Edema
or
apoptosis?
Jackeline
Moraes
Malheiros
a,d,
Daniele
Suzete
Persike
b,
Leticia
Urbano
Cardoso
de
Castro
c,
Talita
Rojas
Cunha
Sanches
c,
Lúcia
da
Conceic¸ão
Andrade
c,
Alberto
Tannús
d,
Luciene
Covolan
a,∗aDepartamentodeFisiologia,UniversidadeFederaldeSãoPaulo—–UNIFESP,SãoPaulo04023-06,SP,Brazil bDepartamentodeNeurologiaeNeurocirurgia,UniversidadeFederaldeSãoPaulo—–UNIFESP,SãoPaulo,SP, Brazil
cDepartamentodeNefrologia,FaculdadedeMedicinadaUniversidadedeSãoPaulo,SãoPaulo,SP,Brazil dCentrodeImagenseEspectroscopiainvivoporRessonânciaMagnética(CIERMag),InstitutodeFísicade SãoCarlos,UniversidadedeSãoPaulo(IFSC-USP),SãoCarlos13566-590,SP,Brazil
Received6November2013;receivedinrevisedform13January2014;accepted4February2014
Availableonline19February2014
KEYWORDS Pilocarpine; Epilepsy; Manganese-enhanced magneticresonance imaging; Edema; Apoptosis; Hippocampus
Summary Manganese-enhancedMRI(MEMRI)hasbeenconsideredasurrogatemarkerofCa+2
influxintoactivatedcellsandtracerofneuronalactivecircuits.However,theinductionofstatus epilepticus(SE)bykainicaciddoesnotresultinhippocampalMEMRIhypersignal,inspiteof itshighcellactivity.Similarly,shortdurationsofstatus(5or15min)inducedbypilocarpine didnotalterthehippocampalMEMRI,while30minofSEevenreducedMEMRIsignalThus,this studywasdesignedtoinvestigatepossibleexplanationsfortheabsenceordecreaseofMEMRI signalafter shortperiodsofSE.Weanalyzed hippocampalcaspase-3activation(toevaluate apoptosis),T2relaxometry(tissuewatercontent)andaquaporin4expression(water-channel
protein) ofratssubjected toshort periodsofpilocarpine-induced SE.For the timeperiods studiedhere,apoptoticcelldeathdidnotcontributetothedecreaseofthehippocampalMEMRI
∗Correspondingauthor.Tel.:+551155792033;fax:+551155792033.
E-mailaddresses:[email protected],[email protected](L.Covolan). http://dx.doi.org/10.1016/j.eplepsyres.2014.02.007
Reducedhippocampalmanganese-enhancedMRI(MEMRI)signalduringpilocarpine-inducedstatusepilepticus 645
signal.However,T2relaxationwashigherinthegroupofanimalssubjectedto30minofSEthan
intheotherSEorcontrolgroups.ThisresultisconsistentwithhigherAQP-4expressionduringthe sametimeperiod.Basedonapoptosisandtissuewatercontentanalysis,thelowhippocampal MEMRIsignal 30minafter SEcanpotentiallybe attributedtolocaledemarather thantocell death.
©2014ElsevierB.V.Allrightsreserved.
Introduction
Statusepilepticus(SE)isdefinedasaseizurethatpersistsfor asufficientlengthoftimeorisrepeatedfrequentlyenough that recovery between attacks does notoccur (CCT-ILAE, 1981).Thisdefinitionwasbasedonthepersistenceorrepe- titionoftheepilepticseizuresratherthanontheduration, althoughexpertsagreedthatitshouldlastatleast30minto evokeachronicepilepticcondition(LemosandCavalheiro, 1995).
Inthepilocarpinemodeloftemporallobeepilepsy(TLE), SE onsetis definedas a seizure that persists for at least 5min,andusuallyitmaypersistfrom4to24h(Leiteetal., 1990).Electroencephalographical(EEG)recordingsimmedi- atelyafterintraperitonialpilocarpineinjectionhaveshown thatlow-voltage,fastactivityappearsintheneocortexand amygdala,whiletheta rhythmisevidentinthehippocam- pus. As the behavioral manifestation of seizures becomes more severe, the theta hippocampal activity is replaced by high-voltage spiking and fast activity. EEG recordings immediatelyafterinjectionhaveshownthatpilocarpinecan induceictalepilepticeventsandthattheseEEGpatternsare correlatedwithbehavioralchangesthatculminateintoSE [forreview,see(Curiaetal.,2008)].
MRIisanoninvasiveandhighresolutionimagingmodality thatisconsideredthemostsensitiveandspecificstructural neuroimaging for epilepsy, allowing several neuropatho- logical studies. There are many MRI techniques: T2- and
T1-weightedimaging,functionalMRI,manganeseenhanced
MRI (MEMRI), arterial spin labeling(ASL), diffusion tensor imaging(DTI)that candetect not onlydamage causedby
statusepilepticusbutalsoplasticchangesinthebrainthat occurinresponsetodamage[forreview,see(Gröhnetal., 2011)].
Manganese-enhanced magnetic resonance imaging (MEMRI)is based onthe fact that Mn+2 is a paramagnetic
substance that changes both transverse and longitudinal relaxation(KorestkyandSilva,2004;Silvaetal.,2004)and thus can act as a contrast agent in magnetic resonance imaging (MRI). The ability of Mn+2 to compete with Ca+2
allowsittobeamarkerof increasedcellularactivity and to trace neuronal connections (Korestky and Silva, 2004; Pautleretal.,1998).
Many animal studies have found a strong correlation betweenlocalbrain activityandmanganeseenhancement usingspecificstimuliandMnCl2systemically injectedwith
andwithout transient breakdown of theblood—brain bar- rier(BBB)(Kuoetal.,2006;LinandKoretsky,1997;Pautler andKoretsky,2002;Wengetal.,2007;Yuetal.,2005).This ability, addedto the fact that Mn+2 clearly enhances the
varioussubfields ofthehippocampus(dentate gyrus(DG), CA1(CornuAmmonis)andCA3(Aokietal.,2004;Watanabe
etal.,2004),suggeststhatMEMRIcouldactasanimaging markerofepilepticfocus.
Consideringthechronicphaseofepilepsyinthekainate or pilocarpine models, recent reports provide evidence that the MEMRI hyperintensity in the DG is mostly corre- lated with mossy fiber sprouting (Immonen et al., 2008; Malheiros et al., 2012b; Nairismägi et al., 2005), or, alternatively, inversely correlated with the frequency of spontaneous recurrent seizures (Dedeurwaerdere et al., 2013).Althoughconflicting,thesefindingsrelatetheMEMRI signalinthehippocampustolocalincreasesofcellactivity. However,ithasbeenshownthatmanganeseenhancement of the MRI signal is significantly decreased in the hip- pocampus in the acute and latent phases of the kainate model(Alvestad etal.,2007). Theselast resultscouldbe eitherattributedtocelldamageor lossfollowingthelong duration of SE (Alvestad et al., 2007; Immonen et al., 2008). In an attempt to circumvent the possible causes of reduction in the MEMRI signal during SE (cell dam- age/loss),thestatuswasfullyblockedatprogressivetime periods after its onset (5, 15 or 30min) and the cell activity andthe MEMRI signal were evaluated. It resulted thatshortdurations ofstatus didnotproducedifferences in the MEMRI signal (30min of SE reduced MEMRI sig- nal) despite increased c-fos expression (Malheiros et al., 2012a).
Basedon thesefindings,the present study willfurther investigatepossible causes (apoptosis cell deathor tissue watercontent)forreductionoftheMEMRIsignalduringSE inducedbypilocarpine.
Methods
Animalsandstudydesign
All protocols were approved by the Animal Care Com- mittee of the Universidade Federal de São Paulo (CEP 0750/07).AdultmaleWistarrats(250—300g)werehoused 4rats/cageandkeptundercontrolledlaboratoryconditions (12hlight/12hdarkcyclewithlightsonat07:00a.m.,tem- perature22±1◦C,airhumidity50—60%,adlibitumaccess
tofoodandwater).Pilocarpinehydrochloride(300mg/kg, i.p.Vegeflora,Parnaiba,Brazil)wassystemicallyinjected, and30min prior, animals were given scopolaminemethyl bromide(1mg/kg,i.p.,Sigma,SaintLouis,MI,US)toreduce systemiccholinergicsideeffects.Pilocarpineanimalsdevel- opedSEonaverage30minaftertheinjection.Onehundred twoanimalswereusedinthisstudy.TheSE-relatedmortal- itywas22.3%and24.7%ofanimalsthatreceivedpilocarpine injectiondidnotdevelopSE.
646 J.M.Malheirosetal.
Fig.1 Temporaldiagramdepictingthegroups,treatments,andproceduresusedinthepresentstudy.Theanimalsweredivided
intosixgroups:Groups1—4receivedasolution ofMnCl2·4H2O(60mg/kg)12hprior topilocarpineinjection. Group1(n=6for
eachtimeperiod)andGroup3(n=4foreachtimeperiod)weredesignedtocompareMEMRIcontrastwithc-fosexpressionand
caspase-3assay,respectivelyafterSE.Groups2(n=8)and4(n=4)servedascontrolsforGroups1and3,respectively.Thesegroups
underwentthesameprotocolbutdidnotreceivepilocarpineinjections.Group5(n=5foreachtimeperiod)receivedpilocarpine
andGroup6(n=5)didnotexperienceSE.ThesegroupswereusedwithoutMnCl2injection,andT2-weightedMRIwasacquiredto
comparetheiraquaporin-4expression.Abbreviations:M-esc—–scopolaminemethylbromide;SE—–Statusepilepticus;AQ4—–aquaporin
4;Pilo—–pilocarpineandthio+dz—–thionembutal+diazepam.
ThestudydesignisoutlinedinFig.1.Ratsweredivided into6experimentalgroups.AwakeanimalsforGroups1—4 received a solution of MnCl2·4H2O (1M) diluted in bicine
solution(100mM indeionized water) withpH adjusted to 7.4usingNaOH.Final concentrationof MnCl2 was100mM.
FreshlypreparedMnCl2(60mg/kg)wasinjected intraperi-
toneally12h prior topilocarpine injectionwhen Mn+2 has
alreadyreached thehippocampusandtheMn+2 accumula-
tionisstillongoing(Leeetal.,2005).
Behavioralseizuresdevelopmentwasobservedbasedon theRacinescale (Racine,1972).TheSEonsetwasdefined after 5min of continuous seizure activity. At the end of differentSEtimeperiods(5,15 or 30min), alltheexper- imentalgroups,includingthecontrols, receivedamixture ofthionembutal+diazepam(30+10mg/kg,i.p.).Behavioral analysisin this papersupports previous encephalographic results(Melloetal.,2006)showingthatthismixturetermi- natestheSEinupto10min.
Group1 (n=6 foreach timeperiod)andGroup 3(n=4 foreachtimeperiod)wasdesignedtocompareMEMRIwith c-fosexpressionandcaspase-3assay,respectively,afterSE. Group 2 (n=8) andGroup 4 (n=4) served as controls for Group 1 and Group 3, respectively. They underwent the sameprotocolbutdidnotexperienceSE(didnotreceivethe pilocarpineinjection).Group5(n=5foreachtimeperiod) receivedpilocarpineandGroup6(n=5)didnotexperience SE.ThesegroupswerenotMnCl2injected,andT2-weighted
MRIwasacquiredtocomparetheiraquaporin-4expression. ForGroups1and2weusedexactlythesameprotocolsas previouslypublished(Malheirosetal.,2012a).
MRI
All MRI (T1 and T2-weighted) were acquired 14h after
Reducedhippocampalmanganese-enhancedMRI(MEMRI)signalduringpilocarpine-inducedstatusepilepticus 647
Fig.2 HippocampalMEMRIforpilocarpineinjectedanimals intheacutephase5, 15and30minafterSE.T1-weightedMEMRI
images(AandC)andMEMRIdata(B).Theregionsofinterest(ROIS)weredrawninthehippocampalsubregionsDG(dentategyrus),
CA1andCA3(CornuAmmonis),asrepresentedinA.TheDGwasenlargedandconvertedfromgrayintoacoloredscaleinCtoshow
differencesbetweentheCTRandSE30groups(*P<0.01).(Forinterpretationofthereferencestocolorinthisfigurelegend,the
readerisreferredtothewebversionofthisarticle.)
(even controls treated with the mixture of thionembu- tal+diazepam,asabovedescribed)wereplacedinasupine position onthe surgical table, tracheotomised, intubated (tubewithapproximately0.5mmdiameter),andconnected toarespirator forsmallanimals(model7025, UgoBasile) andventilatedwithambientair(21%FiO2),atarespiratory
rateof70cycles/minandavolumeof3.5mL/cycle.These ventilatory parameters were monitored during the image acquisitionsaccordingtophysiologicalparametersofeach animal.
Images were obtained in a 2T/30cm superconducting magnet85310HR(OxfordInstruments,Abingdon,UK)inter- facedwith a BrukerAvance AVIIIconsole (Bruker-Biospin, Inc., Billerica, MA, USA) using Paravision 5.0 software. A crossedsaddleradiofrequencycoil(Papoti,2006)wasused asaheadprobe.T1-weightedFLASH(FastLow-AngleShot)
sequence was used in animals (Groups 1—4) that were MnCl2 injected (TR=200ms, TE=5.8ms, flip angle=90◦,
4 means, 40min/animal).A volumeof 40×40×11.2mm3
was covered with a 192×192×16 points, generating a spatial resolution of 208×208×700m3. T
2 MSME (Multi
SliceMultiEcho)sequencewasacquiredinGroups5and6 with208×208m2spatialresolutiontodeterminethehip-
pocampalT2relaxation time(4averages;TR=2000ms,15
equally spaced echoes, TE=15—225ms;FOV=40×40mm, 19min/animal).
MRI data was analyzed using the Paravision 5.0 soft- ware.Oneauthor(JMM),blindedtothegroup’sidentityhas
manuallyoutlinedtheregionsofinterest(ROI).Alterations inthe relativesignal intensity ofthe dentate gyrus (DG), CA1(CornuAmmonis)andCA3werequantifiedfromasingle coronal section in T1-weighted 3D images at the antero-
posteriorlevelof−3.6mmfromthebregma(Fig.2A).The signals were calculated as the ratio between the inten- sity of the mean signal in the ROI and the intensity of themeansignalof theadjacentcorpus callosum(baseline value).Theincreasedintensityoftherelativesignal,when compared to control animals, was determined as MEMRI hyperintensity. A ROI utilized in this study, denominated DG, in reality includes the DG and the proximal portion ofthe CA3,becauseit wasnot possibletoseparate them foranalysis(Immonenetal.,2008).T2relaxationtimewas
determinedbydrawingbilaterally thecontour ofthe hip- pocampustodefinetheregionofinterest(ROI)ontheMSME images.Thehippocampallevelwasthesameasusedinthe analysisofT1-weightedimages(3.6mmcaudaltobregma).
ThesoftwaretoolISA(ImageSequenceAnalysis)wasused andtheT2calculatedfromamonoexponentialcurve.
Braintissue
C-fosimmunohistochemistry
AnimalsforGroups1and2wereperfusedtranscardially,just afterMRIacquisitions,withsalinefollowedby4%formalde- hydein0.1Mphosphatebuffer(PB,Sigma-Aldrich,pH7.4). After perfusion, the brains were then removed from the
648 J.M.Malheirosetal. skullandstoredat 4◦Cin30%sucrosefor3—4days.Coro-
nal sections with 30m were cut on a cryostat and one ofthreeconsecutivecoronalsectionswaspre-treatedwith hydrogenperoxidase,followedbynormalgoatserum(1:200) and 0.3% Triton X-100for 30min. Sections were then (1) incubatedwithprimaryantibody(rabbitantic-Fos1:3000; VectorLaboratories,CA)atroomtemperaturefor24h;(2) incubatedwitha secondaryantibody (goat anti-rabbitIgG 1:200;VectorLaboratories,CA)for2hatroomtemperature; (3) treated with 1:100 avidin—biotin complex for 90min andanickel-intensifieddiaminobenzidinereaction.Thesec- tionswererinsedinphosphatebuffer,driedandmountedon gelatin-coatedslidesandcoverslipped.
Histological imageswerecapturedona high-resolution digital camera (Nikon DXM1200), installed in a Nikon microscope (Eclipse E600FN) with a magnification of 10×. Immunohistochemical labeling of c-Fos in the DG, CA1 and CA3 was evaluated by quantitatively measuring grayscalevaluesusingtheNationalInstitutesofHealth(NIH) Image J software (http://rsbweb.nih.gov/ij/index.html). Thegrayscalevaluesforthehippocampalsubregionswere comparedtothosefortheadjacentcorpuscallosum(base- line value). Sectionswere assessedacross threedifferent levels (rostral, medium, and caudal) of the hippocampus bilaterally,correspondingtolevels2.8,3.8,and4.8mmcau- daltothebregma(PaxinosandWatson,1998)toexcludeany possiblerostro-caudalvariability.
Caspase-3fluorimetricassay
Caspase-3 activity was studied in n=4 animals per group using the method described by Thornberry et al. (1997)
recently modified by Belizário et al. (2001). Rats were decapitated just after MRI acquisitions with animals still under the effects of anaesthetic. The hippocampi were dissected at 4◦Cand immediately added to20mMHEPES
buffer(pH7.4)thatcontained2mMEDTA,0.1%CHAPS,10% sucrose,0.1%PMSF, 0.1% benzamidin, 0.1%antipain, 0.1% TLCK, 0.1% chemostatin and 0.1% pepstatin (5ml homog- enization buffer/mg tissue). Homogenates were obtained by mechanically disrupting the tissue three times on dry-ice, with thawing in an ice bath, interpolated by 1min of moderate vortex shaking. Samples were cen- trifugedat12,000×gfor40minat4◦Ctoremovecellular
debris.Totalproteinsweredeterminedinthesupernatants usingtheBio—RadProteinAssay(Bio-RadLabs,Germany). Homogenates(100mg/protein)wereincubatedat37◦Cwith
thetetrapeptidesubstrate:AspGlu-Val-Asp(Ac-DEVD-AMC, 4mM)forcaspase-3,inafinalvolumeof150ml.Foranega- tivecontrol,homogenateswerepre-incubatedfor10minat 37◦Cwithcommercialinhibitortocaspase-3(Ac-DEVD-CHO,
1mM),followedbytheadditionoftherespectivesubstrate. Activitywasmeasuredcontinuouslyover2honaFlexStation 3(MolecularProbes)Spectrofluorimeter,usingex=360nm and em=465nm. Results are expressed in activity (nmol AMC).