Short
communication
Participation
of
bone
marrow-derived
cells
in
hippocampal
vascularization
after
status
epilepticus
Simone
A.
Romariz,
Karina
de
O.
Garcia,
Daisyle´a
de
Souza
Paiva,
Simone
Bittencourt,
Luciene
Covolan,
Luis
Eugeˆnio
Mello,
Beatriz
Monteiro
Longo
*
DepartmentofPhysiology,FederalUniversityofSa˜oPaulo,RuaBotucatu,862,04023-062Sa˜oPaulo,SP,Brazil
1. Introduction
In neurological disorderssuchas hypoxia/ischemia, epilepsy andbraintumors,theproliferationofendothelialcellsinducesthe growthofnewbloodvesselsinthebrain.1,2Recentstudieshave
shownthatvascular alterationsoccur afterepilepticseizuresin both humans and animals.3–6 Blood–brain barrier permeability
andincreasedblood flowintothebrainareassociatedwiththe increased vascular density and angiogenesis that occur in the epileptic brain.3 In particular, hippocampal vascularization is
severelycompromisedduringepileptogenesisinanimalmodelsof temporal lobe epilepsy.4,6 In addition, after brain damage,
inflammatory and endothelial progenitorcells withinthe bone marrowcellpopulationinfiltrate intothebrainand proliferate, increasingdramaticallyatthesiteofinjury.7–10Thesecirculating
bonemarrowendothelialcellshavebeensuggestedtoparticipate inneovascularizationafterbraininjury.11
According to theseobservations, bone marrow-derived cells canparticipateinstatusepilepticus(SE)-inducedangiogenesisand recruitcirculatingendothelialprogenitorcellsfrombonemarrow to the brain after SE. To test this possibility, we proposed to investigate the role of bone marrow-derived cells in the hippocampal vasculature at various time-points after pilocar-pine-induced SE animals. Chimeric mice engrafted with bone marrow cells expressing enhanced green fluorescent protein (eGFP) were used to visualize and easily track bone marrow-derivedcellsincorporatedintothebloodvessels.
2. Materialsandmethods
AllanimalsweremaintainedinaccordancewiththeGuidefor the Care and Use of Laboratory Animals (National Research Council). All protocols were approved by the Animal Care and EthicsCommitteeoftheUniversity(no.0334/08).
2.1. Chimerapreparation
Wepreparedthechimericmice(n=29)bytransplantingbone marrow from the C57BL/6 eGFP transgenic mice into lethally irradiated(600rad,137Cesiumsourceirradiator)adultmaleC57BL/
Seizure23(2014)386–389
ARTICLE INFO
Articlehistory:
Received16July2013
Receivedinrevisedform24December2013 Accepted22January2014
Keywords:
Hippocampus Bloodvessels Chimericmice Acuteseizures GFP Pilocarpine
ABSTRACT
Purpose:Diseasessuchastemporallobeepilepsy,braintraumaandstrokecaninduceendothelialcell proliferationandangiogenesis inspecificbrain areas.Duringstatusepilepticus (SE),bone marrow-derivedcellsareabletoinfiltrateandproliferate,dramaticallyincreasingatthesiteofinjury.However,it isstillunclearwhetherthesecellsdirectlyparticipateinvascularchangesinducedbySE.
Method: Toinvestigatethepossibleroleofbonemarrow-derivedcellsinangiogenesisafterseizures,we inducedSEbypilocarpineinjectioninpreviouslypreparedchimericmice.Micewereeuthanizedat8h, 7dor15dafterSEonset.
Results:OurresultsindicatedthatSEmodifiedhippocampalvascularizationandinducedangiogenesis. Further,bonemarrow-derivedGFP+cellspenetratedthroughtheparenchymaandparticipatedinthe
formationofnewvesselsafterSE.Wedetectedbonemarrow-derivedcellscloselyassociated with vesselsinthehippocampus,increasingthedensityofbloodvesselsthathaddecreasedimmediatelyafter pilocarpine-inducedSE.
Conclusion:We conclude thatepilepticseizuresdirectlyaffectvascularization in thehippocampus mediatedbybonemarrow-derivedcellsinatime-dependentmanner.
ß 2014BritishEpilepsyAssociation.PublishedbyElsevierLtd.Allrightsreserved.
* Correspondingauthorat:DepartamentodeFisiologia,UNIFESP,RuaBotucatu, 862,04023-062Sa˜oPaulo,SP,Brazil.Tel.:+551155792033/55764513.
E-mailaddresses:beatriz.longo@unifesp.br,beatrizlongo@gmail.com
(B.M.Longo).
ContentslistsavailableatScienceDirect
Seizure
j o urn a l hom e pa g e : ww w . e l se v i e r. c om / l oca t e / y se i z
1059-1311/$–seefrontmatterß2014BritishEpilepsyAssociation.PublishedbyElsevierLtd.Allrightsreserved.
6mice.Bonemarrow-derivedcellswereobtainedfromadult eGFP-donormice(20–25g)byflushingthefemursandtibiaewithsterile medium.The cells werewashedin Dulbecco’s modified Eagle’s medium (DMEM, Gibco, San Diego, CA, USA), counted, and resuspendedin sterilesaline. Approximately 3107 cells were administeredintoeachirradiatedrecipientanimalviaintravenous injection. The chimeric mice were allowed to recover for one monthpriortoSEinduction.
2.2. SEinduction
Chimeric mice were injected with pilocarpine (280mg/kg, intraperitoneal,i.p.,Merck,Brazil)toinduceSE.Fifteenminutes afterthepilocarpineadministration,theanimalsbeganshowing stereotypicbehaviorsandacuteseizures,asdescribed
previous-ly.12SEwascharacterizedbycontinuousepilepticseizure,rearing
andfalling,straubtail,andrepeatedheadtwitches.Becauseofthe highmortalityrateofthechimericmiceduringSE,we adminis-tered thionembutal (25mg/kg, i.p.) 30min afterthe SE onset.9
Thus,thetotalSEdurationvariedfrom30to50min,withRacine stage 3–5 in the first 30min going down to stage 2–3 after thionembutaladministration.Atthefollowingtime-points post-SE, the animals were deeply anesthetized by overdose of thionembutal (200mg/kg, i.p.): 8h (n=5), 7d (n=7) or 15d (n=7)andcontrolno-SEchimericanimals(n=10).
2.3. Immunofluorescence
Mice were deeply anesthetized and perfused through the heartwith50mLofphosphate-bufferedsaline(PBS)followedby
Fig.1.Photomicrographsshowingimmunofluorescencerepresentativesectionsofco-expressionGFPandlamininintheCA1ofchimericSE-inducedmiceofthefourgroups, (A–C)controlgroup,(D–F)8hafterSE,(G–I)7daysafterSEand(J–L)15daysafterSE.NotethelownumberofGFP+cellsco-localizedwithvesselsinmergedfiguresat8h(F)
comparedwith7and15daysafterSE(IandL).Scalebarsrepresent100mm.
150mLof4%paraformaldehyde(Sigma–AldrichCorporation,St. Louis, EUA). The brains were removed and processed for immunohistochemistry on free-floating brain slices. Coronal brainsections (30
mm
thick)weremade betweenbregma0.98 andbregma 3.28mm,targetingthehippocampusaccordingto thestereotaxiccoordinatesofthemousebrainatlas.13TovisualizeGFP+cellsinsertedinbloodvesselwalls,sectionswereincubated withanti-GFPAlexaFluor488(1:600;Molecular Probes/Invitro-gen,Eugene,USA)andwithanti-laminin(1:500,Sigma–Aldrich Corporation, St. Louis, EUA) conjugated with Alexa Fluor 546 (MolecularProbes/Invitrogen,Eugene,USA).Laminin,a constitu-entof the endothelial basal lamina, is a vascular marker that delineatesbloodvessels.4,14,15Lamininstainingby
immunofluo-rescenceallowstheidentificationofGFPcellspresentinthewall andanynewbranchesformed.Allsectionsweremountedusinga nuclear-counterstaining mounting medium containing DAPI (Vector,Burlingame,CA,EUA).Theco-localizationofGFP+cells and laminin-stained microvessels was quantified in dorsal hippocampal regions CA1, CA3 and hilar (polymorphic layer, PoDG)under20magnificationin8hippocampalserialslicesin 10randomnon-overlappingfieldsforthethreetime-pointsafter SE(8h,7dand15d)andinnon-SEchimericanimals.GFP+cells presentintheparenchymawerealsocountedonthesamecapture images.Theslideswereexaminedusingafluorescentmicroscope (Nikon80i)andconfocal(Leica SP5TS).Imageswerecaptured usingaNikonACT-1v.2systemandanalyzedusingtheImageJ imaging system. Statistical analyses were performed using ANOVA followed by the Tukey–Kramer post hoc test and Kruskal–Wallisfollowed bytheDunn test.Asignificancelevel of5%wasassumed.
3. Resultsanddiscussion
Inthepresentwork,theinfiltrationofbonemarrow-derived cells into the brain after pilocarpine-induced SE modified
the pattern of vascularization in the hippocampus. After SE, brightpointsofGFPstainingweredetectedinthevesselwalls, which suggeststhatbonemarrow-derived GFP+cells incorpo-rated into the walls of pre-existing blood vessels. These cells werealsodetectedinnewbranchessproutedfromoldones,as observedbycontinuouslaminin-GFP+stainedformedbranches (Fig.1).
Regardingtheearlystagespost-SE(8h),thenumberof laminin-stained blood vessels with inserted GFP+ cells immediately decreasedinresponsetoseizuresinallthreeanalyzed hippocam-palregions(p<0.001).Ontheotherhand,thenumberof double-stainedlaminin-GFP+bloodvesselsincreasedinthehippocampus at7daysafterSE(p<0.001)andcontinuedincreasinguntil15 daysafterSE(p<0.001),whichwasthelasttimepointexamined. Whenanalyzingthethreehippocampalregionsindividually,this patternwasalsofoundintheCA1andCA3,withasharpreduction soonafterSE(8h;p<0.001)followedbyaslightincreasein7days thatstilldifferedfromcontrol(p<0.001),reachingitslevelsat15 days afterSE. In thePoDG,however,the numberof GFP+ cells presentinthebloodvessels’wallswassignificantlyreducedinall analyzed time points, compared to the control chimera group (p<0.001)andthetotalGFP+bloodvesselscountedinCA1and CA3(p<0.001)(Fig.2A–C).
As proposed by Ndode-Ekane et al.,6 endothelial cell
proliferation and angiogenesis are responsible for recovering from the vascular injury induced by SE by restoring vascular lengthwithin2weeksafterSE.Infact,weobservedthat15days afterSE,thenumberofvesselsformedbybonemarrow-derived cells in the CA1 and CA3 (not the PoDG) reached the control values(p>0.05nscomparedtocontrolgroup;Fig.2).However, the15thdaywasthelasttime-pointweexamined,incontrast
to Ndode-Ekane’ long-lasting experiment (two months)
whichsuggested thatthegenerationof epileptogeniccircuitry was not correlated with the intensity of vascular injury or angiogenesis.
Fig.2.QuantificationofbloodvesselscontainingGFP+/laminin+double-labeledcellsinthehippocampusofchimericmiceat8h,7daysand15daysafterSE.(A)CA1;(B)PoDG
and(C)CA3.Asignificantandlownumberofdouble-labeledvesselsweredetectedat8hand7daysafterSEcomparedtoCTRLinthethreehippocampalareasandafter15 daysonlyinthePoDG(*p< 0.05versusCTRLgroup).IntheCA1andCA3,thenumberofdouble-labeledvesselsincreasedat7and15dayscomparedto8hafterSE(#p< 0.05
versus8hgroup).Inthehippocampalparenchyma,thenumberofGFP+cells(D)increasedat8hand15daysafterSE(*p< 0.05versuschronicgroup).Nodifferencewasfound
betweenthegroupsforlamininquantificationinthehippocampus(E)(one-wayANOVAfollowedbyTukey’multiplecomparisonstest;Kruskal–WallistestfollowedbyDunn’ test).DatarepresentthemeansSEM.
S.A.Romarizetal./Seizure23(2014)386–389
Theproliferationofendothelialcellsandtheirmigrationfrom bone marrow and adjacent tissue give rise to new blood vessels.11AspresentedinFig.2,controlnon-SEanimalsshowed
anelevatednumberofGFP+cellsinsertedinthevesselwalls.As suggested by Galimi and coleagues,16 in a physiological
situation, bone marrow-derived cells have been shown to participate in the vasculature of the adult central nervous system.ByanalyzingthebrainandothertissuesofchimericGFP+ animals,Galimietal.detectedalargenumberofbone marrow-derivedcellstightlyassociatedwithbloodvesselsinthebrain. However,thosecellswerenotidentifiedasendothelialcellsbut were positive for monocytic and microglial markers. In the presentstudy,incontrasttothehighnumberofbone marrow-derived cells present in the blood vessels’ walls of control animals, there were a low number of GFP+ cells in the hippocampalparenchyma ofthisgroup. Ontheotherhand, in epileptic animals, the GFP+ cells detected in the parenchyma significantly increased after SE induction (p=0.002; Fig. 2D). Thus, it is possible that when SE is induced, bone marrow-derivedGFP+cellsemerge fromthevesselsandmigrate tothe parenchyma,althoughwecannotaffirmthattheseGFP+cellsare thesamecellsfoundintheparenchyma.Accordingly,whennot associatedwithbloodvessels,cellsderivedfrombonemarrow gradually migrate to the brain parenchyma, particularly the hippocampusofepilepticanimals.9
In summary, SE directly affected vascularization in the hippocampus mediated by bone marrow-derived cells. These results are in accordance with other studies proposing that angiogenesisisassociatedwithblood-brainbarrier permeabili-ty3andbonemarrowcellinfiltrationintheepilepticbrain.9The relationwasevidentbetweenthetimeafterSEandthenumber of double-stained laminin-GFP+ vessels in the hippocampus, indicatingthatthenumberofvesselsformedwithbone marrow-derivedcellsincreasesovertimeafterSE.Likewise,ithasbeen suggestedthatvascularchangesaremostactiveduringthefirst monthafterepileptogenicinsults.4,5Onceinitiated,angiogenesis
progressively increases as the time after SE passes.3 In this
regard, the experimental model of pilocarpine-induced SE combinedwithchimericGFPanimalscontributesto understand-ing the role of bone marrow-derived cells in hippocampal vasculaturein a pathologicalsituation and helps clarifysome important aspects associated with SE-induced angiogenesis. Nevertheless, itremainscontroversialwhether theincrease in vasculardensityoftheepilepticbrainrepresentsaprogressive adaptationtoimproveperfusionduringseizures3oris
implicat-edinthemechanismunderlyingtheoccurrenceofspontaneous seizures.4,5
Conflictofinterest
Noneoftheauthorshasanyconflictofinteresttodisclose.
Acknowledgments
WearegratefultoMariaFernandaValenteandEne´asFerrazolli fortheirtechnicalsupport;toProf.Dr.RenatoMortara,Dept.of Microbiology,ImmunologyandParasitology-UNIFESPforconfocal technicalassistance;andtoVegefloraExtrac¸o˜esdoNordesteLtda, which kindlydonatedpilocarpine hydrochloride.Thisworkwas supportedbyFAPESPandCNPq.
Weconfirmthatwehavereadthejournal’spositiononissues involved in ethical publication and affirm that this report is consistentwiththoseguidelines.
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