Rev. Bras. Anestesiol. vol.66 número1

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REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

OfficialPublicationoftheBrazilianSocietyofAnesthesiology

www.sba.com.br

SCIENTIFIC

ARTICLE

Comparative

effects

of

vitamin

C

on

the

effects

of

local

anesthetics

ropivacaine,

bupivacaine,

and

lidocaine

on

human

chondrocytes

Jun

Tian

∗

_,

_Yan

_Li

DepartmentofAnesthesiology,XinxiangCentralHospitalofHenanProvince,Xinxiang,China

Received16January2015;accepted26January2015 Availableonline9October2015

KEYWORDS

Chondrocytes; VitaminC; Localanesthetics; Ropivacaine; Bupivacaine; Lidocaine

Abstract

Background: Intra-articularinjectionsoflocalanestheticsarecommonlyusedtoenhance post-operativeanalgesiafollowingorthopedicsurgeryasarthroscopicsurgeries.Nevertheless,recent reportsofseverecomplicationsduetotheuseofintra-articularlocalanesthetichaveraised concerns.

Objectives: ThestudyaimstoassessuseofvitaminCinreducingadverseeffectsofthemost commonlyemployedanesthetics---ropivacaine,bupivacaineandlidocaine---onhuman chon-drocytes.

Methods:The chondrocyteviabilityfollowingexposureto0.5%bupivacaineor0.75% ropiva-caine or1.0%lidocaineand/or vitaminCatdoses 125,250and500␮Mwas determinedby LIVE/DEADassayandannexinVstaining.Expressionlevelsofcaspases3and9wereassessed usingantibodiesbyWesternblotting.Flowcytometrywasperformedtoanalyzethegeneration ofreactiveoxygenspecies.

Results:On exposure tothelocalanesthetics, chondrotoxicitywas foundintheorder ropi-vacaine<bupivacaine<lidocaine. Vitamin C effectively improved the reduced chondrocyte viabilityanddecreasedtheraisedapoptosislevelsfollowingexposuretoanesthesia.Athigher doses,vitaminCwasfoundefficientinreducingthegenerationofreactiveoxygenspeciesand aswelldown-regulatetheexpressionsofcaspases3and9.

Conclusions: VitaminCwasobservedtoeffectivelyprotectchondrocytesagainstthetoxicinsult oflocalanestheticsropivacaine,bupivacaineandlidocaine.

∗_{Corresponding}_author.

E-mail:[email protected](J.Tian).

http://dx.doi.org/10.1016/j.bjane.2015.01.006

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PALAVRAS-CHAVE

Condrócitos; VitaminaC; Anestésicoslocais; Ropivacaína; Bupivacaína; Lidocaína

EfeitoscomparativosdevitaminaCsobreosefeitosdosanestésicoslocais ropivacaína,bupivacaínaelidocaínaemcondrócitoshumanos

Resumo

Justificaiva:Injeçõesdeanestésicoslocaisporviaintra-articularsãocomumenteusadaspara melhoraraanalgesianoperíodopós-operatóriodecirurgiaortopédicacomoartroscopias.No entanto,relatos recentesdecomplicações gravesdevidoao usodeanestésico localporvia intra-articularcausoupreocupações.

Objetivos: OobjetivodoestudofoiavaliarousodevitaminaCparareduzirosefeitosadversos dosanestésicosmaiscomumenteusados(ropivocaína,bupivacaínaelidocaína)sobre condróc-itoshumanos.

Métodos: Aviabilidadedoscondrócitosapósaexposiçãoàbupivacaínaa0,5%ouropivacaína a0,75%oulidocaínaa1,0%e/ouvitaminaCemdosesde125,250e500␮Mfoideterminada peloensaioVIVO/MORTOecoloraçãocomanexinaV.Osníveisdeexpressãodascaspases3e9 foramavaliadosusandoanticorpospelatécnicaWesternblot.Citometriadefluxofoirealizada paraanalisarageraçãodeespéciesreativasaooxigênio.

Resultados: Naexposiçãoaosanestésicoslocais,condrotoxicidadefoiencontradanaseguinte ordem:ropivacaína<bupivacaína<lidocaína.AvitaminaCefetivamentemelhorouaredução daviabilidade doscondrócitosediminuiuosníveis elevadosdeapoptoseapósaexposiçãoà anestesia.Emdosesmaisaltas,avitaminaCfoieficienteparareduzirageraçãodeespécies reativasaooxigênioeassimregularnegativamenteaexpressãodascaspases3e9.

Conclusões:Observamos que a vitamina C foi eficaz na protec¸ão dos condrócitos contraa agressãotóxicadosanestésicoslocaisropivacaína,bupivacaínaelidocaína.

Introduction

Localanesthetics arecommonlyemployedinvarious clini-calsettingsforeither preventionor for symptomatic pain relief.Duringorthopedicpractice,theyareadministeredby manyroutesasspinal, epiduralor intra-articularfor post-operative pain relief, or as a modality in the treatment inosteoarthritis.Multipleclinical studieshave shownthat intra-articularinjectionsoflocalanestheticshavehigh suc-cessrateswhenusedforpost-operativeanalgesia.1---3

Concernaboutthedetrimentaleffectsoflocal

anesthet-icscametonotewhenmultiplecasestudiesdemonstrated

severe toxicity to cartilage and chondrolysis after local

anesthetic infusions into the glenohumeral joint post

arthroscopy.4---8 _As _the _evidence _of _{chondrotoxicity} _due _to thelocalanestheticshasbeenincreasing,investigationson theuseofothersubstancestoeitherinhibitorreducethe toxicityarebeingdone.Inorthopedicsurgeries,addingto theeffectsoflocalanestheticslimitedhealingpotentialof hyalinearticularcartilageisalsoawell-knownproblem.9

Among local anesthetics that are often used for pain

relief, lidocaine and bupivacaine are members of amide

group, but the duration of action of lidocaine is about

one-halfasthatofbupivacaine.10_Ropivacaine_is_a_long

act-ingaminoamidememberofthepipecoloxylididegroupof

localanesthetics andisa promisingsubstitute for bupiva-cainefor spine anesthesia.1 _However,_recent_studies _have

presented thedeleterious effects of theseanesthetics on

chondrocytes.11---14 _Lo _et _al.14 _demonstrated _that bupiva-caine,ropivacaine,andlidocainehaveanegativeeffectin

adose-andduration-dependentmannerontheviabilityof

chondrocytes.

Invitro studies have shown that lidocaineand

bupiva-cainecanhavecytotoxiceffectsonneuronsandmyocytes,

withcelldeathoccurringbybothnecrosisandapoptosis.15,16

In chondrocytes,exposure to theseanesthetics also leads

to cell death by both necrosis and apoptosis in a

dose-andduration-dependentmanner.1,17_Fedder_et_al.18_reported

that exposure to ropivacaine, lidocaine and bupivacaine

reduced the viability of fibroblasts which is linked to

generation of reactive oxygen species (ROS).

Apopto-sis is a highly regulated process distinct from necrosis

that occursin response totrauma, toxins, cytokines,and

pathogens.19,20_Thus,_inhibition_of_chondrocyte_apoptosis_has

beendescribedasaprospectivemeanstoreduce

chondro-cyteloss.20,21

Vitamins have been considered to be vital for living.

Vitamin C, an exogenous water-soluble substance,22 _is _a

cofactorin buildingbloodvesselsandtakestheroleofan

antioxidant,23 _by _acting _as _an _electron _donor _and

reduc-ing agent and thus preventing lipid, protein and DNA

oxidation.24 _There _is _growing _recognition _of _the

impor-tance of nutritional factors in the maintenance of bone

and jointhealth.25 _Bone_matrix _contains _over_90% _of pro-tein as collagen26 _and _vitamin _C _is _an _essential _cofactor

for collagen formation and synthesis of hydroxyproline

and hydroxylysine.27 _Therefore, _vitamin _C _may _help _in

strengtheningbone and preventfractures.Experimentally

induceddeficiencyofvitaminCinanimalsleadstoimpaired

(3)

follow-up study by Sahni et al.29 _demonstrated _that

vita-min C supplementationdecreases risk of hipfracture and

osteoporosis.ThusconsideringtheeffectsofvitaminC,the presentstudyinvestigatesifvitaminCinhibitschrondocyte loss and protects cartilage against the adverse effects of localanesthetics.

Methods

Chemicalsandcells

Normal human chondrocytes were procured from

Promo-cell, Germany and were cultured in chondrocyte growth

medium (Promocell, Germany) under standard laboratory

conditions(humidifiedatmosphereof 95%air and5%CO2,

37◦_C). _Monolayer _culture _of _chondrocytes _has _long _been

used as a method for assessing in vitro cell response to

treatment. The cells at passage5 wereseeded at a

den-sityof 103_cells/cm2_into_96-well_plates_and_cultured_until

75---80%confluence.Uponreachingthedesiredconfluence, thecells wereexposedtolocalanesthetics. Allchemicals

usedin the studywere obtained fromSigma-Aldrich,MO,

USA;otherwise,theyarementioned.

Anestheticexposure

Chondrocytes were exposedfor 1h to1mL of local

anes-theticsolutions:0.5% bupivacaineor 0.75%ropivacaineor 1.0%lidocaine,21,30_obtained_from_{Sigma---Aldrich,}_MO,_USA.

Allsamplesweretreatedusingthesameprotocol.

Specif-ically,culturemedium wasaspirated;1mLof 0.9%normal

salinesolutionoranestheticwereaddedtoeachwell; sam-pleswereincubatedin5%CO2 at37◦Cfor60min;andthe

treatmentsolutionwasaspiratedandfreshculturemedium

was added. Samples were returned tothe incubator and

chondrocyteviabilitywasmeasured24hlater.Inseparate

experiments, following exposure to anesthesia, the cells

were incubatedin fresh medium supplemented with

vita-minC(125or250or500␮M)for24h, followingwhichthe cellswereassessedforviability.

Apoptosisanalysis

Chondrocytesexposedtoanestheticsand/orvitaminCwere

rinsedwithPBSandusedforcellviabilityanalysis.To mea-sureapoptosis,theLIVE/DEADcellviability(LIVE/DEADcell

viability kit, Invitrogen) assay was performed. The assay

determines the intracellular esterase activity and plasma

membrane integrityto assess theviability of cells. Chon-drocytes,treatedoruntreatedcellswithanesthesiaand/or

vitaminC,werestainedwith5␮mol/Lethidiumhomodimer

and5␮mol/Lcalcein-AMandincubatedat37◦_C_for₃₀_min.

ThecellswerethenanalyzedunderaNikonlabophot-2

fluo-rescencemicroscope.Livechondrocytesretaincalcein-AM,

anon-fluorescentpolyanionicdye,andproduceagreen fluo-rescencethroughenzymatic(esterase)conversion.Further,

ethidium homodimer enters damaged membranes of the

deadcellsandbindstonucleicacidstherebyyieldingared fluorescence.

Apoptosis was also detected by measuring the loss of

membraneasymmetry by assessing thebinding properties

ofannexinV.ThebindingpropertyofannexinVwas evalu-atedusingannexinVstainingkit(SantaCruzBiotechnology,

SantaCruz,CA,USA).AnnexinVantibody wasconjugated

withaFluoresceinisothiocyanate(FITC)dye.Briefly,1×106

cellswereexposedtobupivacineorropivacaineorlidocaine

for 1h following treatment withvitamin C, or only those

exposedtoanestheticsweresubjected toannexinV

stain-ing.The cellswerewashedin PBS,resuspendedin 100␮L

ofbindingbuffercontainingaFITC-conjugatedanti-annexin

Vantibody,and then analyzed withflow cytometer (FACS

Calibur,BDBiosciences).

Westernblotting

To further analyze as to whether caspase activation was

involved in the initiation of apoptosisfollowing exposure

tolocalanesthetics, western blotanalysis wasperformed

to determine the expression of caspase-3 and caspase-9

usingrespectiveantibodies(CellSignalingTechnology, Dan-vers,MA,USA).Forisolationoftotalcellularproteins,cells werelysedincelllysing buffer(Cell SignalingTechnology,

Danvers, MA, USA) and processed according to the

man-ufacturer’s instructions. Proteins were then fractionated

by SDS-PAGE, electrotransferred to nitrocellulose

mem-branes,blottedwithrespectiveantibodiesanddetectedby

enhanced chemiluminescence (GE Healthcare). The band

signals of other proteins were normalized to those of ␤

-actin using anti-␤-actin at 1:2000 dilution (Cell Signaling

Technology,Danvers,MA,USA).

Determinationofreactiveoxygenspecies(ROS)

Thegenerationofintracellular ROSwasmeasured byflow

cytometryusing 2′_,7′_{-dichlorofluorescein}_diacetate

(DCFH-DA)staining.DCFH-DA isanon-fluorescentcompoundthat

canbeenzymaticallyconvertedtoDCF,ahighlyfluorescent compound,inthepresenceofROS.Cellsfollowingexposure toanestheticand/orvitaminCwerefurtherincubatedwith DCFH-DA(10␮M)for30minat37◦_C_in_dark._The_cells_were

thenwashedtwice withPBSandintensity offluorescence

wasmeasuredbyflowcytometry.31

Statisticalanalysis

Theresultsarerepresentedasmean±SD.Valuespresenting p<0.05areconsideredsignificantasdeterminedbyoneway

analysisofvariance(ANOVA).Theanalyseswereperformed

usingversion17.0SPPSSpackage.

Results

VitaminCimprovesviabilityofchondrocytes followinganestheticexposure

Chondrocyteviabilityinresponsetoanestheticexposurewas

analyzed by LIVE/DEAD assay. After exposure of

chondro-cyteculturestolocalanestheticsfor 24h,chondrotoxicity

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∗

100

75

50

25

0

Control

Cell viability

, %

Control

Bupivacaine + 250 μM vitamin C Bupivacaine + 500 μM vitamin C

Bupivacaine + 125 μM vitamin C 0.5% Bupivacaine

Ropivacaine + 250 μM vitamin C

0.75% Ropivacaine

Ropivacaine + 250 μM vitamin C Ropivacaine + 500 μM vitamin C 1.0% Lidocaine

Lidocaine + 500 μM vitamin C

Lidocaine + 125 μM vitamin C Lidocaine + 250 μM vitamin C

Bupivacaine Ropivacaine Lidocaine

Figure1 InfluenceofvitaminConthecellviabilityofchondrocytes.Valuesarerepresentedasmean±SD;n=6.*Represents

p<0.05comparedwithcontrolasdeterminedbyonewayANOVA.

0.75%ropivacaine(Fig.1).Therewasamarkeddecreasein cellviabilitypercentageafterexposuretolidocaine,

ropiva-caineorbupivacaine.Comparingequipotentconcentrations

ofropivacaine(0.75%)andbupivacaine(0.5%),thecell via-bility rates were significantly higher 24h after treatment

with0.75%ropivacainethanwith0.5%bupivacaine.

Chon-drotoxicitywasmorepronouncedonexposuretolidocaine

followed bybupivacaine. Exposure tovitamin C following

anesthesiaresulted ina significant(p<0.05)improvement incellviabilitypercentage.Theviablechondrocytecounts increasedwithincreasingconcentrationsofvitaminC.The 500␮Mconcentrationcausedamarkedraiseinchondrocyte viabilitywhencomparedtolowerdoses(125and250␮M).

Apoptosisofthechondrocytesfollowinganesthesia

expo-surewasalsodetectedbymeasuringthelossofmembrane

asymmetrybyannexinVstaining.Theapoptoticcellcounts weremarkedlyhigher(p<0.05)followinganesthesia

expo-sure. Ropivacaine at 0.75% caused considerably lower

apoptosis as compared to 1% lidocaine and 0.5%

bupiva-caine.IncubationwithvitaminCresultedinraisedviability percentagewithdecreasedapoptoticcellcounts.VitaminC

at500␮Mconcentrationwasmorepotentthanlowerdoses

in reducing apoptosis after exposure to local anesthetics

(Fig.2).

VitaminCsuppressesapoptosisbydown-regulating expressionofcaspase-3andcaspase-9

Toevaluatethepossibleinvolvementofcaspaseactivationin anesthesiainducedchondrotoxicity,expressionof

caspase-3 and caspase-9 was determined 24h following exposure

to anesthesia. One hour exposure to anesthetics caused

up-regulation of caspase-3 and caspase-9 in the order,

lidocaine>bupivacaine>ropivacaine(Fig.3).Thought

ropi-vacaine at 0.75% caused raised expressions of caspases,

itwas found tobea non-significant raiseascompared to

controlchondrocytesnotexposedtoanesthetics.

Neverthe-less,1% lidocaineresulted inmarked enhancementinthe

levelofexpressionsindicatingcaspasecascadeactivationin

apoptosis. Incubation with vitamin C for 24h markedly

reduced the expression of caspases in a dose-dependent

manner thatwasinlinewiththeresultsoftheLIVE/DEAD

assayandannexinVstaining.VitaminCat 250and500␮M

weremoreeffectiveindown-regulatingtheexpressionsof

caspases3and9.

InfluenceofvitaminConROSgeneration

We determined the levels of ROS by flow cytometry

using2′_,7′_{-dichlorofluorescein}_diacetate_(DCFH-DA)_staining.

Lidocaine, bupivacaine and ropivacaine caused a marked

increase in ROS production. Lidocaine caused twice as

much raised ROS generation as compared to ropivacaine

and almost nearly 10% more than bupivacaine. This raise

however was suppressed in chondrocytes incubated with

vitamin C as against cells without vitamin C. Treatment

withvitaminCsignificantlyreducedROSproductioninthe order125<250<500␮M(Fig.4).The antioxidantcapacity

of vitamin C couldhave been responsible for the marked

suppressionofROS.

Discussion

Intra-articularinjectionsoflocalanestheticsarefrequently

employed in peri-operative and ambulatory settings.32

Intra-articular injections of local anesthetics enhance

post-operative analgesia.1,2,33 _Lidocaine, _bupivacaine _and

ropivacaineareamide-typelocalanesthetics.Recent

pub-licationshavesuggestedpotentialadverseeffectsofthese threelocalanestheticsonarticularchondrocytesinvitro.1,11

Severalstudies have described the detrimental effects

of local anesthetics on chondrocyte viability. Chuet al.34

demonstrated severe toxicity of bupivacaine. The study

evidencedonexposureto0.5%bupivacaineand>99%ofthe bovinechondrocyteswerefoundtobekilledinallexposed cultures.Gomoll etal.35 _reported_marked _{histopathologic}

andmetabolicchangesinrabbitshoulderswithcontinuous

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100 ₁₀1

a

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

b

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

c

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

d

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

e

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

f

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

g

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

h

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

i

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

j

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

k

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

100 ₁₀1

l

102 ₁₀3 ₁₀4

10 0 10 1 10 2 10 3 10 4

∗

_∗

∗

_∗

∗

100

b

75 50 25 0 Control

Apoptotic cell count, %

Control

Bupivacaine + 125 μM vitamin C 0.5% Bupivacaine

0.75% Ropivacaine

Ropivacaine + 250 μM vitamin C Ropivacaine + 500 μM vitamin C 1.0% Lidocaine

Lidocaine + 125 μM vitamin C Lidocaine + 250 μM vitamin C

Figure2 Chondrotoxicityinducedbyanestheticexposure:(a)0.5%bupivacaine;(e)1%lidocaine;(i)0.75%ropivacaine.Vitamin Coneffectofanesthetics: (b) 0.5%bupivacaine+125␮MvitaminC; (c)0.5%bupivacaine+250␮MvitaminC; (d) 0.5% bupiva-caine+500␮MvitaminC;(f)1%lidocaine+125␮MvitaminC;(g)1%lidocaine+250␮MvitaminC;(h)1%lidocaine+500␮Mvitamin C;(j) 0.75%ropivacaine+125␮M vitaminC;(k)0.75% ropivacaine+250␮MvitaminC; (l)0.75% ropivacaine+500␮MvitaminC. Apoptoticcellcounts.Valuesarerepresentedasmean±SD;n=6.*Representsp<0.05comparedwithcontrolasdeterminedbyone wayANOVA.

Lidocaine at 1% and 2% doses was reported to exhibit

chondrocyte toxicity.11 _Further, _chondrocyte _toxicity _was

reported by Piper and Kim1 _following _exposure _of _human

cartilage explants and chondrocytes to local anesthetics,

bupivacaine and ropivacaine. In line with the previous

reports,inourstudy,0.5%bupivacaine,0.75% ropivacaine and1%lidocaineinducedapoptosisandreducedtheviability

(6)

∗ ∗

∗

∗ ∗

∗ ∗ 200

150

100

50

0

Relative expression, %

Control

Bupivacaine + 250 μM vitamin C Bupivacaine + 500 μM vitamin C 0.5% Bupivacaine

0.75% Ropivacaine Ropivacaine + 125 μM vitamin C Ropivacaine + 500 μM vitamin C

Control Bupivacaine

Bupivacaine L1

a

b

c

d

L2 L3 L4 L5

Ropivacaine Ropivacaine

Lidocaine Lidocaine

Bupivacaine

L1 L2 L3 L4 L5

Ropivacaine

Lidocaine

Control

Bupivacaine + 125 μM vitamin C

Ropivacaine + 250 μM vitamin C 1.0% Lidocaine

∗ ∗ _∗

∗ ∗

∗ _∗ 200

150

100

50

0

Relative expression, %

Control

Bupivacaine + 250 μM vitamin C Bupivacaine + 500 μM vitamin C 0.5% Bupivacaine

0.75% Ropivacaine Ropivacaine + 125 μM vitamin C Ropivacaine + 500 μM vitamin C

Lidocaine + 125 μM vitamin C Control

Lidocaine + 250 μM vitamin C Control

Ropivacaine + 250 μM vitamin C 1.0% Lidocaine

Figure3 EffectofvitaminConcaspase-3(aandb)andcaspase-9(candd)expressionsinchondrocytesfollowinganesthetic expo-sure.L1,control;L2,anestheticalone;L3,anesthesia+125␮MvitaminC;L4,anesthesia+250␮MvitaminC;L5,anesthesia+500␮M vitaminC.(bandd)Valuesarerepresentedasmean±SD;n=6(bandd).*Representsp<0.05comparedwithcontrolasdetermined byonewayANOVA.

more toxic that bupivacaine and ropivacaine. Similar to

earlierstudies,ropivacainewasfound tobeless chondro-toxicthanbupivacaineinhumanchondrocytes.1,36 _Vitamin Csignificantlyimprovedcellviabilityofthechondrocytes. StudieshavedemonstratedthepositiveeffectofvitaminC onbonehealth.29

Apoptosiscanbeinducedthroughintrinsicandextrinsic

pathways, both involving the activation of cellular

cas-pases. Caspases are key enzymes involved in regulating

thehighlyspecificproteolyticcleavageofcellularproteins

leading to cell death. Evidence suggests that caspase-3

mayalsobeinvolvedinDNAfragmentation.21 _In_our_study,

∗ ∗

∗

∗ ∗

∗

200

150

100

50

0

Relative oxygen species, %

Control

0.5% Bupivacaine

0.75% Ropivacaine Ropivacaine + 125 μM vitamin C

Control

1.0% Lidocaine

Figure4 InfluenceofvitaminConROSgenerationbyflowcytometry.Valuesarerepresentedasmean±SD;n=6.*Represents

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localanesthetics---lidocaine,ropivacaineandbupivacaine

were observed to induce caspase expression levelswhich

isindicative ofinduction ofapoptosisin thechondrocytes viacaspaseactivation. Perez-Castroetal.16 _demonstrated

activation of caspase-3/-7 in human neuroblastoma cells

followingincubation withlidocaine,ropivacaineand bupi-vacaine.Nevertheless,vitaminCsuppressedtheexpression ofcaspases3and9,markedlysuggestingitsanti-apoptotic efficacy.

Themechanismsthatleadtothechondrotoxicityoflocal

anestheticsarenotcompletelyunderstood.Toanalyzethe

involvementofROS-mediatedapoptosis,ROSgenerationin

the chondrocytes wasassessed. Marked raise in ROS

gen-erationfollowingexposuretoanestheticswasconsiderably

normalizedtoalmostnormallevelsonincubationof

chon-drocytes with various concentrations of vitamin C. This

couldbeduetothepotentantioxidantpropertyofvitamin

C.VitaminC wouldhaveeffectivelyprevented generation

of ROS and/or neutralized ROS. However,we observed a

strikingcorrelationbetweencell viability,caspase expres-sion and the levelsof ROS production. Park etal.37 _have

shown increased ROS concentration correlating with cell

deathofSchwanncells afterincubationwithbupivacaine.

Inourstudy,ROSconcentrationsincreaseduponexposureto lidocaineandbupivacaine, butnon-significant levelswere

observed following ropivacaine exposure. Similar raise in

ROSgenerationfollowingexposuretolocalanestheticswas reportedbyGrishkoetal.38

To summarize, we have shown that 0.75% ropivacaine,

though toxictohuman chondrocyte cells following 60min

exposure, is considerably on the lower toxic range as

against 0.5% bupivacaine and 1% lidocaine. Lidocaine at

1%concentrationpresentsmorechondrotoxicityamongthe

anesthetics studied. Enhanced ROS production evidenced

in our study suggests that chondrotoxicity could be

pos-sibly due to ROS and ROS-mediated apoptosis. However,

incubationwithvitaminCsignificantlyofferedchondrocyte

protection, which we could possibly be attributed to its

antioxidantcapacity.

Conclusion

Vitamin C was able to markedly protect the condrocytes

againstthetoxiceffectsoflocalanesthetics---ropivacaine, bupivacaineandlidocaine.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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