0software

(Statistical Package for the Social Sciences, Chicago,

Illi-nois). Kruskall Wallis and Chi-square tests were used for

comparisonof thegroups. Mann---WhitneyUtest wasused

forcomparisonofthegroupsseparatelyandBonferroni

cor-rectionwasusedwhenneeded.p-Valueslessthan0.05were

consideredstatisticallysignificant.

Results

Thisstudywascompletedwith28rats,eachgroup

contain-ingseven.Thetimeintervalforthestudywasthesameas

indicatedinMethodssection(Fig.1).

Table1representsTNF-␣andIL-6valuesandhistological scoresofthepulmonarytissue.Intergroupanalysisrevealed nostatistically significant differenceregarding TNF-␣ and IL-6valuesneitherinthe1st,norinthe3rdhoursof reper-fusion.

Statistical analysis of the pulmonary injury

histologi-cal scores was made using median values of the scores

obtainedfrom6microscopicareasexamined.Therewas

sta-tisticallysignificantdifferenceamongthegroupsregarding pulmonaryinjuryscores(p=0.001).Therewasobserved

dif-fuseinjury (score4) inall the specimensof theGroup 2.

HistologicalscoresobtainedfromtheGroup2were

signifi-cantlydifferentfromtheGroups1,3and4withp-valuesof

0.001foreachcomparison.Moreover,Group1scoreswere

foundtobesignificantlydifferentfromtheGroups3and4

withp-valuesof0.001and0.011,respectively.Nosignificant

differencewasobservedbetweentheGroups3and4.

In the microscopical examination; samples from the

Group1demonstratednormalalveolarsequence,

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Group 1 (SHAM)

∆

---

--- n=7

6. hour

4. hour

3. hour

0. hour

6. hour

4. hour

3. hour

0. hour

6. hour

4. hour

3. hour

2. hour

0. hour

6. hour

4. hour

3. hour

0. hour

Histopathology

and Biochemistry

Biochemistry

Histopathology

and Biochemistry

Biochemistry

Histopathology

and Biochemistry

Biochemistry

Group 2 (IR)

ISCHEMIA

REPERFUSION

∆

---

--- n=7

Group 3 (D 50)

ISCHEMIA REPERFUSION

∆

---

--- n=7

Dexmedetomidine

50 µ

g/kg

Histopathology

and Biochemistry

Biochemistry

Dexmedetomidine

100 µ

g/kg

Group 4 (D 100)

ISCHEMIA

REPERFUSION

∆

---

--- n=7

Figure1 Schematicrepresentationofthestudyprotocol.

Figure2 Group1,normalstructureofventilatedpulmonary tissue.

SamplesfromtheGroup2showedinterstitialand

intraalve-olar neutrophile infiltration, hemorrhage, interalveolar

septalthickening, interstitial edema, diffuse

emphysema-tous areas and congestion. Large magnification revealed

mostly atelectatic areas and areas with capillary stasis

withdecreasedventilation,collapsedalveoli,nonfunctional

parenchymal areas, dense parenchymal neutrophile and

macrophageinfiltrationintheseregions,diffuse

intraalve-olar hemorrhage in the capillary stasis areas and patchy

fibrosisareastogetherwithsparsenormallyventilatedfields (Figs.3and4).SamplesfromtheGroup3demonstrated dif-fuseinterstitialneutrophileinfiltration;interalveolarseptal

thickening,atelectasis,congestion,hemorrhageand

emphy-semalessthanintheGroup2.Normalpulmonarytissuewas

observedattheneighboringareas,despitemild

parenchy-malenlargementandneutrophileinfiltration.Someregions

revealed normal pulmonary tissue together with intense

parenchymalhemorrhage,emphysematous areasand

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Table1 TNF-␣andIL-6valuesandhistologicalscores.

Group1 (SHAM)

n=7

Group2 (IR)

n=7

Group3 (D50)

n=7

Group4 (D100)

n=7

p

TNF-˛(pg/mL)

Reperfusion1h 9.40 (7.40---11.50)

10.10 (9.10---25.30)

9.70 (8.30---10.90)

9.70 (7.70---10.60)

0.55

Reperfusion3h 9.30 (8.20---10.90)

9.80 (9.30---22.90)

10.20 (8.70---11.80)

9.00 (8.90---12.10)

0.26

IL-6(pg/mL)

Reperfusion1h 27.10 (21.60---41.30)

17.30 (13.20---52.60)

19.00 (15.80---58.00)

25.20 (20.00---48.90)

0.11

Reperfusion3h 32.70 (21.60---64.70)

61.20

(17.40---120.30)

24.40 (14.00---91.80)

31.80 (21.40---47.70)

0.83

Histologicalscore 0.5b

(0.0---1.0)

4.0a

(4.0---4.0)

2.5 (2.0---3.0)

2.0 (0.5---3.5)

0.001

a _Significant_difference_in_the_Group₂_compared_with_the_Groups_1,₃_and_4,_p_<_0.05. b _Significant_difference_in_the_Group₁_compared_with_the_Groups₃_and_4,_p_<_0.05.

Thevaluesarerepresentedinmedian(minimum---maximum).

Figure3 Group2,alveolarstructurehaslostitspattern(↑), emphysematous changes (A), parenchymal inflammatory cell infiltration(P˙I).

4 demonstrated mild interstitial neutrophile infiltration,

interalveolarseptalthickening,atelectasis,congestionand

hemorrhagetoa muchlesserextentthan inthe Group 2.

Someregionsrevealedcompletelynormalpulmonarytissue

(Fig.6).

Discussion

The main outcome of the study was that,

dexmedetomi-dinedecreasedhistologicalinjuryscoresrelatedtoIRinrats

experiencingischemiaandreperfusion,thatwasgenerated

by extremitytourniquet application. Although therewere

notobserveddifferencesamongthegroupsasregardsblood

inflammatorymediatorsTNF-␣andIL-6, therewere

signif-icant differenceregardinghistologicalappearancesof the

tissues.HistologicalscoresoftheGroup2werefoundtobe

high,indicatingtheeffectsoftheIRinjurytothepulmonary

Figure4 Group 2,denseneutrophileinfiltration, parenchy-mal enlargement, non-ventilated pulmonary areas ( ) and atelectaticareas(A).

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Figure6 Group4,mildinflammatoryinfiltrationand thick-enedinteralveolarseptum(*)togetherwithnormalpulmonary tissue.

tissue.Groups 3and 4 demonstratedlower injury scores,

indicatingprotectiveeffectofdexmedetomidineagainstIRI.

Although some authors offer multiple ligation method

instead of tourniquet application because of the concern

ofvenous and lymphatic obstruction, muscle,nerve

dam-ageafterprolongedmechanical compression,17 _tourniquet

methodisnon-invasiveand seemstobemore practicalin

theclinicalapplication.7,18

Different ischemia and reperfusion times had been

usedfor IRcreation in the literature. We had taken into

account resistance of the tissue against ischemia in our

study. For instance, prominent morphological changes in

the muscle tissue appear after 2h of ischemia and so,

thestudiesdealingwithmuscleIRIhadusedlongischemia

periods.19,20_Important_correlation_between_ischemia_period

and ischemic injury was found.21 _Taking _into _account

thattherehadbeen demonstratedirreversiblehistological

injury20 _and_diffuse_neutrophile_infiltration18 _in_the_muscle tissueafter3hofischemiaperiodinthestudiesdealingwith

IRIinthemuscle,wehadchosenourischemiaperiodtobe

3hinthestudy.

Reperfusionperiodalsowasfoundtobeanimportant

fac-torintheformationoflocalanddistanteffectsofIR.2,7,19

Yasin et al.7 _had _used _different _reperfusion _times _after

3hischemiaandfoundpositivecorrelationbetween

reper-fusionperiodanddistantorgandysfunctions.Theyhadalso

demonstratedhistological changes after the 3h of

reper-fusion,thatwerenotapparentinthefirst2h.On account

ofthiswehadchosenthereperfusiontimetobe3h. Promi-nenthistologicalchanges,asdiffuseneutrophileinfiltration,

alveolarsequencedistortion,edema,inthepulmonary

tis-sueoftheIRgrouphadsupportedourchoice.Thesefindings

were coherentwith the studies that usedthe same time

periods.7,18

TNF-␣ was shown to play an important role in the

pathogenesisofSystemicInflammatoryResponseSyndrome

(SIRS)andMultipleOrganFailure(MOF).5,22,23_Similar

mech-anisms weresuggested in the localand distanteffects of

IRIandstudiesconductedhadshownTNF-␣toplayrolein

theorgandamagepathogenesis.3,7 _Similarly, _IL-6_also_was

showntoplayroleincomplexinflammatoryreactionrelated

totrauma,shock andinfection.24 _Positive _correlation _was

demonstrated between plasma IL-6 concentrations and

magnitudeofsofttissuedamage.24_IL-6_levels_were_shown_to increaseincorrelationwithTNF-␣inthestudiesdealingwith

lower extremity IRmodels.5,6 _Yasin _et_al.7 _had _suggested

that IRrelated inflammatoryresponse can beshown with

serumTNF-␣andIL-6levelsandthatthereispositive corre-lationbetweenthisincreaseanddistantorganinjuries.They hadevaluatedbloodTNF-␣andIL-6levelsatthe1st,2ndand

3rdhoursofreperfusionafter3-hourischemiaperiod.

TNF-␣wasfoundtoincreasesignificantlyattheendofthe1st

hourandreturntocontrolvaluesatthe2ndhour;whereas

IL-6wasfoundtoincreaseprogressivelyafterthe1sthour

ofreperfusionwithpeakattheendofthe3rdhour.Hence,

we hadevaluatedblood TNF-␣andIL-6levelsbothat the

endofthe1stand3rdhours.

There have been conducted many clinical and

experi-mentalstudieswithdexmedetomidineasitshowsanalgesic,

anxiolytic and sedative properties alltogether.25,26

More-over, it has been shown that dexmedetomidine possesses

antiinflammatory properties in the studies investigating

its effects in ischemic and toxic inflammatory models.11

Taniguchi et al.11 _have _demonstrated _that

dexmedetomi-dinealleviateslunginjuryanddecreasesmortalityratesby

decreasing TNF-␣ and IL-6levels and neutrophile

infiltra-tioninthealveolarwallsintheexperimentalendotoxemic

septicshock model.Shen etal.27 _have_investigated_TNF-_␣

andIL-6levelsinlunginjurycausedbyexperimental

intesti-nal IRI model and demonstrated decreased production of

thesecytokinesinpulmonarytissueafterdexmedetomidine

administration.

Dexmedetomidine has been used via intraperitoneal

route before28 _or _during12---14 _IR_in _rat_IR_models._Although

dexmedetomidine’s protective effects had been shown

regardless of its time of administration, there are

no studies in the literature comparing its application

periods.Furthermore,somestudiesinvestigatingtheeffects

of intraperitoneally administered dexmedetomidine have

shown that, IRperiods were notlonger than the

elimina-tiontimeofdexmedetomidine.13,28_Taking_into_{consideration} thatthemainculpritintheformationoflunginjuryis

reper-fusion process, and that TNF-␣ levels begin to rise near

the end of the ischemiawith peaklevels at the 1st hour

ofreperfusion,7_it_was_relevant_to_apply_{dexmedetomidine}

1hourbeforethereperfusion.

We have not come across certain dose intervals

of dexmedetomidine for its antiinflammatory effects in

the literature. The dose for dexmedetomidine, applied

intraperitoneally, wasdecided tobe 100␮g/kg, a highest

dose safely applied in the current literature,16,28 _and _the

second dose was decided to be 50␮g/kg, to evaluate its

possibledosedependenteffects.

Despite similar IR periods in the studies regarding rat

pulmonaryIRIafterextremitytourniquetapplication, differ-entresultsregardingTNF-␣levelshavebeenobserved.5,18,29

Seekampetal.5_have_demonstrated_that_TNF-_␣_levels_were

notdetectableinthebloodthroughoutthe4-hourischemia

period, but started torise at the 1st hour of reperfusion

with decline after the 2nd hour. In addition, IL-6 levels

started to rise at the 30th minute with peak at the end

of the 4th hour. Yassin et al.7 _have _stated _that, _TNF-_␣

and IL-6levelsreachtheir peaklevelsat the 1st and3rd

hoursofreperfusion,respectively;TNF-␣levelsdropbelow

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IL-6reachdetectablelevelsinplasmafromthe1sthourof reperfusion.

Duru etal.,18 _Harkin _et_al.,30 _Gaines _et_al.31 _have_used

similar IR models and time periods; despite they have

demonstrated histopathological changes due to IR injury,

theycouldnotobservechangesinTNF-␣levels.Similarly,

Welbourn et al.29 _could _not _observe _TNF-_␣ _level _changes

after reperfusion; they have concluded that TNF-␣ had

role in the IRI pathogenesis as histopathological changes

decreasedwiththeapplicationofanti-TNFserum.Wehave

alsofoundnodifferencesintheTNF-␣andIL-6levelsinthe

dexmedetomidineappliedgroups,comparedtoSHAMgroup,

despitesignificanthistopathologicaldifferences.This

condi-tionmaybeattributedtoinsufficientnumberof subjects.

Duruetal.18_have_stated_that,_the_reason_they_could_not_have

demonstratedcytokine levelchanges wasthattheymight

havesparedTNF-␣peaklevels,astheyobtainedblood

sam-plesattheendofthe2ndhourofreperfusion.Gainesetal.31

attributedthistothebloodsampleinsufficiency.Welbourn

etal.29_have_proposed_that_TNF-_␣_can_stay_in_the_tissue_after

productioninthepulmonarymacrophagesandsocouldnot

alwaysbedetectedintheblood;itcanmediatepulmonary

tissueinjuryactivatingendotheliumlocally.

IRI generated bythetourniquet modelwas

histopatho-logically demonstrated in this study. The changes in the

IRgroupweresignificant comparedtotheSHAMandboth

dexmedetomidinegroups, andthisverifiedthatthemodel

wasappropriatelyconducted.Significantdifferencesinthe

findings in the dexmedetomidine groups comparedto the

IR group was interpreted as that dexmedetomidine had

decreasedpulmonaryinjury.

Although histopathological scores between the groups

withdexmedetomidine 50 and100␮g/kg were not

signif-icantly different, some normal pulmonary tissue areas in

themicroscopicalexaminationintheD100groupsuggested

thatthedrugmighthaveactedindosedependentmanner.

Studieswithlargersamplesizeswithdifferentdoses,

admin-istrationtimeandroutesmaybeconductedtoevaluatethis

effect.

Nolimitationsforthestudywereencountered.

In conclusion, in our experimental study of lower

extremitytourniquetinduced IRmodel,dexmedetomidine

was effective in preventing the IR induced lung injury.

Histopathological and IL-6 level differences between the

two groups with different doses of dexmedetomidine,

although not statistically significant, suggests to conduct

studieswithlargersample sizestoevaluatetheeffectsof

differentdoses.

Implication

statement

This experimental study demonstrates the ability of

dexmedetomidine to reduce experimental

ischemia---reperfusionrelatedinjuryin rats.Controlledtrials canbe

conductedinhumantoobservetheclinicalsignificanceof

thiseffect.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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