REVISTA
BRASILEIRA
DE
ANESTESIOLOGIA
PublicaçãoOficialdaSociedadeBrasileiradeAnestesiologiawww.sba.com.br
SCIENTIFIC
ARTICLE
The
effects
of
intra-cerebroventricular
administered
rocuronium
on
the
central
nervous
system
of
rats
and
determination
of
its
epileptic
seizure-inducing
dose
Mehmet
Baykal
a,
Necati
Gökmen
a,
Alper
Do˘
gan
a,
Serhat
Erbayraktar
b,
Osman
Yılmaz
c,
Elvan
Ocmen
a,
Hale
Aksu
Erdost
a,∗,
Atalay
Arkan
aaDokuzEylulUniversity,DepartmentofAnesthesiology,Izmir,Turkey bDokuzEylulUniversity,DepartmentofNeurosurgery,Izmir,Turkey
cDokuzEylulUniversity,DepartmentofAnimalResearchCenter,Izmir,Turkey
Received12December2014;accepted23February2015 Availableonline19June2016
KEYWORDS Rocuronium; Seizure; Centralnervous system; Rat
Abstract
Background: Theaimofthisstudywastoinvestigatetheeffectsofintracerebroventricularly
administeredrocuroniumbromideonthecentralnervoussystem,determinetheseizure thresh-olddoseofrocuroniumbromideinrats,andinvestigatetheeffectsofrocuroniumonthecentral nervoussystemat1/5,1/10,and1/100dilutionsofthedeterminedseizurethresholddose.
Methods:Apermanentcannulawasplacedinthelateralcerebralventricleoftheanimals.The
studywasdesignedintwophases.Inthefirstphase,theseizurethresholddoseofrocuronium bromidewasdetermined.Inthesecondphase,GroupR1/5(n=6),Group1/10(n=6),andGroup 1/100(n=6)wereformedusingdosesof1/5,1/10,and1/100,respectively,oftheobtained rocuroniumbromideseizurethresholddose.
Results:Therocuroniumbromideseizurethresholdvaluewasfoundtobe0.056±0.009moL.
The seizure threshold, as a function of the body weight of rats, was calculated as 0.286moL/kg−1.Adoseof1/5oftheseizurethresholddoseprimarilycausedsplayedlimbs,
posturing,andtremors ofthe entirebody, whereasthedoseof1/10oftheseizure thresh-olddosecaused agitationandshivering.A doseof1/100oftheseizurethresholddosewas associatedwithdecreasedlocomotoractivity.
Conclusions: Thisstudyshowedthatrocuroniumbromidehasdose-relateddeleteriouseffects
onthecentralnervoussystemandcanproducedose-dependentexcitatoryeffectsandseizures. PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeAnestesiologia.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).
∗Correspondingauthor.
E-mail:hale.erdost@deu.edu.tr(H.A.Erdost). http://dx.doi.org/10.1016/j.bjane.2015.02.010
PALAVRAS-CHAVE Rocurônio;
Convulsão; Sistemanervoso central; Rato
Efeitosdaadministrac¸ãointracerebroventricularderocurôniosobreosistema
nervosocentralderatosedeterminac¸ãodadoseindutoradecriseepiléptica
Resumo
Justificativa:Oobjetivodesteestudofoiinvestigarosefeitosdobrometoderocurônio
admin-istradointracerebroventricularmentesobreosistemanervosocentral,determinaradosedo limiarconvulsivoderocurônioemratoseinvestigarosefeitosderocurônionosistemanervoso centralemdiluic¸õesde1/5,1/10e1/100dadosedolimiarconvulsivodeterminada.
Métodos: Umacânulapermanentefoicolocadanoventrículolateraldocérebrodosanimais.O
estudofoiprojetadoemduasfases.Naprimeirafase,adosedolimiarconvulsivodobrometode rocurôniofoideterminada.Nasegundafase,oGrupoR1/5(n=6),Grupo1/10(n=6)eGrupo 1/100(n=6)foramformadosusandodosesde1/5,1/10e1/100,respectivamente,dadosedo limiarconvulsivodebrometoderocurônioobtida.
Resultados: Descobrimos que o valor do limiar convulsivo de brometo de rocurônio é
0,056±0,009moL. Olimiar convulsivo, como uma func¸ãodo peso corporal dos ratos, foi
calculadocomo0,286moL/kg−1.Umadosede1/5dadosedolimiarconvulsivocausou
princi-palmenteaberturaposturaldosmembrosetremoresemtodoocorpo,enquantoumadosede 1/10dadosedolimiarconvulsivocausouagitac¸ãoetremores.Umadosede1/100dadosedo limiarconvulsivofoiassociadaàdiminuic¸ãodaatividadelocomotora.
Conclusões:Esteestudomostrou queobrometo derocurôniopossuiefeitosdeletérios
rela-cionados com a dose sobre o sistema nervoso centrale pode produzir efeitos excitatórios dependentesdadoseeconvulsões.
PublicadoporElsevierEditoraLtda.emnomedeSociedadeBrasileiradeAnestesiologia.Este ´
eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND(http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Introduction
In cases in which neuromuscular blocking drugs have beenadministeredaccidentallyintothecerebrospinalfluid (CSF),1myotonia,autonomicchanges,andconvulsionshave
occurred.2Prolonged administrationofrocuroniumin
crit-ical care patients has resulted in the drug entering the CSF.3 Alsoinpatients withimpairedfunction ofthe blood
brainbarrierthesemolecules mayreachtheCSF.Tassonyi etal.4 has reportedthat some neuromusculeragents like
atracurium and itsmetabolite laudanosine present in the CSF,vecuroniumcould notbedetected inCSFin the sub-arachnoidhemorrhage.Andalsoatracuriumpersistedinthe CSFforseveralhoursaftertheendoftheinfusion.Although itisanon-depolarizing,ionizedmedicationwithrelatively lowlipophilicity,itisknownthatrocuroniumcanpermeate theCSFafterintravenousinjection.5Accidental
administra-tionof rocuronium intothe central nervoussystem (CNS) duringregional anesthesia hasbeen reported.6 There are
limiteddataregardingtheeffectsofthesedrugsontheCNS. The aim of this study was to investigate the effects ofintracerebroventricularly administeredrocuronium bro-mideontheCNS,determinetheseizurethresholddoseof rocuroniumbromideinrats,andinvestigatetheeffectsof rocuroniumontheCNSat1/5,1/10,and1/100dilutionsof theseizurethresholddose.
Methods
The study protocol wasapproved by the Animal Research Committee of Dokuz Eylul University. Thirty-six female
Wistar albino rats that were bred in the Experimental ResearchLaboratoryofDokuzEylulUniversitywereusedin thestudy.Theratsweighed180---260gandwere87% homo-geneous, wereaged 18---24 months, andexhibited normal activity.Theanimalswerekeptunderstandardlaboratory conditionsforoneweekbeforetheexperimentsbegan(12h light: 12h light, 20---22◦C roomtemperature) to adapt to
theenvironment.Theyhadfreeaccesstowaterandpellet food, andthe study wasconducted in a noise-attenuated environmentandatthesametimeeachday.
Surgery
Anesthesia was induced by intraperitoneal injection of 50mg/kg−1sodiumthiopental(Pental® SodyumI.E.Ulugay
Ilac¸San. TAS, Istanbul,Turkey) while maintaining sponta-neousrespirationandtheblinkreflex.Theratswereplaced intheproneposition,anda4cm×2cmareabetweenthe earsandextendingtowardthenasuswasshaved.Thearea was cleaned withpovidon iodine, 1mL of sterile 1% lido-caine (Aritmal® 2%amp,Biosel Ilac¸ Sanayive TicaretA.S,
Istanbul,Turkey)wasinfiltratedunderthescalp,anda mid-linesurgicalincisionwasmade.Theheadoftheanimalwas placedinastereotaxicframewheretheheadwassecuredin aflexed-forwardposition.Theskinwaslaterallyreflected, and theexposed cranium wasgently blunt-dissectedwith thebregmaexposed.A2mmholeforthe intracerebroven-tricular rocuronium bromide injection was bored at the stereotaxiccoordinates,determinedaccordingtotheatlas of Paxinos and Watson,7 to access the lateral ventricle.
(C311/G, 20G, Plastics OneInc., VA, USA) and a suitable stylet(C311/DC,PlasticsOneInc.,VA,USA)toprevent tis-sueand/orforeignbodiesfrommovingintothecannulawere placed. The placementof the cannulain the lateral ven-triclewasconfirmedbyCSFdrainagethroughthecannula. Theedgesoftheincisionwereapproximated,suturedwith 2/0silkthread,andcleanedwith10%povidoniodine.These procedureswereconductedundersterileconditions. Forty-eighthourswereallowedfortheusualactivityoftheratsto berestoredafterthisintervention.Thepurposeofplacing apermanentcannulawastoenableICVdrugadministration whiletheratswereawakeandmobile.
Preparationofthedrug
A stock solution with 0.016moL rocuronium bromide
(Esmeron®,OrganonCorp,Oss,Holland)in10
Lwasused
todeterminetheseizurethresholddoseofrocuronium bro-mide.
Following the determination of the seizure threshold dose ofrocuronium,the1/5,1/10,and1/100 dilutionsin 10L Ringer’slactate solution(Ringer lactate, Biosel Ilac
San.TAS,Istanbul,Turkey)wereprepared.
The pHvaluesoftherocuroniumbromide,Ringer’s lac-tate,androcuroniumbromide-Ringer’slactatesolutionsat 23◦CweremeasuredwithapHmeter (InoLab® 720,WTW
Wissenschaftlich-Technische Werkstätten GmbH, Munich, Germany).
Experimentalprotocol
Determinationoftheseizurethresholddoseofrocuronium bromide (Pilotstudy):To determinethe seizurethreshold dose of rocuroniumbromide,a totalof 12 rats were ran-domizedinto2groups.
Group rocuronium (n=6): To determine the seizure threshold dose, a vinyl-coated polyethylene adapter was attachedtotheICVcannulaandaHamiltonmicro-syringe was attached to this adapter (Hamilton® 710SNR 100
L
Syringe Hamilton 710 series syringe, NV, USA). Rocuro-niumbromide(0.016moL×10L−1,total0.08moL)was
injectedviatheHamiltonsyringeindivideddosesof5L.
Each dose wasadministered consecutively over 60s, dur-ing which timethe effects of the dose on the rats were observed.Thetotaldoserequiredtoinduceatonic-clonic seizurewasrecorded.
Groupcontrol(n=6):Atotalof50LofRinger’slactate
solution,individeddosesof5L,wasadministeredviathe
ICVcannula,asexplainedabove.Theratsthatdidnothave seizuresweresacrificedaftera6hobservationperiod.
Dose-responsestudygroups(experimentalstudy):After thedeterminationoftheseizurethresholddoseof rocuro-niumbromide,theratswererandomizedinto4groupsfor thedose-responsestudy.
Group1(GroupC)(n=6):Tenmicrolitersofamixtureof Ringer’slactatesolutionandaceticacidwithapHidentical tothatoftherocuroniumsolutionthatinducedseizureswas administeredwiththeHamiltonsyringeover60s.
Group2(GroupR1/5)(n=6):Usingthesamemethodas thatusedforGroupC,1/5oftheseizuredoseofrocuronium
wasaddedtotheRinger’slactatesolutiontoobtainatotal solutionvolumeof10Landadministeredtotherats.
Group3(GroupR1/10)(n=6):Usingthesamemethod asthatusedforGroupC,1/10oftheseizuredoseof rocuro-niumwasaddedtotheRinger’slactatesolutiontoobtaina totalsolutionvolumeof10Landadministeredtotherats.
Group4(GroupR1/100)(n=6):Usingthesamemethod asthatusedforGroupC,1/100oftheseizuredoseof rocuro-niumwasaddedtotheRinger’slactatesolutiontoobtaina totalsolutionvolumeof10Landadministeredtotherats.
Assessment of the effects of intracerebroventricu-lar rocuronium bromide: A five-point scale was used to assesstheCNSeffectsofrocuroniumbromideadministered intracerebroventricularly.8
0=noobservableeffects
1=decreasedlocomotoractivityand/orpiloerection 2=agitationorshivering
3=entirebodytremors,posturing,orsplayedlimbs 4=tonic-clonicconvulsionsorseizures
Terminationofthestudy
Theanimalsthathadseizureswereimmediatelysacrificed with an intraperitoneal injection of 120mg/kg−1 sodium
thiopental.The animalsthat didnot have a seizurewere observedfor6handsacrificedwith120mg/kg−1
intraperi-tonealsodiumthiopentalattheendofthestudy.
Alltheanimalswereinjectedpostmortemwith50Lof
methyleneblueviathepermanentcannulaintothecerebral ventricle, and the brain was bisected along the longitu-dinal fissure to examine whether the dye was uniformly distributedwithintheventricle.
Statisticalanalysis
The statistical analyses were performed using the SPSS for Windows v. 11.0 statistical package. The results are expressed as the mean±standard deviation. The Kruskal---Wallistest,followed by theMann---WhitneyUand Fisher’sexacttests,wasusedforthebetween-group com-parisons. The within-group comparisons were conducted usingFriedmanand Wilcoxon tests,and p<0.05 was con-sideredstatisticallysignificant.
Results
Determinationofseizurethreshold(pilotstudy)
Theratsinthecontrolgroup,whichreceivedatotalof50L
ofRinger’slactatesolution(pH=5.2)inthelateralcerebral ventricles,didnotexhibitanybehavioralalterations.
In the rocuronium group, the seizure threshold for ICV rocuronium bromide (pH=3.6) was found to be 0.056±0.009moL. The volume necessary to induce a
seizurewasfoundtobe35.0±5.48L.Theseizure
Table1 Rocuroniumbromidedosesandvolumes.
Weight (g)
Dose (moL)
Dose (moL/kg)
Volume (L)
201 0.064 0.326 40
198 0.064 0.331 40
212 0.064 0.308 40
206 0.048 0.239 30
198 0.048 0.248 30
184 0.048 0.267 30
Theseizurethreshold,asafunctionofthebodyweightofrats, wascalculatedas0.286moL/kg.
Table2 CNSeffectsofrocuroniumat1/5oftheseizure thresholddose.
Weight(g) CNSeffect Score
189 Seizure 4
266 Extremitygetsaposture 3
232 Extremitygetsaposture 3
238 Extremitygetsaposture 3
199 Extremitygetsaposture 3
184 Seizure 4
Table3 CNSeffectsofrocuroniumat1/10oftheseizure thresholddose.
Weight(g) CNSeffect Score
194 Shivering 2
180 Extremityposture 2
182 Extremityposture 2
217 Agitation 3
187 Agitation 3
189 Shivering 2
Evaluationofthedose-responseresultsstudy (experimentalstudy)
ThepHvaluesoftheGroupC,GroupR1/5,GroupR1/10, andGroupR1/100weredeterminedtobe3.6,3.9,4.1,and 4.8,respectively.
In thisstudy,we found that 1/5of the seizure thresh-old dose typically caused splayed limbs, posturing, and entirebodytremors,whereas1/10oftheseizurethreshold dosecausedagitationandshivering(Tables2and3). One-hundredthoftheseizurethresholddosewasassociatedwith decreasedlocomotoractivity(Table4).Theeffectsobserved
Table4 CNSeffectsofrocuroniumat1/100oftheseizure thresholddose.
Weight(g) CNSeffect Score
218 Decreasedlocomotoractivity 2 196 Decreasedlocomotoractivity 1 192 Decreasedlocomotoractivity 3 202 Decreasedlocomotoractivity 1
186 Shivering 2
191 Decreasedlocomotoractivity 1
at these doses graduallyimproved over a 1-h period and were then maintainedas reducedlocomotor activity.The animals wereobserved for a totalof 6h, during thetime whenfeedingandmotor behaviorsbecamecomparableto thoseofthecontrolgroup.
Discussion
Inthis study,wefound thatrocuroniumadministeredinto the central nervous system of rats through the cerebral ventricles caused seizures when the rats were not under anesthesiaand that theseizure thresholddose of rocuro-nium was 0.286moL/kg−1. We determined during the dose-response study that 1/5 and 1/10 of the seizure thresholddoseofrocuroniumyieldedexcitatoryresponses, while 1/100 of the dose resulted in decreased locomotor activity.
Inthepilotstudy,behavioralchangeswerenotobserved in the rats when we administered 100L of Ringer’s lac-tate solution at a rate of 5L/min−1. We administered a maximum of40Lwhiledeterminingtheepilepticseizure thresholddose.Wedonothypothesizethatthevolumeof thedrugisresponsiblefortheCNSchangesobservedinrats. Inthisstudy,wedeterminedthatthepHofthedrug admin-isteredviatheICVcannulawasnotacontributingfactorto the inductionof seizures becauseonlyreduced locomotor activitywasobservedinGroup1.
Inasimilarstudy,Szenohradszkyandcolleagues admin-isteredatracurium,pancuronium,andvecuroniumintothe CNS and observed the side effects, finding the epileptic seizurepotenciesofatracurium>pancuronium>vecuronium (0.12, 0.26, and 0.46moL/kg−1, respectively).8 In the
present study, the potency of rocuronium necessary to cause seizures was found to be near that of pancuro-nium.
Incasesinwhichneuromuscularblockingdrugshavebeen administered accidentally into the CSF,1 the diffusion of
rocuroniumintotheneuronaltissuefromcerebrospinalfluid islikelyafunctionofmolecularweight,lipidsolubility,and time.9Basedonthephysicalpropertiesofrocuronium,itis
likelytoremainin thecerebrospinal fluidafterinjection, withrapidpenetrationintoneuronaltissuesbeingunlikely.9
Drugsinthecerebrospinalfluidcanberedistributedintothe peripheral circulation bytransportacross theblood---brain barrier. An ICV infusion may, in some instances, mimic a slowintravenousinfusion.10Wedidnotobservesuchan
inci-denceatanystageofthestudy,whichmayhaveoccurred becausethedoseofrocuroniumthatenteredthesystemic circulation was not sufficient to reach the concentration necessary toreach theneuromuscular junctionand cause musclerelaxation.
ICV-administered drugs may exert receptor-mediated effects at or near the brain-cerebrospinalfluid interface. Rocuronium is a quaternary ammonium compound. The positive charge of this molecule mimics that of the qua-ternary nitrogen atom of Acetylcholine (Ach). This atom is the major contributor to the effects of rocuronium on the neuronal nAchr.11 There are insufficient data on the
kineticsof thedistributionofmuscle relaxantsin theCSF when administered intothe cerebral ventricles,8,12 and it
mediatetheinhibitoryandexcitatoryeffectsobservedwith rocuronium.
Aswithallmusclerelaxants,thesiteofactionof rocuro-niumisthepostsynapticAChreceptorattheneuromuscular junction.Inanexperimentalstudy,Jonssonetal.13
demon-strated that rocuronium inhibited muscular and neuronal nAchr. Neuronal nAchr is irreversibly inhibited by rocuro-niumatthemicro-molarlevelinaconcentration-dependent manner.Thesefindingssuggestthatrocuroniumexertsonly inhibitoryeffectsontheAChreceptorsintheneuromuscular junction,whileitexertsinhibitoryandexcitatoryeffectson thenicotinicAChreceptorsintheCNS.
Fuchs-Buder etal.6 and Tassonyiet al.14 reported that
neuromuscular blocking agents may affect the CNS, as evidencedbytheinductionofapnea.
Shaoetal.15 demonstratedthatthecholinergicnervous
systemisimportantintheregulationofrespiratorypatterns, andinhibitionofnAchmaycausecentraldepressionof res-piration.Inourstudy,weobservednosignofdepressionof respirationorapnea.
Thebehavioralchangesinratsmanifestedasdecreased locomotoractivityafter1handrecoveryofnormal move-mentandfeedingbehaviorafter6hwereattributedtorapid CSFclearance,whichis2.83L/min−1inrats.16Becausethe
CSFvolumeinratsis500L,thetotalCSFclearancetimeis
176min.4,8Meulemansetal.17reportedthat45%oftheCSF
ofa290gratwasclearedin1h,whichisinaccordancewith ourfindings.
The exactmechanismsofactionofmusclerelaxantson thecentralnervoussystemhavenotbeenestablished.This studyshowedthatrocuroniumiseffectiveontheCNSand hasdose-related deleteriouseffects. Basedonthe results of this study,it is not possible toestablish a relationship betweentheeffectsofrocuroniuminhumansandrats.The amount of rocuronium accumulation in the cerebrospinal fluidinpatientsreceivinglong-termrocuroniuminfusionis unknown.
ItshouldbeconsideredthatCNSeffectscanbeobserved ifrocuroniumisinfusedoverlongerperiodsoftimeandin casesin which the blood---brain barrieris compromised or rocuroniumisadministeredinadvertentlyduringaregional block.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
References
1.PedutoV,GunguiP,DiMartinoM,etal.Accidentalsubarachnoid injectionofpancuronium.AnesthAnalg.1989;69:516. 2.SparrH,WierdaJ,ProostJ,etal.Pharmacodynamicsand
phar-macokinetics of rocuronium in intensive care patients. BJA. 1997;78:267---73.
3.Tobias J. Continuous infusion of rocuronium in a paediatric intensivecareunit.CanJAnaesth.1996;43:353---7.
4.Tassonyi E, Fathi M, Hughes GJ, et al. Cerebrospinal fluid concentrationsofatracurium,laudanosineandvecuronium fol-lowing clinical subarachnoid hemorrhage. Acta Anaesthesiol Scand.2002;46:1236---41.
5.SavareseJJ, CaldwellJE, Lien CA,et al.In: Miller RD, edi-tor.Pharmacologyofmuscle relaxantsand theirantagonists, anesthesia.5thed.Philadelphia:ChurchillLivingstone;2000. p.412---90.
6.Cardone C, Szenohradszky J, Yost S, et al. Activation of brain acetylcholine receptors by neuromuscular blocking drugs:apossiblemechanismofneurotoxicity.Anesthesiology. 1994;80:1155---9.
7.Fuchs-BuderT,StrowitzkiM,RentschK,etal.Concentrationof rocuroniumincerebrospinalfluidofpatientsundergoing cere-bralaneurysmclipping.BJA.2004;92:419---21.
8.MatteoRS, PuaEK,Khambatta HJ,etal.Cerebrospinalfluid levelsofd-tubocurarineinman.Anesthesiology.1977;46:396. 9.SegredoV,MatthayMA,SharmaML,etal.Prolonged
neuromus-cularblockadeafterlong-termadministrationofvecuroniumin twocriticallyillpatients.Anesthesiology.1990;72:566---70. 10.HennisPJ,FaheyMR,CanfellPC,etal.Pharmacologyof
lau-danosineindogs.Anesthesiology.1986;65:56---60.
11.Szenohradszky J,TrevorAJ,BicklerP,et al.Central nervous systemeffectsofintrathecalmusclerelaxantsinawakerats. AnesthAnalg.1993;76:1304---9.
12.VassilikosD,TsakiliotisS,VeronikiF,etal.Inadvertentepidural administrationofcisatracurium.EJA.2004;21:663---72. 13.Kostopanagitou G, Mylona M, Massoura L, et al.
Acci-dental epidural injection of vecuronium. Anesth Analg. 2000;91:1550---1.
14.Cesur M, Alıcı H, Erdem A, et al. Accidental caudal injec-tion of rocuronium in an awake patient. Anesthesiology. 2005;103:444---5.
15.StoeltingRK,Miller RD.Neuromuscularblockingdrugs, anes-thesia.4thed.Philadelphia:ElsevierHealthSciences;2000.p. 89---106.
16.BartkowskiRR,WitlkowskiTA,AzadS,etal.Rocuroniumonset of action: a comparison with atracurium and vecuronium. AnesthAnalg.1993;77:574---8.
