Mast cells improve functional recovery of transected peripheral nerve:
A novel preliminary study
Behrooz Ilkhanizadeh
a, Leila Zarei
b,*, Negin Farhad
c, Mehran Bahrami-Bukani
c, Rahim Mohammadi
daDepartmentofPathology,FacultyofMedicine,UrmiaUniversityofMedicalSciences,Urmia,Iran
bDepartmentofAnatomicalSciences,FacultyofMedicine,LorestanUniversityofMedicalSciences,Khorramabad,Iran
cSolidTumorResearchCenter,UrmiaUniversityofMedicalSciences,Urmia,Iran
dDepartmentofSurgeryandDiagnosticImaging,FacultyofVeterinaryMedicine,UrmiaUniversity,Urmia,Iran
ARTICLE INFO
Keywords:
Nerveregeneration BMMCs
Chitosanconduit
ABSTRACT
Background:Employmentofregenerativepropertiesofcellsattheserviceofnerverepairhasbeen initiatedduringrecentdecades.Effectsoflocaltransplantationofbonemarrow-derivedmastcellson peripheralnerveregenerationwerestudiedusingaratsciaticnervetransectionmodel.
Materialsandmethods:A10-mmsciaticnervedefectwasbridgedusingaconduitchitosan-basedhybrid conduitfilledwithBMMCsinBMMCgroup.Inpositivecontrolgroup(Pos),theconduitwasfilledwith phosphate-bufferedsaline alone.Theregeneratednervefiberswerestudied within12weeksafter surgery.Insham-operated group,thesciatic nervewasonlyexposedand manipulated.Innegative control(Neg)a10-mmsciaticnervedefectwascreatedandthenervestumpsweresuturedtothe adjacentmuscles.Theregeneratednervefiberswerestudiedfunctionally,biomechanically,histologically andimmunohiscochemically.
Results:FunctionalandbiomechanicalstudiesconfirmedfasterrecoveryofregeneratedaxonsinBMMCs transplantedanimalscomparedtoPosgroup(p<0.05).Morphometricindicesoftheregeneratedfibers showedthatthenumberanddiameterofthemyelinatedfibersweresignificantlyhigherinBMMCs transplantedanimalsthaninPosgroup(p<0.05).Inimmunohistochemistry,locationofreactionstoS- 100inBMMCstransplantedanimalswasclearlymorepositivethanthatinPosgroup.
Conclusions:BMMCstransplantationcouldbeconsideredasareadilyaccessiblesourceofcellsthatcould improvefunctionalrecoveryoftransectedsciaticnerve.
©2017ElsevierLtd.Allrightsreserved.
Introduction
A commonly encountered clinical problem in regenerative medicineisperipheralnerveinjurythatoftenendsuplong-term functionaldeficits[1].Widespreadresearchiscontinuingtoward thedevelopmentofmethodstoimproveregenerationfollowing nerveinjuryespeciallywhereatransectioninjuryisthecase[1].
Employment of regenerative properties of the cells at the serviceofnerverepairhasbeeninitiatedduringrecentdecades[2– 4]. Mast cells are fascinating, multifunctional, bone marrow- derived,tissuedwellingcells.Theycanbeactivatedtodegranulate in minutes,not onlyby IgEand antigensignaling viathe high affinityreceptorfor IgE,but alsobya diversegroupof stimuli.
These cells can release a wide variety of immune mediators, includinganexpandinglistofcytokines,chemokines,andgrowth factors[5].Mast cellshaveanarmamentariumofinflammatory mediatorsinterleukinssuchasIL-6andIL-8,andgrowthfactors, suchasvascularendothelialgrowthfactor,plateletderivedgrowth factorandproteasesthatarereleasedindegranulation[6].Asa result of extra cellular matrix degradation and changes in the microenvironmentfollowinginitialmastcellsecretion,themast cellpopulationsmaychangesignificantlyinnumber,phenotype andfunction.Thereis,moreover,strongevidencethatmastcells significantlyinfluenceangiogenesis[7,8].
Thesecharacteristics ofthemast cells hasencouraged usto conduct a study to assess local mast cell therapy in site of transectionofsciaticnervetoobservewhetherthecellscouldbeof benefitinperipheralnerveregeneration.Theaimofthepresent preliminary study was a single local transplantation of bone marrow-derived mast cells after sciatic nerve transection and entubulationusingchitosan-basedhybridconduitinrat.
*Correspondingauthorat:SolidTumorResearchCenter,UrmiaUniversityof MedicalSciences,Urmia,Iran.
E-mailaddress:[email protected](L.Zarei).
http://dx.doi.org/10.1016/j.injury.2017.05.015 0020-1383/©2017ElsevierLtd.Allrightsreserved.
ContentslistsavailableatScienceDirect
Injury
j o u r n al h o m ep a g e: w w w . el s e v i e r . c o m / l o c at e / i n j u r y
Materialsandmethods Animalsandsurgery
SixtyadultmaleSpragueDawleyrats300gwererandomized intofourgroups(n=15).Positivecontrolgroup(PC).Animalswere housed five animals per cage in a temperature and humidity controlledroomwith12:12hlight/darkcycles,andwereallowed normalcageactivitiesunderstandardlaboratoryconditions.The animals were fed with standard chow and water ad libitum.
Adequatemeasuresweretakentominimizepainanddiscomfort taking intoaccount human endpoints for animal suffering and distress.Animalswerehoused for2 weeks before entering the experiment.Allprocedureswereperformedwiththeapprovalof theEthicalCommitteeofUrmiaUniversityofMedicalSciences.The surgicalproceduresweredescribedinourpreviousworkindetail [5]. A 10-mmsciatic nerve defectwas bridged using a conduit chitosan-basedhybridconduitfilledwith20
m
LBMMCs(1106 cells/20m
L)inBMMCgroup.Inpositivecontrolgroup(Pos),theconduitwas filledwiththesamevolumeofphosphate-buffered salinealone.Theregeneratednervefiberswerestudied4weeks,8 weeks,and 12weeksaftersurgery.In Sham-surgerygroup,the sciatic nerve was only exposed and manipulated. In negative control(Neg)a10-mmsciaticnervedefectwascreated andthe nervestumpsweresuturedtotheadjacentmuscles.
Preparationoftheconduit
Thechitosan-basedhybridconduitwas preparedbasedona methodsdescribedbyothers[9].Chitosanconduitwasmadeby80 gentleinjectionofthesolutionintoahome-mademold[10].The prepared conduit was 2mmin external diameter, 1.8mm in internal diameter,and 10mm in length.This internal diameter compliedwithoptimalfunctioninratmodels.
Pokeweedmitogen-stimulatedspleencellconditionedmedium (PWM-SCM)
Spleen cells froma donorrat werecultured at a density of 2106cells/ml inRPMI 1640medium containing4mM L-gluta- mine, 510 5M 2-mercaptoethanol, 1mM sodium pyruvate, 100U/mlpenicillin,100mg/mlstreptomycin,and0.1mMnones- sentialaminoacids(completeRPMI1640)containinglectin(8mg/
ml) and placed in 75-cm2 tissue culture flasks.The cells were incubatedat37–8Cina5%CO2humidifiedatmosphere.After5– 7days,medium was collected,centrifuged for15minat 3200g, filteredthrougha0.22
m
mMilliporefilterandusedasPWM-SCM.Preparationofthebonemarrowderivedmastcells(BMMCs) Bonemarrowofadonormaleratwasusedtogeneratemast cells based on a method described by others [11]. Briefly, the animalwasanesthetized,euthanized(seeabove)andintactfemurs were removed. Sterile endotoxin-free medium was repeatedly flushedthroughtheboneshaftusinga needleand syringe.The suspension of bone marrow cells was centrifuged at 320g for 10min,andculturedataconcentrationof0.5106nucleatedcells/
mlinRPMI1640with10%FCS,100units/mlpenicillin,100mg/ml streptomycin (Life technology), 10mg/ml gentamycin, 2mM L- glutamine and0.1mMnonessential amino acids(referredtoas enrichedmedium).PWM-SCM20%(v/v)wasaddedtotheenriched medium. Flasks were then incubated at 37C in a 5% CO2 humidifiedatmosphere. Non-adherent cells weretransferredto freshmediumatleastonce aweek.After3–4 weeks,mastcell purityof>90%wasachievedasassessedbytoluidinebluestaining andflowcytometery.
Stainingofthemastcells
ThegranularityofthemastcellswasdeterminedbyToluidine blue, Alcian blue and Gimsa stainings. In brief, the cells were cytospun,fixedwithCarnoy’sfluid,andinToluidinebluestaining specimensstainedby either2min withacidtoluidineblue(pH=2.7).
Cells wereexamined by light microscopy.Staining procedure was the same for Alcian blue staining on cytospun. Briefly, slides were incubatedin3%aceticacid,3minalcianbluesolutionmicrowave:Hi power,30sand Washed inrunning water for 2min, rinsedindistilled waterand counterstainedin nuclearfastredsolution for5min, dehydrated,clearedandcoversliped[12].
Characterizationofmastcells
Mastcellswereharvested,andafterwashingwithcoldPBS,the cell-surfaceFc receptorswereblockedwith2.4G2(PharMingen, SanDiego,CA,USA)beforestaining.WeusedaPE-conjugatedanti- mousec-kit(PharMingen,USA)tostainc-kit,andmouseFc
e
RIwasstained with an FITC-conjugated anti-mouse Fc
e
RI antibody(PharMingen,USA)andcomparedwithmatchedisotypecontrol antibodies.Thecellswereincubatedwithantibodiesin50
m
LofPBSfor1hat4C,washedwithPBS,andanalyzedonBDFACSCanto IIflowcytometer(BectonDickinson,SanJose,CA,USA).Deadcells weregatedoutwhenperformingtheanalysis[12].
Walkingtrackanalysis
Theanimalsweretestedina restrictedfootpathwithadark chamberattheend.Awhitepaperwasplacedonthefloorofthe footpathandthepawsofthehindlimboftheratswerepressed downontoaink-soakedsponge,andtheywerethenallowedto walkdownthepathleavingitshindpawprintsonthepaper[13].
Thewalkingtrackswereobtainedduringthehealingperiodof12 weeksonaweeklybasis.
Distance from theheel tothe third toe knownas theprint length(PL),distancefromthefirsttothefifthtoeknownasthetoe spread(TS)anddistancefromthesecondtothefourthtoeknown as the intermediary toe spread (ITS) were obtained. All three measurementsweretakenfromtheexperimental(E)andnormal (N) sides. The Sciatic function index (SFI) in each animal was calculatedusingthefollowingformula:
SFI= 38.3(EPL-NPL)/NPL+109.5(ETS-NTS)/NTS+13.3(EIT- NIT)/NIT-8.8
AnSFIof0and 100indicatednormalandtotalimpairment, respectively.
Biomechanicaltesting
Thenervesamples wereharvestedandfixed betweenfrozen fixturesinamechanicalapparatus.TheTA.XTPlusTextureAnalyzer mechanicaltestdevicewasusedfortheassessment(StableMicro Systems,SurreyGU71YL,UK).After5min,thefrozenfixtureswere tightenedtoensurethatnoslippageoccurredduringtesting.The initiallength wassetto10mm.Eachsamplewasstretchedata constant rate of 1mm/min. The loadand displacement were sampled 5timespersecond.Eachsamplewasstretchedtocompletetensile failure.Sampleswerekeptwetmoistduringtestingusingadropof normalsalinesolutiontothenervesegments.
Histologicalassessments
Theregeneratednervesfromallgroupswereisolatedandpost fixedinOsO4(2%,2h),dehydratedthroughanethanolseriesand
embeddedinEpon.Semithintransverse(5
m
m)sectionswerenextstainedwithtoluidineblue.Animageanalyzingsoftware(Image- ProExpress,version6.0.0.319,MediaCybernetics,SilverSprings, MD,USA)wasusedtoperformmorphometricanalysis.
Immunohistochemicalassessments
For myelin sheath determination anti-S-100 (1:200, DAKO, USA)was used asa marker basedona methoddescribed in a previous study [9]. In brief, samples were post fixed with 4%
paraformaldehyde for 2h and embedded in paraffin. Before immunreaction the samples were dewaxed and rehydrated in PBS(pH7.4).Thesampleswerethenincubatedwith0.6%hydrogen peroxide for 30min. After blocking of non-specific immuno- reactions,sectionswerethenincubatedinS-100proteinantibody solutionfor1hatroomtemperature.FollowingwashingwithPBS andincubatinginbiotinylatedanti-mouserabbitIgGsolutionfor 1h, horseradish peroxidase-labelled secondary antibody was appliedfor1h.Thesampleswerethenincubatedwithchromogene substratesolution (DAB,DAKO, USA)for 10min. The results of immunohistochemistrywereexaminedunderalightmicroscope andassessedqualitatively.
Statisticalanalysis
The Results were analyzed using repeated measures and a factorial ANOVA with two between-subjects factors and the Bonferroni test was used to examine the effect of time and treatments.ExperimentalresultswereexpressedasmeansSD.
Statisticalanalyses wereperformedusingPASW18.0(SPSSInc., Chicago,IL,USA)andthesignificantdifferencewassetatP<0.05.
Results
Findingsofmastcellstaining
Bone marrow stromal cells of the mice were successfully harvested and cultured. In the first culture of the cells both adherentandconfluentcellswereobservedthatwereappearedas heterogeneouscells.Withinthefirstweektheadherentcellswere observedasconfluentcells.Incontrarytoothercommonculture media,theconfluentcellscouldlivelonger.Inthesecondpassage, becauseoflimitedspaceinthesmallerflasks(T25),theconfluent cellswereappeareddenselyandondays18and19thefirstculture cellswereappearedmorehomogenous.Afewdividingcellswere alsoobserved.Following3to4passagesandchangeoftheculture media on day 21, the cells were homogenous enough to be harvested.Theharvestedcellswerecountedandtheirviabilitywas
assessedusingtrypanbluewithNeubauermethod.Fromeachflask 12,000,000cellswithviabilityrate of90%wereharvested.After centrifugation,thesupernatantwasdiscardedandthepelletwas resuspendedin a1mLculturemediaand spreadonslides. The slideswereairdriedatroomtemperature.Theywerefixedusing carnoy and stained using toluidine blue, alcin blue and gimsa stains.Thegranulesofmastcellswerepurpletoredwherestained withtoluidineblue.Thesecellswerematachromatic.Thegranules wereblueandthenucleiwereredwherestainedwithalcinblue andvioletwherestainedwithgimsa(Fig.1).
Findingsofcharacterizationofmastcells
To characterize the harvested cells, 100000 cells were also assessedbasedontheirsurfacemarkers.Inthepresentstudy,for theharvestedcellsfromdifferentiatedbonemarrowstromalcells specificmarkers,CD117(c-kit)andFC
e
RI, wereusedusingflow cytometers.The resultsshowedthat thecells werepositivefor Mastcell-relatedantigensforeachofCD117and FCe
RI95%and93%,respectively,andfor bothofthemarkers90%(Fig.2).This resultwasconsistentwithsuccessfuldifferentiationofthecells.
Analysisofthewalkingtracks
The analysis of the walking tracks showed that the MMCs treatedanimalswereimprovedinmovementsignificantlybetter thanothernon-treatedanimals(P=0.001)(Fig.3).
Findingsofbiomechanicaltesting
Maximumpullforce(Fmax)ofnormalsciaticnervewasfoundto be5.400.33.BiomechanicalparametersincludingFmax,tensile strength,ultimatestrainandtoughness ofthenervesamplesin experimental groups are shown in Fig. 4. The biomechanical Fig1.Bonemarrowmastcellsfromratwereculturedinthemediumduringthe thirdweekofculturingbonemarrowcells.(A)Alcianblue,(B)Toluidineblueand(C) Gimsastaining.Scalebar:10mm.
Fig.2.Characterizationofbonemarrowderivedmastcells(BMMC)after3weeks.Flowcytometricanalysesofcellsurfacemarkersshowedthatthecellswerepositivefor BMMC-relatedantigensofFCeRI(93%),CD117(c-kit)(95%)andforBMMC-relateddoublepositivecells(90%).
findingsindicatedthattheparametersweresignificantlyimproved intheBMMCstreatedanimalsthannon-treatedones(P=0.032)
Histologicalstudies
TheanimalsinBMMCgroupdemonstratedsignificantlygreater nervefiber,axondiameter,andmyelinsheaththicknessduringthe studyperiodcomparedtonon-treatedanimals(Table1)(P<0.05).
Immunohistochemistry
The qualitative analysis of immunohistochemistry of regen- eratednervefibersshowedextensiveimmunoreactivitytoS-100 proteininBMMCgroup.TheexpressionofS-100proteinsignalwas locatedmainlyinthemyelinsheath.Theaxonalsoshowedaslight expressionshowingthattherewasSchwanncell-likephenotype aroundthemyelinatedaxons(Fig.5).
Discussion
Thefindingsofthepresentpreliminarystudyindicatedthatthe local transplantation of BMMCs improved functional recovery, biomechanical and histomorphometric indices in animals with transectedsciaticnerves.
Analysisofthewalkingtrackinthepresentstudyshowedthat localtreatmentoftheanimalswithBMMCsendedupsignificant improvement in locomotion. Walking track analysis has been reportedtobereliableinevaluationof repairprocessin sciatic nervesmainlybecauseinthistestsensoryinput,motorresponse andcorticalintegrationareinvolved[14].
InthepresentstudylocaladministrationofBMMCsresultedin the enhanced biomechanical properties. Peripheral nerves are remarkabletissuesthatnotonlyconductelectricalimpulses,but also must bendand stretch toaccommodatethemovement of limbs. In order to achieve this theyhave a complex structure consisting of bundles of neurons packed into fascicles and surrounded by connective tissue layers, the perineurium and epineurium. Both neural and connective tissue elements are tetheredproximallyatthespinalcordandhavenumerousbranch pointsallowingneuronsfromasinglenervetrunktosynapsewith various targetorgans. The strongest connectivetissue layers in peripheralnervesaretheperineuriumand,toalesserextent,the epineurium.Changesintheepineuriumand perineuriumextra- cellularmatrixcompositionarelikelytohavesignificanteffectson thebiomechanicalpropertiesofacellularnerve[15].Theconnec- tivetissuefromtheepineuriumformsalayeroffibermembraneat the3rddaypostoperativelyandthenformscollagenatthe8thday.
The keypoint influencingfunctionalrecoveryis thenumber of axons throughout the suture that enhances the anti-tension capacityofthenerve[16].Identifyingthemaximumtensionwhich nerves can withstand and understanding the origin of their mechanical resilience is of great importance to improve the Fig.3.Bargraphindicatingstaticfunctionalindex(SFI)valuesineachexperimentalgroupduringthestudyperiod.LocaladministrationofBMMCsgavebetterresultsin functionalrecoveryofthesciaticnervethaninPOSgroup.DataarepresentedasmeanSD.*P<0.05vsPosgroup.
Fig.4.Thegraphindicatingbiomechanicalanalysesofregeneratednervesforeach oftheexperimentalgroups.MPF:MaximumPullForce.TS:TensileStrength.US:
UltimateStrain.
Table1
Morphometricanalysesofsciaticnerveineachoftheexperimentalgroups:ValuesaregivenasmeanSD.
Groups Axoncountsfb/mm2 Axondiameter(mm) Myelinsheaththickness(mm)
Sham 286741982 11.350.15 2.600.03
Neg 51231746 3.340.12 1.030.04
Pos 182932005 6.270.16 1.340.02
BMMC 229802076* 7.720.15* 1.380.03
* Themeandifferenceissignificantatthe0.05levelvs.Posgroup.
outcomeofsurgicalnerverepairs.Theirbehaviourunderloadingis viscoelasticandislikelytobedependentuponanumberoffactors suchastheinternalfluidpressuremaintainedbytheimpermeable perineurium, the outer-inner layer integrity, the number and arrangementoffasciclesandthemolecularstructuralelementsof theextracellularmatrixsuchascollagenandelastin[16].
Morphometric analysisoftherepairednervefibersindicated that there was significant difference between BMMC and Pos animals.Regardingbetterfunctionalandmorphometricindicesin BMMCgroup,itcouldbestatedthatcelltherapybothaccelerated andimprovedtheprocessofnerveregeneration.
In immunohistochemistrytheexpressionofaxonandmyelin sheath special proteins was apparent in cell treated animals demonstratingthenormalstructureinhistology.Theresponseto S-100inBMMCstreatedanimalswasevidentlymorepositivethan inPosgroup.Thisfurtherimplied that bothrepaired axonand Schwann cell-like cells were present and accompanied by the process of myelination and the structural recovery of repaired nervefibers.
Dependingonthemastcellphenotypeandstimulus,mastcells initiatethetranscription,translationandsecretionofavariedarray ofcytokinesincludingPDGF,VEGF.Ithasalreadybeenshownthat PDGF,VEGFbearbeneficialeffectsonperipheralnerveregenera- tion[17–20].
Mastcellshavebeenproposedasangiogenesispromotersand themastcellcountappearstobeareliableprognosticmarkerin some tumors [21,22]. Mast cells cause neovascularization by producingangiogenicfactors,suchasVEGF,or substances with angiogenicproperties,suchastryptase,FGF,TNF,interleukin(IL)-8, histamineandheparin.
Angiogenesisisacomplexprocessgovernedbymanydifferent variables.Growthfactors,includingVEGF,plateletderivedgrowth factor(PDGF)andfibroblastgrowthfactor(FGF),playimportant roles.Consequentlytheirgenerationwithinnerveconduitsisvital toachievingpositiveclinicaloutcomes[23].
Theregulatoryinfluenceofthemastcellsisalsodemonstrated bythenerverepairphenomenacharacteristicoftheproliferation stage[24].Therelease ofspecificmediators(vasoactiveamines, tryptase,IL-4andNGF)ofmastcelloriginisessentialtoinitiate regeneration of damaged nerve fibres, and lead to temporary hyperinnervationofthescaratthisstage[21–29].
Mastcell-derivedcytokines,includingTNF,andgrowthfactors, suchasNGF,lowerthethresholdforactivationoflocalneuronsand promotenervefibergrowth.Thereisanatomicalevidenceformast cell associations with peripheral myelinated and unmyelinated nerves[30].
The mast cells were introduced in the site of injury in the presentpreliminary studyregardingthis factthat changingthe mastcellmicroenvironmentalterssignificantchangesinpheno- typeofthemastcellsandtheymayactasgrowthfactorspackages that only degranulate in situ and do not induce inflammatory responses[4].
Asthetitleindicatesthispreliminarystudywasconductedto assess effects of in situ transplantation BMMCs at the site of peripheral nerve injury and motor testing modalities like electrophysiology and/or electromyography are needed in the futureexperimentstosupportotherresultsincludingfunctional assessments.Sincemastcellsbeararmamentariumofinflamma- torymediators,theauthorsaimedtoassesswhethertheBMMCs couldpositivelyaffectthenerverepairprocess.Weaimedtouse the cells as package of mediators and growth factors and we expectedautolysisof thecellsinsituand releaseof theagents.
Whetherthereleaseofthesefactorsonlyisresponsiblefornerve regenerationofthetransectedsciaticnerveisnotabundantlyclear andfurtherstudyonproliferationanddifferentiationofthecells remaintobeconductedinthefuture.
Future experiments could possibly use the same chitosan hybridconduitbutcoatedwithgrowthfactorsinadditiontomast cellstoexamineifthisacceleratesregeneration.
Themajorlimitationofthepresentstudywascomparisonof thecellswithextracellularmatrix,microtubules,fibroblasts,and Schwanncellsandothernervesegmentconstituentsandconduits withgivingthehistologicaland molecularevidencesforneuro- protectiveactionofBMMCs.Thiswouldbeconsideredforfurther studies.Therefore,theauthorsstressthatthecurrentinvestigation was conducted to evaluate a single local dose and clinical treatment potential of BMMCs on nerve repair and precise mechanismsofneuroprotective actionof BMMCsintransection modelsremaintobeinvestigated.
Inconclusion,thisalterationinthebehaviorofmastcellscould befavorableincelltherapywherereadilyaccessibleandinstant sourceofcellsinlargequantitiesarerequiredandcouldbetaken intoconsiderationintheemergingfieldofregenerativemedicine andsurgery.Itcouldbeconsideredclinicallyasatranslatableroute towards new methods to enhance peripheral nerve repair in clinicalapplications.
Conflictsofinterest
Therearenoconflictsofintereststodeclare.
Authorcontributions
Behrooz Ilkhanizadeh: Analysis of data and writing of the manuscript.
LeilaZarei:StudyDesignandAnalysisofdata.
NeginFarhad:CollectionofData.
MehranBahrami-Bukani:CollectionofData.
RahimMohammadi:SurgicalProcedures.
Acknowledgements
TheauthorswouldliketothankDr.Hasanpour,Departmentof MechanicalEngineeringofBiosystems,forbiomechanicaltesting Fig.5.Immunohistochemicalanalysisoftheregeneratednerves12weeksaftersurgeryfrommiddlecable(A)Sham,(B)Neg,(C)Posand(D)BMMC.Thereisclearlymore positivestainingofthemyelinsheath-associatedproteinS-100(arrow)withintheperipheryofnerve,indicatingwellorganizedstructuralnervereconstructioninBMMSc treatednerve.Scalebar:20mm.
andKeyvanAmini,UniversityofSaskatchewanfortheirtechnical expertise.
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