Technical Reference #1627

1627.

Nerve Fibroblast Impact on Schwann Cell Behavior Lars Dreesmann; Ursual Mittnacht; Martin Lietz; Burkhard Schlosshauer, Universitat Tubingen, European Journal of Cell Biology, 88(1627), (2009)

Abstract:
In ordertorevealnon-neuronalcellinteractionsafterperipheralnervelesionswebegantoanalyzetheimpactofsciatic nervefibroblastsonSchwanncellsinvitro.Bothcelltypesareconsideredtohaveoppositeeffectsonaxonalregeneration.Fewdataareavailableonhowrepulsivenerve

Keywords:
cell interactions in vitro; migration; nerve fibroblast; nerve regeneration; proliferation; schwann cell; SiRNA; time lapse video recording

Materials & Methods:
Materials Fetal calfserum(FCS)penicillin/streptomycin(pen/ strep)gentamycinand L-glutaminewerepurchased from PAA(LinzAustria).Recombinantneuregulin- 1b1 wasfromR&DSystemsUSA.DNA/nucleusstain 46-diaminido-2-phenylindol(DAPI)andlaminincame from Sigma-Aldrich(DeisenhofenGermany).Poly-Dlysine(PDL)wasfromBecton-Dickinson(Bedford USA)Dulbecco’smodifiedEaglemedium(DMEM) was fromBioWhittaker(VerviersBelgium)andparaf- ormaldehyde(PFA)wasfromMerck(Darmstadt Germany). Cell preparations Schwanncellswerepurifiedfromsciaticnervesof Lewisrats(Brockes etal.1979). Brieflyasciaticnerve was cutinto1-mmpiecesandenzymaticallydigested withcollagenaseanddispase(1mg/ml1h37 1C each). Cellswerecollectedbycentrifugation(800rpm10min) andthencultivatedonPDL-coatedPetridisheswith normalmedium(DMEM10%FCS2mM L-glutamine 100U/mlpenicillin/100 mg/ml streptomycinor 50 mg/ml gentamycin).Threedayslatercytosinearabi- noside(10mM)wasaddedtoeliminatefibroblasts.On days5and10remainingfibroblastswerelysedwith Thy-1antibodytogetherwithbabyrabbitcomplement. To enhanceSchwanncellproliferation2 mM forskolin was addedtonormalmedium(Lietz etal.2006). Fibroblastswereisolatedfromthesciaticnervesof Lewisrats.Dissectedtissuefragmentswereputonto non-coatedPetridisheswithaminimumoffluidfor 20minat37 1C. Thennormalmediumwasadded (DMEM10%FCS2mM L-glutamine100U/ml penicillin/100 mg/ml streptomycinor50 mg/ml gentamy- cin).Aftercultivation(7days37 1C 5%CO2) tissue explantswereremovedandfibroblaststhathademi- gratedfromtheexplantswereharvestedforexpansion. Bromo-deoxy-uridine (BrdU)labeling Schwanncellproliferationwasdeterminedeither onlinebytime-lapsevideorecording(seebelow)orby metaboliclabelingwithBrdUaddedtothenormal culturemediumfor5–24hasspecifiedbelow(Schlos- shaueretal.1991). Theimmunologicaldetectionof BrdU-labeledcellswasperformedwithaBrdUlabeling kit (RocheGermany).Inco-culturesofnervefibro- blastsandSchwanncellsthetwocelltypeshadnodirect cellcontactduetospatialsegregation.Inorderto determinethepossibleproliferativeeffectofneuregulin on Schwanncellsthecellculturewastreatedwith1or 81ng/mlofneuregulin-1b1 for8hfollowedbyBrdU treatmentfor24h.Cellproliferationwasalsoanalyzed in two-fieldassays(seebelow). Time-lapse videorecording The migrationofcellswasanalyzedusingfour differentapproaches.Thefirstassaywasperformed using time-lapsevideorecording(ZeissAxiovert35with climate chamberTRZ3700temperaturecontrollerand CTI 3700CO2 controller)(Stier andSchlosshauer1998 1999). Glass-bottomPetridishes(MattekCorp.USA) were coatedwithPDL(50 mg/ml1h37 1C) andlaminin (33 mg/ml1h37 1C) andseededwith10000Schwann cells/cm2. Toperformplanartwo-compartmentco- cultureexperimentsthecentralarea(+ 4 mm)ofthe coverslipwasencircledwithagreasepen(PAP-PEN Roth Germany)toformaphysicochemicalbarrier. PurifiedSchwanncells(10000 cells)wereseededinthe centralareaonlyandnervefibroblasts(500000cells)were positionedintheareaoutsidethecirclebarrier.For monoculturesonlySchwann cells wereemployed.Cells were allowedtoadherefor2hbefore3mlnormalmedium were added.Cultivationwascontinuedfor24hat37 1C 5% CO2 beforetime-lapsevideorecordingwasstarted. Every tenminutesaframewascapturedwiththeaidof CaptureEzeProsoftware(ApplicationTechniquesInc. USA) foranadditional24h.Theresultingimagesequence was analyzedwithImageJsoftware. Two-field assay Thisassaywasperformedasanend-pointexperiment allowingahigherthroughput.Uncoatedcopyfoils (2 cm1cmand1cm1.5 cmX-70foleximaging Switzerland)weretreatedwithoxygenplasma(Plasma Cleaner/SterilizerHarrickUSA)for30sandthencoated with PDL(50 mg/ml1h37 1C). Thelargerfoilwas turnedupsidedownsothattheuncoatedsurfacewason top.Arazorbladewasusedtoimprintamarkerlinein the middleofthefoil.Thetwopointswheretheline reachedthefoiledgeswerecompletelytransectedfor 3mmwhichmadeitpossibletobendupsmalltriangular segments.ThePDL-coatedfoilsurfacewasthencoated with laminin(33 mg/ml1h37 1C). Thesmallerfoilwas then placedontothefirsttouchingthebent-uptriangles so thathalfofthelargerfoilwascovered.Thefoil sandwichwasseededwithSchwanncellsallowingthe cellstoadherefor2h.Theuppersmallerfoilwasthen removedgivingthecelltheopportunitytomigratetothe cell-freehalf.After48hinvitrothecellswerefixedand stainedwithDAPI.Thenumberofthecellswhichhad migratedtothecell-freeareaandthedistancetheyhad coveredwereanalyzedwithImageJsoftware.Therazor blade-imprinted lineservedasthezerolinetodetermine migrationdistances.Inordertoanalyzeproliferative effectsofneuregulinonSchwanncellsBrdUwasadded to theseculturesfor24h(seeabove). Gap assay To addresschangesinthemigrationvelocityof neuregulin-stimulatedcellswithorwithoutinhibitionof the RhoApathwaythetechnicallylessdemandinggap assay wasemployed.Inthisassayadefinedcell-freearea was createdwithinaconfluentcellmonolayeras describedearlier(Etienne-Manneville2006). Small rectangularmetalblocks(V4Asteel; 1.5mm1.5mm0.8 cm)wereputontoPDL/lami- nin-coatedcoverslipsin24-wellplates.Subsequently Schwanncells(50000cells/cm2) wereseededontocover- slips andallowedtoadhere for 24hbeforethemetal blocks wereremovedandpharmacologicalinhibitors addedtothemedium.Sixteenhourslaterneuregulin-1b1 was addedtothemediumfor4hfollowedbyfixationand DAPIstaining.Theremainingcell-freeareaattheoriginal siteofthemetalblockwasquantifiedusingImageJby measuring the(decreasing)distancebetweenthelong- itudinalcellfrontsataminimumoffivedifferentpositions. Transwell filterassay To studychemoattractionatranswellfilterassay (Boydenchamber)wasemployedinwhichaporous membraneservedastheinterfaceseparatingtwoculture chambers.Transwellmembranes(CorningCostar; 8 mm poresize)werecoatedonbothsideswithlaminin (33 mg/ml 1h37 1C). Schwanncells(1.5105) pre- stainedwithPKH26(Sigma)wereloadedontothe surfaceofthemembraneintheupperchamber. Differentcombinationsofmediawereusedintheupper (up) andlower(lo)chambers: (1) culture mediumplus 10% serumin(up)and(lo)(negativecontrol) (2) culturemediumplus2%serumin(up)andplus10% serum in(lo)(positivecontrol).Allothercombinations had10%serumin(up)and(lo)inadditiontomedia preconditionedfor48hbynervefibroblastsatdensities of (3) 2105 cellsor (4) 4105 cellsperwellinthe lowerchamber.In (3) and (4) nerve fibroblastswere present throughouttheassay.Afterincubationat37 1C for 6htheuppersurfaceofthemembranewascarefully wipedtoremoveallcells.Trans-migratingcellsonthe bottom ofthemembranefilterwerefixedandcounter- stainedwithDAPI.Inthreeindependentexperiments the trans-migratingcellswerecountedatfivelocations permembrane.

Microscopic Technique
Folex Imaging

Cell Type(s)
Schwann