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Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens

Authors:
Christopher Kapitza at Friedrich-Alexander-University of Erlangen-Nürnberg
Christopher Kapitza
Rittika Chunder at Universitätsklinikum Bonn
Rittika Chunder
Anja Scheller at Universität des Saarlandes
Anja Scheller
Katherine Given at University of Colorado
Katherine Given
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Abstract and Figures

Multiple sclerosis (MS) has been considered to specifically affect the central nervous system (CNS) for a long time. As autonomic dysfunction including dysphagia can occur as accompanying phenomena in patients, the enteric nervous system has been attracting increasing attention over the past years. The aim of this study was to identify glial and myelin markers as potential target structures for autoimmune processes in the esophagus. RT-PCR analysis revealed glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), and myelin basic protein (MBP) expression, but an absence of myelin oligodendrocyte glycoprotein (MOG) in the murine esophagus. Selected immunohistochemistry for GFAP, PLP, and MBP including transgenic mice with cell-type specific expression of PLP and GFAP supported these results by detection of (1) GFAP, PLP, and MBP in Schwann cells in skeletal muscle and esophagus; (2) GFAP, PLP, but no MBP in perisynaptic Schwann cells of skeletal and esophageal motor endplates; (3) GFAP and PLP, but no MBP in glial cells surrounding esophageal myenteric neurons; and (4) PLP, but no GFAP and MBP in enteric glial cells forming a network in the esophagus. Our results pave the way for further investigations regarding the involvement of esophageal glial cells in the pathogenesis of dysphagia in MS.
RT-PCR analysis for β-actin, glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), myelin basic protein (MBP), myelin associated glycoprotein (MAG), oligodendrocytespecific protein (OSP) and myelin oligodendrocyte glycoprotein (MOG) of n = 6 mice. (A) Overview of different expression levels of the examined markers with β-actin used as housekeeping gene, shown by one representative mouse. (B) Table summarizing the PCR results of six mice and showing the expression profile of the different tissues investigated with + indicating a "high expression", + indicating a "medium-low expression", o indicating a "very low expression" and-indicating no expression. Since results from individual mice did not differ, data were pooled. C: Cerebrum; Cb: Cerebellum; Bs: Brain stem; Sm: Skeletal muscle (M. tibialis anterior); E: Esophagus; J: Jejunum; Co: Colon ascendens; GFAP: Glial fibrillary acidic protein; PLP: Proteolipid protein; MBP: Myelin basic protein; MAG: Myelin-associated glycoprotein; OSP: Oligodendrocyte-specific protein; MOG: Myelin oligodendrocyte glycoprotein.
RT-PCR analysis for β-actin, glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), myelin basic protein (MBP), myelin associated glycoprotein (MAG), oligodendrocytespecific protein (OSP) and myelin oligodendrocyte glycoprotein (MOG) of n = 6 mice. (A) Overview of different expression levels of the examined markers with β-actin used as housekeeping gene, shown by one representative mouse. (B) Table summarizing the PCR results of six mice and showing the expression profile of the different tissues investigated with + indicating a "high expression", + indicating a "medium-low expression", o indicating a "very low expression" and-indicating no expression. Since results from individual mice did not differ, data were pooled. C: Cerebrum; Cb: Cerebellum; Bs: Brain stem; Sm: Skeletal muscle (M. tibialis anterior); E: Esophagus; J: Jejunum; Co: Colon ascendens; GFAP: Glial fibrillary acidic protein; PLP: Proteolipid protein; MBP: Myelin basic protein; MAG: Myelin-associated glycoprotein; OSP: Oligodendrocyte-specific protein; MOG: Myelin oligodendrocyte glycoprotein.
… 
Expression of βIII-Tubulin, MBP, and α-BT in the esophagus A-E: Triple whole mount staining of βIII-tubulin, MBP, and α-BT (α-BT not shown, N° ⑤, Table 2). Axons (A and B, short arrows) of peripheral vagal nerve branches are only partially myelinated (C, arrowheads), while gracile blood vessel-related nerve fibers, which are tightly wrapped around the outer vessel wall, appear βIII-tubulin-positive (D, arrowheads) but always lack myelin since no MBP signal can be detected (E). The lumen of the blood vessel is marked by the asterisk (D). F-I: Triple whole mount staining of βIII-tubulin, MBP and α-BT (N° ⑤, Table 2) reveals that endplate contacting efferent axons (F, short arrows) are always unmyelinated for a long distance (H) and form a framework in the presynaptic region of the NMJ (G, arrowheads). These findings confirm the results of the ChAT staining protocol (cf. Figure 2E-H). Moreover, βIII-tubulin can also be found in enteric neurons (G, dotted line; nucleus marked by asterisk) of the myenteric plexus and therefore proves to be a suitable neuro-axonal marker for the evaluation of the ENS in the esophagus. ChAT: Cholin acetyltransferase; MBP: Myelin basic protein; Z-step = 1 μm; scale bars 20 μm.
Expression of βIII-Tubulin, MBP, and α-BT in the esophagus A-E: Triple whole mount staining of βIII-tubulin, MBP, and α-BT (α-BT not shown, N° ⑤, Table 2). Axons (A and B, short arrows) of peripheral vagal nerve branches are only partially myelinated (C, arrowheads), while gracile blood vessel-related nerve fibers, which are tightly wrapped around the outer vessel wall, appear βIII-tubulin-positive (D, arrowheads) but always lack myelin since no MBP signal can be detected (E). The lumen of the blood vessel is marked by the asterisk (D). F-I: Triple whole mount staining of βIII-tubulin, MBP and α-BT (N° ⑤, Table 2) reveals that endplate contacting efferent axons (F, short arrows) are always unmyelinated for a long distance (H) and form a framework in the presynaptic region of the NMJ (G, arrowheads). These findings confirm the results of the ChAT staining protocol (cf. Figure 2E-H). Moreover, βIII-tubulin can also be found in enteric neurons (G, dotted line; nucleus marked by asterisk) of the myenteric plexus and therefore proves to be a suitable neuro-axonal marker for the evaluation of the ENS in the esophagus. ChAT: Cholin acetyltransferase; MBP: Myelin basic protein; Z-step = 1 μm; scale bars 20 μm.
… 
PLP + -glial cells in the esophagus (identified by triple staining for DsRed, PLP and PGP 9.5 in PLP-CreERT2 x tdT mice (N° ②, Table 2). A-D (PGP 9.5 not shown): PLP + -glial cells form a meshwork throughout the esophagus. Some of these cells, located in the tunica muscularis, are arranged in parallel with the muscle fibers. While only a few of these cells can be detected by PLP antibody staining (A-D, short arrow), transgenic PLP-CreERT2 x tdT mice reveal their distribution (cf. A-C vs. D). E-I: PLP + -glial cells interact very closely with enteric neurons as the processes of the EGCs are woven around the neurons (E-H). Interconnecting strands of enteric neurons (I, short arrows) are accompanied by PLP + -glial cells (F and G, short arrows). PLP antibody staining indicates the contact zone of these cells (H, arrowheads). J (PGP 9.5 and PLP not shown): Close-up of PLP + -EGCs; these cells are interconnected by their fine, filiform processes and therefore form a network of glial cells. K-L (PGP 9.5 not shown): PLP + -myelin sheaths of a vagal nerve fiber bundle in the tunica adventitia can be identified by the PLP antibody (K, nodes of Ranvier are marked by short arrows). TdT expression shows the cell bodies of the peripheral myelinating Schwann cells (K and L, arrowheads). M-N (PGP 9.5 not shown): Blood vessel-connected EGCs have a delicate morphology (M and N, short arrows) and very long filiform processes, which appear woven around the outer vessel wall (M and N, arrowheads). The lumen of the blood vessel is marked by the asterisk. DsRed: Discosoma sp. red fluorescent protein; PGP 9.5: protein gene product 9.5; PLP: proteolipid protein; tdT: tdTomato. Z-step = 1 μm; scale bars 20 μm (A-I, K-N), 10 μm (J).
PLP + -glial cells in the esophagus (identified by triple staining for DsRed, PLP and PGP 9.5 in PLP-CreERT2 x tdT mice (N° ②, Table 2). A-D (PGP 9.5 not shown): PLP + -glial cells form a meshwork throughout the esophagus. Some of these cells, located in the tunica muscularis, are arranged in parallel with the muscle fibers. While only a few of these cells can be detected by PLP antibody staining (A-D, short arrow), transgenic PLP-CreERT2 x tdT mice reveal their distribution (cf. A-C vs. D). E-I: PLP + -glial cells interact very closely with enteric neurons as the processes of the EGCs are woven around the neurons (E-H). Interconnecting strands of enteric neurons (I, short arrows) are accompanied by PLP + -glial cells (F and G, short arrows). PLP antibody staining indicates the contact zone of these cells (H, arrowheads). J (PGP 9.5 and PLP not shown): Close-up of PLP + -EGCs; these cells are interconnected by their fine, filiform processes and therefore form a network of glial cells. K-L (PGP 9.5 not shown): PLP + -myelin sheaths of a vagal nerve fiber bundle in the tunica adventitia can be identified by the PLP antibody (K, nodes of Ranvier are marked by short arrows). TdT expression shows the cell bodies of the peripheral myelinating Schwann cells (K and L, arrowheads). M-N (PGP 9.5 not shown): Blood vessel-connected EGCs have a delicate morphology (M and N, short arrows) and very long filiform processes, which appear woven around the outer vessel wall (M and N, arrowheads). The lumen of the blood vessel is marked by the asterisk. DsRed: Discosoma sp. red fluorescent protein; PGP 9.5: protein gene product 9.5; PLP: proteolipid protein; tdT: tdTomato. Z-step = 1 μm; scale bars 20 μm (A-I, K-N), 10 μm (J).
… 
Synopsis of applied staining protocols for frozen sections including each antibody setup.
Synopsis of applied staining protocols for frozen sections including each antibody setup.
… 
Characterization of antibodies and toxins used for immunohistochemical staining.
Characterization of antibodies and toxins used for immunohistochemical staining.
… 
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Int.J.Mol.Sci.2021,22,3233.https://doi.org/10.3390/ijms22063233www.mdpi.com/journal/ijms
Article
MurineEsophagusExpressesGlial‐DerivedCentralNervous
SystemAntigens
ChristopherKapitza
1
,RittikaChunder
1
,AnjaScheller
2
,KatherineS.Given
3
,WendyB.Macklin
3
,
MichaelEnders
1
,StefanieKuerten
1,4
,WinfriedL.Neuhuber
1
andJürgenWörl
1,
*
1
InstituteofAnatomyandCellBiology,Friedrich‐Alexander‐UniversitätErlangen‐Nürnberg(FAU),
91054Erlangen,Germany;christopher.kapitza@fau.de(C.K.);rittika.chunder@fau.de(R.C.);
michi.enders@fau.de(M.E.);stefanie.kuerten@fau.de(S.K.);winfried.neuhuber@fau.de(W.L.N.)
2
DepartmentofMolecularPhysiology,CenterforIntegrativePhysiologyandMolecularMedicine(CIPMM),
UniversityofSaarland,66421Homburg,Germany;anja.scheller@uks.eu
3
DepartmentofCellandDevelopmentalBiology,UniversityofColoradoSchoolofMedicine,
Aurora,CO80045,USA;katherine.given@ucdenver.edu(K.S.G.);wendy.macklin@ucdenver.edu(W.B.M.)
4
DepartmentofNeuroanatomy,InstituteofAnatomy,UniversityHospitalsBonn,UniversityBonn,
53115Bonn,Germany
*Correspondence:juergen.woerl@fau.de;Tel.:+49‐913‐1852‐2870
Abstract:Multiplesclerosis(MS)hasbeenconsideredtospecificallyaffectthecentralnervous
system(CNS)foralongtime.Asautonomicdysfunctionincludingdysphagiacanoccuras
accompanyingphenomenainpatients,theentericnervoussystemhasbeenattractingincreasing
attentionoverthepastyears.Theaimofthisstudywastoidentifyglialandmyelinmarkersas
potentialtargetstructuresforautoimmuneprocessesintheesophagus.RT‐PCRanalysisrevealed
glialfibrillaryacidicprotein(GFAP),proteolipidprotein(PLP),andmyelinbasicprotein(MBP)
expression,butanabsenceofmyelinoligodendrocyteglycoprotein(MOG)inthemurine
esophagus.SelectedimmunohistochemistryforGFAP,PLP,andMBPincludingtransgenicmice
withcell‐typespecificexpressionofPLPandGFAPsupportedtheseresultsbydetectionof(1)
GFAP,PLP,andMBPinSchwanncellsinskeletalmuscleandesophagus;(2)GFAP,PLP,butno
MBPinperisynapticSchwanncellsofskeletalandesophagealmotorendplates;(3)GFAPandPLP,
butnoMBPinglialcellssurroundingesophagealmyentericneurons;and(4)PLP,butnoGFAP
andMBPinentericglialcellsforminganetworkintheesophagus.Ourresultspavethewayfor
furtherinvestigationsregardingtheinvolvementofesophagealglialcellsinthepathogenesisof
dysphagiainMS.
Keywords:autoantibodies;entericglia;entericnervoussystem;esophagus;dysphagia;glial
fibrillaryacidicprotein;myelinbasicprotein;motorendplate;multiplesclerosis;proteolipidprotein
1.Introduction
Multiplesclerosis(MS)isachronicinflammatory,neurodegenerativediseaseofthe
centralnervoussystem(CNS)causingmyelinsheathdestruction.Duetotheneuronal
damage,thesignaltransmissionintheCNSgetsdisrupted[1,2].Asaconsequence,
patientstypicallysufferfromabroadvarietyofsymptomslikefatigue,sensory
disturbances,paresthesia,chronicalpain,andspasticitytoparalysis,dependingonthe
affectedareas[3].
Historically,theCNShasbeenconsideredtobetheprimarytargetofautoimmunity
inMSwithevidenceforantibody‐dependentpathomechanismsagainstCNSmyelin‐and
glial‐derivedantigensinagroupofpatients[4].However,morerecentstudieshave
discussedtheroleoftheentericnervoussystem(ENS)asapotentialtargetfor
autoimmunity[5,6],hencechallengingourcurrentunderstandingofthe
Citation:Kapitza,C.;Chunder,R.;
Scheller,A.;Given,K.S.;Macklin,
W.B.;Enders,M.;Kuerten,S.;
Neuhuber,W.L.;Wörl,J.Murine
EsophagusExpressesGlial‐Derived
CentralNervousSystemAntigens.
Int.J.Mol.Sci.2021,22,3233.
https://doi.org/10.3390/ijms22063233
AcademicEditor:FabrizioMichetti
Received:23January2021
Accepted:16March2021
Published:22March2021
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.
Copyright:©2021bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(http://creativecommons.org/licenses
/by/4.0/).
Int.J.Mol.Sci.2021,22,32332of25
immunopathogenesisofMS.Forinstance,dysregulatedgastrointestinalfunctions,
anorectaldysfunction,andfecalincontinenceinMSpatientsareexamplesthatmay
includedisruptionsintheregulationoftheENS[7–9].
Anunderestimatedproblem—affectingmorethanathirdofallMSpatientsandmore
than65%ofadvancedcases—isdifficultyinswallowingandesophagealdysphagia
[10,11].Whiledifficultiesinswallowingmayalreadydevelopinpatientswithmild
impairment,itoccursmorefrequentlyinMSpatientswithseveredisability,suggestinga
directcorrelationbetweendiseaseseverityanddysphagia[12].Swallowingproblemsnot
onlyresultinreducedlifequalitybutesophagealdysphagiaalsoaccountsforahighrisk
factorforaspiration‐associatedpneumonia,acommoncauseformorbidityandincreased
mortalityintheprogressivestageofMS[10,13].
Dysphagiacanresultfromacombinationofseveralfactorsincludinginvolvementof
thetractuscorticonuclearisinthebrainstem,cerebellardysfunctionaswellaslesionsof
thelowercranialnerves[10].WhilemostoftheMSresearchfocusesonpathological
changeswithintheCNS[14,15],onlyalimitednumberofstudiesisaddressingthe
involvementoftheesophagusinthepathophysiologyofMS‐relateddysphagia[11,16].
Furthermore,dysphagiamayalsoresultfromdamagedandsubsequentlylostENS
function[17]withstudiessupportingthehypothesisthatpatientswithesophageal
dysfunctionharboranti‐entericneuronalantibodiesintheirsera[18].
Theinnervationoftheesophagusiscomplex.Striatedmuscleissuppliedby
cholinergicvagalmotorneuronsinthebrainstemnucleusambiguousand
nitrergic/peptidergicneuronsinentericganglia[19–22].Inaddition,thereissensory
innervationfromthenodoseanddorsalrootganglia.Thus,avarietyofglialcells,both
myelinatingandnon‐myelinatingaswellasenteric,wastobeexpectedandmayrepresent
potentialtargetsofimmune‐mediateddamageinMSpatients.Inthiscontext,the
antigeniccharacterizationoftheglialcellsintheesophagusremainsasanimportantstep
tounderstandifandhowtheesophagusisaffectedandundergoespathologicalchanges
inthisdisease.
Whiletherearestudiesprimarilyfocusingonthecharacterizationofentericneurons
intheesophagus[21–24],thereareonlyafewstudiesavailableontheexpressionofglial‐
derivedantigensinthisorgan[25,26].Toaddressthisgapinknowledge,inthepresent
study,wehavefirstcharacterizedtheesophagusonamRNAlevelapplyingapre‐selected
panelofcommonglialmarkers,followedbytheirexpressioninimmunohistochemical
stainingsofhealthyC57BL/6Jmousetissue.Inaddition,weusedmono‐ anddouble
transgenicmouselinesforproteolipidprotein(PLP)andglialfibrillaryacidicprotein
(GFAP)tosupportourimmunohistochemicalinvestigations.Thetibialisanteriormuscle
wasincludedinordertocompareglialcellsassociatedwithmotorinnervationofstriated
esophagealmusclewiththoseinarepresentativeskeletalmuscle.
2.Results
2.1.TheEsophagusExpressesCNSTypicalMarkersatthemRNA‐Level
Forantigeniccharacterizationoftheesophagus,sixC57BL/6Jmice(n=3female,n=
3male)wereusedtocomparetheesophaguswiththeotherregionsoftheENS,withcolon
ascendensandjejunumincludedascontrols.Furthermore,themusculustibialisanterior
wasusedasaskeletalmusclereferencefortheesophagus.Ontheotherhand,different
regionsofthebrain(includingthecerebrum,cerebellum,andbrainstem)servedas
controlsforCNSantigens.Sinceresultsfromindividualmicedidnotdiffer,datawere
pooledforexpressionanalysis.
WescreenedforexpressionofPLP,myelinbasicprotein(MBP),myelinassociated
glycoprotein(MAG),myelinoligodendrocyteglycoprotein(MOG),GFAP,and
oligodendrocyte‐specificprotein(OSP,alsoknownasclaudin‐11).PLPandMBParethe
twomostabundantmyelinproteinsofthemyelinsheathandtheirimportancefor
inducingexperimentalautoimmuneencephalomyelitis(EAE),onemousemodelforMS,
Int.J.Mol.Sci.2021,22,32333of25
hasbeenpreviouslydiscussed[27–29].Furthermore,PLPincombinationwithGFAP
identifiesauniquepopulationofglialcellsintheENSofthelowergastrointestinaltract
[30,31].MOG,whichispredominantlyexpressedinCNSmyelin,isapotentialtargetfor
cellularandhumoralimmuneresponseinEAEandMS[32].MAGisacommonmyelin
antigenthatisexpressedbothintheCNSandthePNS[33]andisalsoanantigenictarget
inperipheralneuropathies[34,35].
Differentexpressionlevelsoftheinvestigatedglialantigensweredetectedatthe
mRNAlevelintheesophagusunderphysiologicalconditions.mRNAexpressionofPLP
andMBPwerethehighestincomparisontolowlevelsofGFAPandOSPexpression
(Figure1.MAGmRNAtranscriptsweredetectedataverylowlevel.Asexpected,MOG,
whichisconsideredtobeaCNSspecificmyelinmarker,wasnotexpressedinthe
esophagus.Wealsofoundthesameexpressionofglialandmyelinmarkersinskeletal
muscle,jejunumandcolon.Theonlydifferenceswere(1)theexpressionofMAGinthe
esophagus,whichwascompletelyabsentinskeletalmuscle,jejunumandcolon;(2)the
differentexpressionlevelsinallinvestigatednon‐CNStissues.Furthermore,thedifferent
regionsofCNStissues,thatwerechosenaspositivecontrols,allshowedahighexpression
ofallinvestigatedmarkers.Basedontheseresults,wedecidedtoprovethepresenceof
MBP,PLPandGFAPintheesophagusbyimmunohistochemistryinafurtherstep.
Figure1.RT‐PCRanalysisforβ‐actin,glialfibrillaryacidicprotein(GFAP),proteolipidprotein
(PLP),myelinbasicprotein(MBP),myelinassociatedglycoprotein(MAG),oligodendrocyte‐
specificprotein(OSP)andmyelinoligodendrocyteglycoprotein(MOG)ofn=6mice.(A)
Overviewofdifferentexpressionlevelsoftheexaminedmarkerswithβ‐actinusedas
housekeepinggene,shownbyonerepresentativemouse.(B)TablesummarizingthePCRresults
ofsixmiceandshowingtheexpressionprofileofthedifferenttissuesinvestigatedwith+
indicatinga“highexpression”,+indicatinga“medium–lowexpression”,oindicatinga“verylow
expression”and–indicatingnoexpression.Sinceresultsfromindividualmicedidnotdiffer,data
werepooled.C:Cerebrum;Cb:Cerebellum;Bs:Brainstem;Sm:Skeletalmuscle(M.tibialis
anterior);E:Esophagus;J:Jejunum;Co:Colonascendens;GFAP:Glialfibrillaryacidicprotein;
PLP:Proteolipidprotein;MBP:Myelinbasicprotein;MAG:Myelin‐associatedglycoprotein;OSP:
Oligodendrocyte‐specificprotein;MOG:Myelinoligodendrocyteglycoprotein.
Int.J.Mol.Sci.2021,22,32334of25
2.2.DistributionofMBP,PLPandGFAPintheCerebellum
CryosectionsofcerebellartissuewerechosenaspositivecontrolsforMBP,PLP,and
GFAPimmunohistochemistry.Forasimpledifferentiationbetweenthethreemain
laminaeofthecerebellarcortex,(1)stratummoleculare,(2)stratumpurkinjense,and(3)
stratumgranulare(SupplementaryFigureS1A),calbindinD28k(CALB)waschosenasa
marker.ThiscalciumbindingproteinwasfoundinPurkinjecellsandthereforestained
theprominent,pykniccellbodieslocatedinthestratumpurkinjenseaswellastheir
ramificationinthestratummoleculare,causedbytheprotrudingdendrites.Thethird
lamina,thestratumgranulare,whichisdirectlyadjacenttothemedullarylayer,however,
showedonlylittlepositivenessforCALBbutincontrastnumerousgranulecells,indicated
withHoechstnuclearstaining(SupplementaryFigureS1A).
Inordertoidentifythedistributionandtheco‐localizationofPLPandMBP,we
combinedthesetwomyelinmarkerswithCALBandHoechstinaquadruplestaining(N°
①,Table1;SupplementaryFigureS1A,B).PLPandMBPlikewiseindicatedaxons
myelinatedbyoligodendrocytesinthestratumgranulareandthewhitematter.Moreover,
somePLP+‐ andMBP+‐fibersreachedthestratummoleculareensheathingCALB+–
afferences,whichmostlikelyrepresentafferencesfromtheinferiorolive[36].Asexpected,
inallcases,MBPandPLPappearedcompletelycongruentduetotheirappearanceinthe
myelinsheath(SupplementaryFigureS1C–E).Toinvestigatedifferencesinthe
organizationofGFAP+‐glialcellsandmyelinatingglialcells,weperformedanother
quadruplestaining(N°②,Table1;SupplementaryFigureS1F–G),combiningPLP,
GFAP,CALB,andHoechst.IncontrasttothedistributionforPLPasdescribedabove,
GFAP+–cellsoccurredwidelyspreadinalldifferentsegmentsofthecerebellumand
appearedtonotbehighlyco‐locatedwithPLP+‐glialcells(SupplementaryFigureS1H–J).
Inthestratumgranulare,thelongish,cross‐linkedprotrusionsofBergmannGlia,unipolar
astrocyte‐likecells,couldbefound,leadingtoanetworksurroundingthePurkinjecells
(SupplementaryFigureS1I)[37].Throughoutthestratummoleculareahighnumberof
prolate,radialalignedfibersweredetectable,whichthenterminatedatthepialsurface,
formingthemembranalimitansgliaesuperficialis(SupplementaryFigureS1G).Taken
together,theseresultsprovethespecificityoftheusedantibodies.
Table1.Synopsisofappliedstainingprotocolsforfrozensectionsincludingeachantibodysetup.
N°StainingProtocolAntibodySetup
①QuadruplestainingofPLP,CalbindinD28k,MBPand
HoechstforCerebellarTissue
PrimaryAntibodies
Ratanti‐PLP
Guineapiganti‐CalbindinD28k
Rabbitanti‐MBP
SecondaryAntibodies
Donkeyanti‐ratAlexa488
Goatanti‐guineapigAlexa555
Donkeyanti‐rabbitAlexa647
Hoechst
②QuadruplestainingofPLP,CalbindinD28k,GFAPand
HoechstforCerebellarTissue
PrimaryAntibodies
Ratanti‐PLP
Guineapiganti‐CalbindinD28k
Rabbitanti‐GFAP
SecondaryAntibodies
Donkeyanti‐ratAlexa488
Goatanti‐guineapigAlexa555
Donkeyanti‐rabbitAlexa647
Hoechst
③TripleStainingofGFAP,Synaptophysinandα‐BTfor
SkeletalMuscleandEsophagus
PrimaryAntibodies
Rabbitanti‐GFAP
Guineapiganti‐Synaptophysin
Int.J.Mol.Sci.2021,22,32335of25
SecondaryAntibodiesand
Toxins
Donkeyanti‐rabbitAlexa488
Donkeyanti‐guineapigAlexa647
α‐BungarotoxinAlexa555
④TriplestainingofMBP,Synaptophysinandα‐BTforSkeletal
Muscle
PrimaryAntibodies
Rabbitanti‐MBP
Guineapiganti‐Synaptophysin
SecondaryAntibodiesand
Toxins
Donkeyanti‐rabbitAlexa488
Donkeyanti‐guineapigAlexa647
α‐BungarotoxinAlexa555
⑤QuadruplestainingofPLP,Synaptophysin,α‐BTand
HoechstforSkeletalMuscleandEsophagus
PrimaryAntibodies
Ratanti‐PLP
Guineapiganti‐Synaptophysin
SecondaryAntibodiesand
Toxins
Donkeyanti‐ratAlexa488
Donkeyanti‐guineapigAlexa647
α‐BungarotoxinAlexa555
Hoechst
⑥TripleStainingofChAT,MBPandα‐BTforSkeletalMuscle
PrimaryAntibodies
Goatanti‐ChAT
Rabbitanti‐MBP
SecondaryAntibodiesand
Toxins
Donkeyanti‐goatAlexa647
Donkeyanti‐rabbitAlexa488
α‐BungarotoxinAlexa555
⑦TripleStainingofChAT,Synaptophysinandα‐BTfor
SkeletalMuscle
PrimaryAntibodies
Goatanti‐ChAT
Guineapiganti‐Synaptophysin
SecondaryAntibodiesand
Toxins
Donkeyanti‐goatAlexa488
Donkeyanti‐guineapigAlexa647
α‐BungarotoxinAlexa555
⑧TripleStainingofβIII‐tubulin,MBPandα‐BTforSkeletal
Muscle
PrimaryAntibodies
Rabbitanti‐βIII‐tubulin
Chickenanti‐MBP
SecondaryAntibodiesand
Toxins
Donkeyanti‐rabbitAlexa488
Goatanti‐chickenAlexa647
α‐BungarotoxinAlexa555
2.3.GlialCellsinNeuromuscularJunctionsoftheTibialisAnteriorMuscle
Toaddressthequestion,howthethreemainglialmarkersofthisstudyareinvolved
intheformationoftheneuromuscularjunction(NMJ)inaskeletalmuscle,whichserves
asacomparisoncontrolforthestriatedesophagealmuscle,cryosectionsofthemusculus
tibialisanteriorwereappliedforimmunohistochemicaltriplestainings(Supplementary
FigureS2A–P).Eachtime,synaptophysinasamarkerforcholinergicvesicles,andα‐
Bungarotoxin(α‐BT),asaspecificmarkerformotorendplatesbindingtopostsynaptic
acetylcholinereceptors,wereused,andshowedalinkeddistributionintheendplate
Int.J.Mol.Sci.2021,22,32336of25
region:Synaptophysinasapresynapticmarkerwassurroundedbythecontourofα‐BT,
whichtracedthepostsynapticsideofNMJ.Ingeneral,synaptophysinwasabundantin
theendplateregion,whileitsexpressionalongtheaxondecreasedproximallywith
distancetotheendplate(SupplementaryFigureS2D,H,L).
Furthermore,GFAP(N°③,Table1;SupplementaryFigureS2A–D)couldbefound
inmyelinatingglialcellsofnervefibers,whichimpliesthatperipheralSchwanncells
containGFAPasacytoskeletoncomponent[38].Alongtheirwaytothemotorendplate,
thecaliberofthemyelinatedfibersdecreaseduntiltheendplatewasreached.Atthis
point,glialcellschangedfrommyelinatingtonon‐myelinatingGFAPpositive
perisynapticSchwanncells(PSC),resultinginaframe‐likestructurethataccompaniedthe
motoraxon,butwaspredominantlylocatedinthepresynapticregion(Supplementary
FigureS2B).Theassumptionofmyelinlossofglialcellsclosetotheendplateregionwas
confirmedbythestainingresultsofMBP(N°④,Table1;SupplementaryFigureS2E–H)
andPLP(N°⑤,Table1;SupplementaryFigureS2I–P):Inbothcases,theaxonthatwas
eventuallycontactingtheendplatewasensheathedbyamyelinatingSchwanncell—but
shortlybeforetheendplate,themyelinsheathendedandfromthereon,anunmyelinated
synaptophysin+‐axoncontinuedtotheendplateregion(SupplementaryFigureS2Eand
S2I).WhilePLPcouldbefoundinthemyelinsheathinallcases,wefurthermorenoticed
aheterogeneousdistributionofPLPintheterminalendplateregion:Inmostcases,afaint
stainingforPLPcouldbeseen,suggestingthatthePSCaredoublepositiveforGFAPand
PLP(SupplementaryFigureS2BandS2N).However,insomecases,thiscouldnotbe
detected(SupplementaryFigureS2J).
Inordertoconfirmthestainingresultsofthepresynapticmarkersynaptophysin,we
usedtheneuro‐axonalmarkerβIII‐tubulinallowingtotraceaxonsoveralongperiod.
Therefore,weappliedatriplestainingofβIII‐tubulin,MBP,andα‐BT(N°⑧,Table1;
SupplementaryFigureS3A–L).Asexpected,βIII‐tubulinandsynaptophysinshowedthe
samedistributionintheareaaroundtheNMJ(cf.SupplementaryFigureS2D,H,L,Pand
SupplementaryFigureS3J).However,inaddition,βIII‐tubulinmadeitpossibleto
examineaxonalstructurestotheirfullextent(SupplementaryFigureS3B,E,G,J).In
combinationwiththeMBPantibody,wethereforecouldestablishausefulpanelof
markersfortheevaluationofaxonalmyelination(SupplementaryFigureS3A–F).Again,
wefoundanabruptlossofmyelinofthecontactingefferencepriortotheendplate,which
provedtheresultsofthesynaptophysinstainings(cf.SupplementaryFiguresS2Eand
S3I).
Together,theseresultssuggestthatintibialisanteriormusclesubsequentlyafter
myelinatingGFAP+/PLP+‐glialcells,abundantnon‐myelinatingGFAP+/PLP+‐glialcells
(PSC)accompaniedtheterminalmotoraxonasaframe‐likestructure.Incontrast,MBP
distributioninthismusclewasrestrictedtothemyelinsheaths,whichdisappearedshortly
beforetheendplateareaandcouldnotbefoundintheendplateregion.
2.4.GlialCellsinNeuromuscularJunctionsoftheEsophagus
TodeterminewhetherglialcellsintheNMJoftheesophagusareinteractingwiththe
endplatesimilarlytotheonesintheskeletalmuscle,weperformedmultilabel
immunohistochemistryoncryosectionsandwholemounts.
GFAP(N°③,Table1;Figure2A–D)showedasimilardistributionasintheskeletal
muscle.However,motoraxonswereconsiderablysmallerthanintheskeletalmuscle,
supportingtheimpressionthatefferentsintheesophagusalwayslackmyelinbefore
reachingthemotorendplate.Inaddition,motorendplatesappearedtobecontactedbya
moredelicatewebofGFAP+‐glialcells,surroundingtheendplateregion(Figures2A,B).
Int.J.Mol.Sci.2021,22,32337of25
Figure2.ExpressionofGFAP,MBP,andPLPinneuromuscularjunction(NMJ)oftheesophagus
A–D:TriplestainingforGFAP,α‐bungarotoxin(α‐BT),andsynaptophysin(SYN)(N°③,Table1)
showedasimilardistributionastheskeletalmusclewiththeexceptionthatefferenceswere
unmyelinatedandhadasmallercaliber.E–H:QuadruplestainingforMBP,α‐BT,cholin
acetyltransferase(ChAT),andHoechst(Hoechstnotshown;N°①,Table2).ChAT
+
–efferences
whichcontactthemotorendplatealwayslackmyelin(EandH,longarrow).Twofurthertypesof
fiberscouldbedetected:(1)MyelinatedChAT
+
–efferences(EandF,arrowheads)and(2)
myelinatedChAT
‐
‐fibers(EandF,shortarrow)—thelattercanbebroughtinlinewithesophageal
afferences.IandL:QuadruplestainingforPLP,α‐BT,SYN,andHoechst(Hoechstnotshown;N°
⑤;Table1).Incontrasttotheskeletalmuscle,PLPwaspresentinallinvestigatedNMJsas
groupedPLP
+
‐glialcellsaroundtheendplateindicate(IandJ).NucleiofthesePLP
+
‐PSCsare
markedbyasterisks(J;confirmedbyHoechstnuclearstaining(notshown)).Contrarytothe
distributionofMBP,PLPcouldalsobefoundthroughoutthePlexusmyentericusasPLP
+
–
efferencesshow(IandJ,longarrow).M–N:TriplestainingforDiscosomasp.redfluorescent
protein(DsRed1),PLP,andproteingeneproduct9.5(PGP9.5)inPLP‐CreERT2xtdTomato(tdT)
mice(anti‐DsRedandPGP9.5notshown;N°②,Table2).ThefaintsignalofPLPintheendplate
regionwasconfirmedbytdTexpressionasbothPLPandtdTshowthesamedistribution(Mand
N).α‐BT:α‐bungarotoxin;ChAT:Cholinacetyltransferase;DsRed:Discosomasp.redfluorescent
protein;GFAP:Glialfibrillaryacidicprotein;MBP:Myelinbasicprotein;PGP9.5:Proteingene
product9.5;PLP:Proteolipidprotein;SYN:Synaptophysin;tdT:tdTomato.Z‐step=1μm(A–D;I–
N)and0.8μm(E–H);scalebars10μm(A–D,I–N),30μm(E–H).
Int.J.Mol.Sci.2021,22,32338of25
Table2.Synopsisofappliedstainingprotocolsforwholemountsincludingeachantibodysetup.
N°StainingProtocolAntibodySetup
①QuadrupleStainingofMBP,ChAT,α‐BTandHoechst
PrimaryAntibodies
Rabbitanti‐MBP
Goatanti‐ChAT
SecondaryAntibodiesand
Toxins
Donkeyanti‐rabbitAlexa488
Donkeyanti‐GoatAlexa647
α‐BungarotoxinAlexa555
Hoechst
②TripleStainingofDsRed,PLPandProteinGeneProduct9.5
(PGP9.5)inPLP‐CreERT2xtdTmice
PrimaryAntibodies
Rabbitanti‐DsRed
(Thisantibodyalsobindsto
tdTomato)
Ratanti‐PLP
Guineapiganti‐PGP9.5
SecondaryAntibodies
Donkeyanti‐rabbitAlexa647
Donkeyanti‐ratAlexa488
Donkeyanti‐guineapigDYE405
③TripleStainingofGFAP,PGP9.5andHoechst
PrimaryAntibodies
Rabbitanti‐GFAP
Guineapiganti‐PGP9.5
SecondaryAntibodiesand
Toxins
Donkeyanti‐rabbitAlexa488
Goatanti‐guineapigAlexa555
Hoechst
④DoubleStainingofGFPandDsRedinGFAP‐EGFPxPLP‐
DsRed1Mice
PrimaryAntibodies
Chickenanti‐GFP
Rabbitanti‐DsRed
SecondaryAntibodiesand
Toxins
Goatanti‐chickenAlexa647
Donkeyanti‐rabbitDYE405
⑤TriplestainingofβIII‐Tubulin,MBPandα‐BT
PrimaryAntibodies
Rabbitanti‐βIII‐tubulin
Chickenanti‐MBP
SecondaryAntibodiesand
Toxins
Donkeyanti‐rabbitAlexa488
Donkeyanti‐chickenAlexa647
α‐BungarotoxinAlexa555
Toverifytheimpressionofanearliermyelinlossincomparisontoskeletalmuscle,
weperformedaquadruplewholemountstainingofMBP,cholineacetyltransferase
(ChAT),α‐BT,andHoechst(Staining①,Table2;Figure2E–H).Weusedthewhole‐
mountapproachtofacilitatetrackingofnervefibersoveralongerdistance.Theseresults
demonstratethatefferentaxons(identifiedasChAT‐positive)loseMBPlongbeforethey
reachtheendplate(Figure2E,longarrow).Moreover,themajorityofnervefibersinthe
myentericplexuswasalreadyunmyelinatedwithonlyafewmyelinatednervefibers
present(Figure2E,F).SomeofthemyelinatedaxonswereChAT+,thusidentifiedasmotor
axons(Figure2E,arrowheads).OthersappearedtobenegativeforChAT,assumingthat
thesenervefiberswereafferentscrossingtheplexusontheirwaytotheCNS(Figure2E,
Int.J.Mol.Sci.2021,22,32339of25
shortarrows).TodemonstratethespecificityoftheusedChATantibody,weapplied
positivecontrolexperimentsinM.tibialisanterior(Staining⑥and⑦,Table1,
SupplementaryFigureS4A–N).InafurthertriplewholemountstainingofMBP,the
neuro‐axonalmarkerβIII‐tubulin,andα‐BT(Staining⑤,Table2)wecouldvalidatethe
resultsofthedescribedChAT‐stainingprotocolaswefoundendplatecontactingefferent
axonsintheesophagusunmyelinated(Figure3F–I).Moreover,smallerperipheral
branchesofthevagalnervecontainedonlysomemyelinatednervefiberswhilemostof
theaxonslackedmyelin(Figure3A–C).Incontrasttothat,blood‐vessel‐relatednerve
fibers,whichappearedwrappedaroundtheoutervesselwall,alwaysprovedtobe
unmyelinated(Figure3D–E).
Figure3.ExpressionofβIII‐Tubulin,MBP,andα‐BTintheesophagusA–E:Triplewholemount
stainingofβIII‐tubulin,MBP,andα‐BT(α‐BTnotshown,N°⑤,Table2).Axons(AandB,short
arrows)ofperipheralvagalnervebranchesareonlypartiallymyelinated(C,arrowheads),while
gracilebloodvessel‐relatednervefibers,whicharetightlywrappedaroundtheoutervesselwall,
appearβIII‐tubulin‐positive(D,arrowheads)butalwayslackmyelinsincenoMBPsignalcanbe
detected(E).Thelumenofthebloodvesselismarkedbytheasterisk(D).F–I:Triplewholemount
stainingofβIII‐tubulin,MBPandα‐BT(N°⑤,Table2)revealsthatendplatecontactingefferent
axons(F,shortarrows)arealwaysunmyelinatedforalongdistance(H)andformaframeworkin
thepresynapticregionoftheNMJ(G,arrowheads).Thesefindingsconfirmtheresultsofthe
ChATstainingprotocol(cf.Figure2E–H).Moreover,βIII‐tubulincanalsobefoundinenteric
neurons(G,dottedline;nucleusmarkedbyasterisk)ofthemyentericplexusandthereforeproves
tobeasuitableneuro‐axonalmarkerfortheevaluationoftheENSintheesophagus.ChAT:
Cholinacetyltransferase;MBP:Myelinbasicprotein;Z‐step=1μm;scalebars20μm.
Inthenextstep,wesetouttoinvestigatewhetherthereweredifferencesinthe
distributionofMBPandPLP.Therefore,weatfirstusedthesamestainingprotocolfor
esophaguscryosectionsasfortheskeletalmuscle(Staining⑤,Table1;Figure2I–L).The
datashowthesamedistributionpatternforPLPasforMBP.However,PLPwasalso
presentinthemyentericplexusandintheendplateregion.
Theovalshapesofthemotorendplates,labeledbyα‐BT,weresurroundedby
groupedPLP
+
‐cells(Figure2I,J).UnlikeMBP,PLPwasfoundthroughoutthemyenteric
plexus,asshownbyPLP
+
‐axonsterminatingonmotorendplates(Figure2I,J,longarrow).
Therefore,weassumedthatPLPwasontheonehandpresentinthemyelinsheathof
Int.J.Mol.Sci.2021,22,323310of25
myelinatingSchwanncellsandontheotherhandalsoinMBP–/PLP+–cellscontactingthe
motorendplates.Inordertoconfirmthishypothesisandtoprovespecificbindingofthe
usedPLPantibody,weestablishedastainingprotocolusingwholemountsoftransgenic
tamoxifen‐induciblePLP‐CreERT2xtdTomatomice.Thistransgenicmousestrainshows
tdTomato(tdT)fluorescenceinPLP+‐cellsaftertamoxifeninducedrecombination.Hence,
wecombinedthePLPandanti‐DsRedantibodystaining—thelatterreactingagainsttdT
forsignalamplification—withproteingeneproduct9.5(PGP9.5)staining(N°②,Table
2).Inthisway,wecouldshowacompleteco‐localizationofthePLPantibody‐staining
andtdT(Figure2M,N).
Insummary,wewereabletoshowthatintheesophagus(1)GFAPandPLPwere
presentinnon‐myelinatingglialcellsofaxons,whichterminatedonesophagealmotor
endplatesandinPSCsinmotorendplatesareas.(2)Inagreementwiththeresultsinthe
tibialisanteriormuscle,MBPwasabsentfromendplateareasintheesophagus,butin
contrasttotheskeletalmuscle,MBPwasnotpresentinSchwanncellsofmotoraxons
alreadylongbeforetheyreachedthemotorendplatearea.
2.5.TransgenicMiceRevealthePresenceofDifferentTypesofEntericGlialCellsinthe
Esophagus
ToassessthedistributionofPLPinglialcellsoftheesophagealENS,wecontinued
examiningthewholemountstainings(N°②,Table2;Figure4A–N)ofPLP‐CreERT2×
tdTmice.ForafurthercomparisonofGFAPandPLPweintroducedastainingprotocol
forwholemountsofdoubletransgenicGFAP‐EGFPxPLP‐DsRed1mice(N°③,Table2;
Figure5D–O).Intheseanimals,EGFPisdrivenbythehumanGFAPpromotersand
DsRed1expressioniscontrolledbythemurinePLPpromoter.Inaddition,weanalyzeda
triplestainingforGFAP,PGP9.5,andHoechst(N°④,Table2;Figure5A–C)inwhole
mountsofC57BL/6Jmice.
Int.J.Mol.Sci.2021,22,323311of25
Figure4.PLP
+
‐glialcellsintheesophagus(identifiedbytriplestainingforDsRed,PLPandPGP
9.5inPLP‐CreERT2xtdTmice(N°②,Table2).A–D(PGP9.5notshown):PLP
+
‐glialcellsforma
meshworkthroughouttheesophagus.Someofthesecells,locatedinthetunicamuscularis,are
arrangedinparallelwiththemusclefibers.WhileonlyafewofthesecellscanbedetectedbyPLP
antibodystaining(A–D,shortarrow),transgenicPLP‐CreERT2xtdTmicerevealtheirdistribution
(cf.A–Cvs.D).E–I:PLP
+
‐glialcellsinteractverycloselywithentericneuronsastheprocessesof
theEGCsarewovenaroundtheneurons(E–H).Interconnectingstrandsofentericneurons(I,
shortarrows)areaccompaniedbyPLP
+
‐glialcells(FandG,shortarrows).PLPantibodystaining
indicatesthecontactzoneofthesecells(H,arrowheads).J(PGP9.5andPLPnotshown):Close‐up
ofPLP
+
‐EGCs;thesecellsareinterconnectedbytheirfine,filiformprocessesandthereforeforma
networkofglialcells.K–L(PGP9.5notshown):PLP
+
‐myelinsheathsofavagalnervefiberbundle
inthetunicaadventitiacanbeidentifiedbythePLPantibody(K,nodesofRanvieraremarkedby
shortarrows).TdTexpressionshowsthecellbodiesoftheperipheralmyelinatingSchwanncells
(KandL,arrowheads).M–N(PGP9.5notshown):Bloodvessel‐connectedEGCshaveadelicate
morphology(MandN,shortarrows)andverylongfiliformprocesses,whichappearwoven
aroundtheoutervesselwall(MandN,arrowheads).Thelumenofthebloodvesselismarkedby
theasterisk.DsRed:Discosomasp.redfluorescentprotein;PGP9.5:proteingeneproduct9.5;PLP:
proteolipidprotein;tdT:tdTomato.Z‐step=1μm;scalebars20μm(A–I,K–N),10μm(J).
Int.J.Mol.Sci.2021,22,323312of25
Figure5.DistributionofPLPandGFAPinesophagealglialcells.A–C:TriplestainingforGFAP,
PGP9.5,andHoechst(N°③,Table2)inwildtypemiceshowstheweb,whichisformedaround
theentericgangliaofthemyentericplexusbyGFAP
+
‐EGCs.Processesofthesecellsarewoven
around(AandB,shortarrow)everyneuron(A–C,asterisk).D–F:DoublestainingforDsRedand
GFPinGFAP‐EGFPxPLP‐DsRed1mice(N°④,Table2;anti‐DsRednotshown).Twodifferent
typesofglialcellscanbefound:(1)GFAP
+
/PLP
+
‐glialcells,whichappearmostabundantlyin
myentericganglia(D–F,exemplarilyindicatedbyshortarrows).(2)GFAP
‐
/PLP
+
‐glialcells,which
canbeonlyfounddirectlysurroundingtheentericneurons(DandF,dottedline;silhouettesof
neuronsaremarkedbyasterisks).G–I:DoublestainingforDsRed1andGFPinGFAP‐EGFPx
PLP‐DsRed1mice(N°④,Table2;DsRed1andanti‐DsRednotshown).Longitudinalsectionofa
vagalnervefiberbundleofthetunicaadventitia;GFAPcanbedetectedinperipheralSchwann
cells(arrowheads:cellbodies;shortarrow:longishprocess).J–LandM–O:Doublestainingfor
DsRed1andGFPinGFAP‐EGFPxPLP‐DsRed1mice(N°④,Table2;anti‐DsRednotshown).
Detectedbloodvessel‐connectedEGCsshowthesamedelicatemorphologyastheonesinPLP‐
CreERT2×tdTmice(cf.Figure3MandN).Remarkably,(1)GFAP
+
/PLP
+
–(J–L)and(2)
GFAP
+
/PLP
−
‐glialcells(M–O)canbefoundastwodifferenttypesofbloodvesselconnectedglia.
Inallcasestheyshowasimilardistributionpattern,astheirfine,longprocessesappearwoven
around(arrowheads)andthecellbodiescloselylocated(shortarrows)totheoutervesselwall.
Luminaofthevesselsaremarkedbyasterisks.DsRed(1):Discosomasp.redfluorescentprotein
(1);EGFP:Enhancedgreenfluorescentprotein;GFAP:Glialfibrillaryacidicprotein;GFP:Green
fluorescentprotein;H:Hoechst;PGP9.5:Proteingeneproduct9.5;PLP:Proteolipidprotein;Z‐
step=1μm(A–I)and0.5μm(J–O);scalebars20μm(A–F),25μm(G–O).
Int.J.Mol.Sci.2021,22,323313of25
Generalarrangementofglialcells.First,wescrutinizedthedistributionpatternof
esophagealentericglialcells(EGCs).Wefoundastrikingdifferenceintheirgeneral
arrangement:WhileGFAP+‐glialcellsweremostlyrelatedtoentericgangliaandmotor
endplatecontactingefferences,PLP+‐glialcellsformedameshworkofcellspervadingthe
wholeorgan.Moreover,theseglialcellswerearrangedinparallelwiththemusclefibers
ofthetunicamuscularis,givingtheimpressionofenwrappingthemusclecells(Figure
4A–C).AswecouldnotdetectGFAPintheseglialcells,themeshworkseemedtoconsist
ofsingleGFAP–/PLP+–cells,whichwerecross‐linkedbytheirprocesses.Duetotheir
numerousfinelybranchingprotrusions,arisingfromtheirprominentround‐ovalsoma,
thesecellsshowedamorphologyreminiscentofastrocyteresemblingglialcells(Figure
4J).
Entericganglia.Wethenfocusedonthemorphologyofglialcellssurroundingenteric
neuronsintheesophagus.ThroughtdTexpressioninPLP‐CreERT2×tdTmice,PLP+‐glial
cellscouldbedetectedaroundtheentericneurons(Figure4F),evenbetterdistinguished
bytheDsRedantibodystaining(Figure4G).Inaddition,PLP+–glialcellsalsofollowed
interconnectingstrandsofentericneurons(Figure4FandG,shortarrows).ThePLP
antibodyshowedasimilardistributionpattern,thusconfirmingthefluorescentprotein
results(Figure4H).Asanextstep,wecomparedthesefindingsinGFAP‐EGFP×PLP‐
DsRed1miceandGFAPwholemountstainings.Inbothcases,entericgangliawere
surroundedbyGFAP+‐glialcellsandembeddedinawebformedbytheirfiliform
processes(Figure5A,B).Moreover,theGFAP‐EGFP×PLP‐DsRed1micerevealedthe
presenceofatleasttwodifferentcelltypeswithintheentericganglia:Mostabundantly,
GFAP+/PLP+–glialcellswerefound,astheco‐localizationofEGFP–andDsRed1indicated
(Figure5D–F).Inaddition,glialcellssurroundingtheentericneuronsappearedtobe
positiveforPLPonly(Figure5D,F).
Glialcellsofvagalnervefibers.Byscreeningwholemountpreparationsofthe
esophagus,wecouldalsofindbundlesofvagalnervefibers,adherenttothetunica
adventitia.InPLP‐CreERT2×tdTmiceovalcellbodiesofPLP+‐glialcells,representing
theperipheralSchwanncells,werepresentwithinthebundleaswellastheirgently
stainedcytoplasmaticprotrusions.ThePLPantibodystainingshowedthecharacteristic
distributionofPLPinthemyelinsheatharoundtheaxon(Figure4K)andalsoallowedto
identifyensheathingSchwanncellsbynodesofRanvier(Figure4K).Moreover,itwas
noticedthatthePLPantibodyledtostrongstainingofthemyelinsheath,whereascell
bodiesofSchwanncellsshowedonlylittlepositivityincontrasttoastrongcytosolic
expressionoftdT.WecomparedtheseresultstothedistributionofGFAPinGFAP‐EGFP
xPLP‐DsRed1mice(Figure5G–I).Wecouldseethatcellswiththesamemorphologyas
describedabovewerealsopositiveforGFAP,confirmingthatSchwanncellsinthePNS
containGFAPasanintermediatefilament.
Glialcellsaroundbloodvessels.Furthermore,weobservedglialcellsenwrapping
bloodvessels(Figure4M,N;Figure4J–O).ItwasnoticedthatbothGFAP+/PLP+–cells
(Figure5J–L)andGFAP+/PLP–‐cellscouldbefound(Figure4M–O).Remarkably,vessel‐
relatedcellsshowedadelicatemorphology:Closetothetunicaadventitiaoftheblood
vessels,theirsmall,ovalcellbodiescouldbefound,emittinglong,filiformprotrusionsof
differentcalibers.Theseprotrusionsfollowedthecourseofthevesselinthetunica
adventitia,formingglialnetworks.
Inaggregate,wewereabletodemonstratethreedifferentglialcelltypesinthe
esophagususingPLPandGFAPasamarker:GFAP+/PLP–‐,GFAP+/PLP+–,and
GFAP−/PLP+–glialcells.Furthermore,thedistributionofthesecellsintheesophagus
showedsomevariabilitywithGFAP–/PLP+–cellsbeingthemostabundantones.
3.Discussion
Thisstudyrepresentsthefirstdetailedexaminationofdifferentglialmarkersinthe
ENSofthemurineesophagus,withspecialfocusonGFAP,MBP,andPLP.Asascreening
methodforglialcellmarkerexpression,weusedRT‐PCR.Inafurtherstep,weapplied
Int.J.Mol.Sci.2021,22,323314of25
immunohistochemicalstainingprotocolsinhealthyC57BL/6Jmicetoshowthepresence
ofGFAP,MBP,andPLPattheproteinlevelintheesophagus.Inordertospecify,confirm,
andenlargeourfindings,wefinallycompletedourexplorationbytheuseofmono‐and
doubletransgenicmouselinesforPLPandGFAP.
3.1.PresenceofGlialCellSpecificMarkersintheEsophagealNeuromuscularJunction
GFAP,knownasacommonglialmarker,especiallyforastrocytes,wasdetectedin
themotorendplateregionoftheskeletalmuscle.Thedelicatewebformedbytheseglial
cellscanbereconciledwiththemorphologyofperisynapticSchwanncells[39].Whilethe
expressionofGFAPandPLPhasalreadybeendescribedforNMJintheskeletalmuscle,
thesituationinthestriatedmuscleoftheesophagusisstillnotwellunderstood[39,40].
Herewecouldconfirmthefindingsintheskeletalmusclefortheesophagusand
demonstratethatesophagealPSCshowasimilardistributionofGFAPandPLP.
AlthoughthefunctionofthesePSCisstillnotfullyunderstood,thereisevidencefor
theircrucialroleinaxonalgrowthandregenerationaswellasinthelong‐term
maintenanceofmatureNMJ[39,41,42].Moreover,duetotheirhighexpressionofion
channelsandneurotransmitterreceptorstheybothresembleCNSastrocytesandtakepart
insynapticneurotransmission[43,44].Therefore,theymayinteractinthesocalled
tripartitesynapseoftheNMJasthethirdpartner,besidesthepresynapticmotorterminal
andthepostsynapticmusclefiber[41].
3.2.DifferentDegreesofMyelinationintheMyentericPlexus
Inourstudy,wewereabletoshowthatthereare(1)myelinated(MBP+)ChAT+–
fibers,(2)unmyelinated(MBP–)ChAT+–fibers,and(3)myelinated(MBP+)ChAT––fibers
presentinthemyentericplexus.ChATisawell‐knownmarkerformotorefferentsand
henceallowstodistinguishbetweenmotorefferentsandafferents,whicharebothpresent
inthemyentericplexus.Whilenumerousunmyelinatedefferentfiberswerefound,only
fewmyelinatedefferentswerelocatedinthemyentericplexus.Inaddition,inallcases,
terminalmotoraxons,contactingthemotorendplates,provedtobeunmyelinated.These
findingsagreewithresultsofpreviousstudies[45,46],butthequestionconcerningwhere
theswitchfrommyelinatedtounmyelinatednervefibersoccursremainsstilltobesolved.
Onepossibleexplanationcouldbethatmyelinatedfiberslosetheirmyelinsheathafter
enteringthemyentericplexus[45,46].Anotherpossibilitycouldbealingeringlossof
myelinalongthenervefibers’course,endingupintheunmyelinated,terminalaxon
accompaniedbyPSCs.Furtherinvestigationstracingmyelinatednervefibersontheirway
throughtheesophagealmyentericplexusarerequiredinordertoanswerthisquestion.
However,thespeedofsignaltransmissionseemstobeirrelevantintheterminalsections
ofefferents,astheyareunmyelinatedforlongdistanceswithintheplexus.
Asthethirdtypeofnervefibersinvestigatedinthemyentericplexusofthe
esophaguslackedChATbutshowedamyelinsheath,wecametotheconclusionthatthey
canbeclassifiedasafferentnervefibers.Possibleoriginsfortheseafferentsare
intraganglioniclaminarendings(IGLEs)andintramusculararrays(IMAs)[47–49],
playingacrucialroleinthesensoryphysiologyoftheesophagus:Asfarasisknown,
primaryafferentfibersserveasreceptorsfor(1)muscletension,(2)mucosalmechanical
andchemicalstimuli,and(3)mucosaltension,andthereforearesignificantfor
swallowing.Inthisregard,severalstudiescouldconfirmthecorrelationofsensorynerve
impairmentandfunctionaldisordersoftheesophagussuchasfunctionalglobus,
noncardiacchestpain,anddysphagia[50–52].
3.3.DifferentiationofEsophagealEntericGlialCellsbytheUseofGlialMarkers
Inthepresentstudy,wepursuedthegoaltocharacterizeentericglialcellpopulations
oftheesophagus.WecouldshowthatthecommonmyelinmarkerPLPiswidely
expressedinthemurineesophagusand,moreover,isnoticeablymoreabundantthan
Int.J.Mol.Sci.2021,22,323315of25
GFAP.Asaconsequence,wecouldidentifythreedifferentglialcelltypes,dependingon
theirexpressionofmarkers:GFAP+/PLP−,GFAP+/PLP+,andGFAP−/PLP+‐cells.These
findingsharmonizewiththesituationinthelowergastrointestinaltract[30].
Referringtomorphology,HananiandReichenbachintroducedaclassification
systemofEGCsinthemyentericplexusoftheguineapigsmallintestine[53].Basedon
microscopicinvestigations,theyfoundfourtypesofEGCs[53,54]:TypeIcellsare
characterizedbyastar‐shapedsomawithshortandirregularlybranchedprocesses,
thereforealsocalled‘protoplasmic’.TypeII(‘fibrous’)gliocytesappearaselongatedEGCs
withininterganglionicfibertracts.TypeIII(‘mucosal’)glialcellsshownumerouslong‐
branchedprotrusionsandcanbefoundinsubepithelialareas.‘Intramuscular’glialcells,
representingtypeIV–gliocytes,featurealong‐shapedcellbodyandaccompanymuscle
fibersinthetunicamuscularis.OurresultsrevealthatEGCsintheesophagusare
compatiblewiththisclassificationsystem.WhileGFAP+/PLP+–glialcellssurroundingthe
myentericgangliacanbeconsideredtobetypeI–EGCs(Figure5A,B),tdT‐positivecells
directlyadjacenttotheentericneuronsshowsimilaritiestotypeI‐IIIcells(Figure4E–H).
Meshwork‐formingGFAP–/PLP+–cells,pervadingthewholeorgan,showseveral
branchedprocessesresemblingtypeIIIglialcells(Figure4J).Incontrast,the
intramuscularEGCsthatwerefoundinthetunicamuscularisandthatalwayslacked
GFAPcorrespondtotypeIVgliocytesduetotheirelongatedshapeandtheirarrangement
betweenthemusclefibers(Figure4A–D).ThesefindingscanbebroughtinlinewithRao
etal.,underliningthesimilaritiesofEGCsthroughoutthegastrointestinaltract[30].
Lastly,wefoundglialcellsadjacenttobloodvesselsintheesophagus.Ontheone
hand,wecoulddetectthatnotallthesecellsexpressedPLP,asGFAP+/PLP––gliocytes
show.Ontheotherhand,thesecellsvariedintheirmorphologyfromtheonesdescribed
aboveduetotheirelongatedsomaaswellastheirlong,gracileprocesses.Asaresult,they
partiallyborearesemblancewithtypeIIandtypeIVglialcellsandthereforemight
possiblyrepresentafifthtypeofEGCsintheesophagus.
3.4.EntericGlialCellsasanImmunologicalTarget
Ingeneral,therearetwopossiblewaysthatEGCscanbeaffectedbytheimmune
system:ItisconceivablethatadirectcellresponsetakesplaceastheactivationofCD8+‐
cellsinCrohn’sdiseaseshows[55].SinceEGCsexpressabatteryofglialandmyelin
markers,itiswellpossiblethatthesestructurescanontheotherhandserveas
immunologicaltargetsforaB‐cell‐ andantibody‐dependentimmuneresponse,
respectively.Thisisevenmorelikelyasthereareseveralstudiesthatrevealthepresence
ofautoantibodiesagainstmyelincomponentsinMS,includingMBPandPLP[56–59].
Moreover,theclinicalrelevanceofGFAPasamarkerforongoinginflammationin
patientswithMSisdisputed,underliningtheinvolvementofthisglialmarkerin
pathologicalprocesses[60,61].Tofindouthowtheseautoantibodiesaffecttheesophageal
ENSandthereforemightbeinvolvedinthepathogenesisofdysphagiainMS,further
studiesmustbeapplied.Therefore,theEAEanimalmodelofMSisaneligiblepossibility
forfurtherinvestigations,asithasalreadybeenproventhattheENSrepresentsapotential
targetforautoimmuneprocessesinMSinthelowergastrointestinaltract[5].Asthereare
differentwaystoinduceEAEinmice,animalsimmunizedwithMP4,afusionproteinof
MBPandPLP,resembletheetiopathologyofMSmorecloselyincomparisontootherEAE
models[27,62–64].Hence,wesuggesttheMP4‐EAE‐modelasanidealtoolforfuture
studiesconcerningesophagealpathologyinMS.
Interestingly,autoimmunityagainstglialandmyelincomponentsisnotrestrictedto
demyelinatingdiseaseslikeMSbutisalsopresentinotherdiseasepatternsshowing
gastrointestinalmotilitydisordersanddysphagia.Recentstudiesdemonstratethe
presenceofMBP‐andPLP‐autoantibodiesinpatientswithstroke[65–67],wheremore
than50%ofallsurvivorssufferfromdysphagia.Inaddition,GFAP‐autoantibodiescould
notonlybefoundinpatientswithtraumaticbrainorspinalcordinjury[68,69],butalso
inthosewithautoimmuneGFAP‐astrocytopathy,whereatleast20%showautonomic
Int.J.Mol.Sci.2021,22,323316of25
dysfunctionsincludingdysphagia[70].Thisfactmightopenanewperspectiveonthe
pathogenesisofdysphagiaandshowsthepossibleimplicationforavarietyof
neurodegenerativediseases[65–70].
Inourstudy,wecouldidentifypossibletargetstructuresforautoimmuneprocesses
intheesophagus.SincetheesophagealENSanditspossibleimplicationfordysphagia
remainsenigmatic,moreemphasisshouldbeputonthisneglectedareaofresearch.
4.MaterialsandMethods
4.1.Mice
Immunohistochemicalinvestigationswerebasedontheusageofn=22C57BL/6J
miceofeithersex.Furthermore,threetamoxifen‐inducibleTgN(PLP‐CreERT2)mice[71]
crossbredwithTgH(Rosa26‐CAG‐lsl‐tdTomato)mice[72])andthreedoubletransgenic
TgN(hGFAP‐EGFP)GFECmice[73]crossbredwithTgN(mPLP‐DsRed1)PRDBmice[74])of
eithersexwereapplied.ForRT‐PCRexperimentssixadditionalC57BL/6Jmiceofeither
sexwererequired.Allanimalswereagedbetween10and15weeks.Allmicewere
euthanizedwithalethaloverdoseofsodiumthiopental(500mg/kgi.p.).C57BL/6Jmice
usedinthepresentstudieswereobtainedfromTheJacksonLaboratories(CharlesRiver,
Sulzfeld,Germany)andmaintainedasinbredlinesbyfullsiblingmatingsunderspecific
pathogen‐freeconditionsattheexperimentalanimalfacility(‘Präklinisches
ExperimentellesTierzentrum’(PETZ))oftheUniversityErlangen‐Nürnbergwhile
transgenicmicewereheldattheanimalfacilityoftheCenterforIntegrativePhysiology
andMolecularMedicine(CIPMM)oftheUniversityofSaarland.PLP‐CreERT2xRosa26‐
tdTomato(PLP‐CreERT2xtdT)micewereheldinC57BL/6NandGFAP‐EGFPxPLP‐
DsRed1(GFAP‐EGFP×PLP‐DsRed1)miceinFVB/Nbackground.Humidityand
temperatureweremaintainedat45–65%and20–24°Candthefacilitykeptundera12‐h
light‐darkcycle.Allmicehadfreeaccesstoastandardautoclavedrodentdiet(Ssniff
Spezialdiäten,Soest,Germany)andautoclavedtapwater.
Tamoxifeninjection.Tamoxifensolutionwaspreparedaspreviouslydescribed[75].
Briefly,toinducereporter(tdTomato)expressioninPLP‐CreERT2mice,tamoxifen
(Carbolution,Neunkirchen,Germany)wasintraperitoneallyinjected(10μg/mLin
Mygliol®812(CaesarandLorentzGmbH,Hilden,Germany),100μL/10gbodyweight)to
miceonceperdayforthreeconsecutivedays.Analysiswasexecuted14daysafter
injection.
FortheuseofallanimalstheEuropeanandGermanCommunitiesDirectiveand
animalwelfareprotocols,endorsedbythelocalgovernment,andthe“ARRIVEguidelines
forreportinganimalresearch”[76]werefollowed.Animalexperimentsandtheremoval
oforganswereapprovedbythelocalveterinaryinspectionofficesoftheUniversityof
Erlangen‐NürnbergandtheUniversityofSaarland(filereference:TS‐99/20‐AnatomieI
(15.12.1999)and36/2016(08.11.2016)).
4.2.RT‐PCR
4.2.1.TissuePreparation
Directlyaftereuthanasia,micewerecarefullydissected.Thecerebrum,cerebellum,
brainstem,esophagus,segmentsofjejunum,andcolonascendensaswellastheanterior
tibialmuscles(bothsides)wereremoved.TissueswererinsedinsterileRingersolution
(B.Braun,Melsungen,Germany)andsnap‐frozeninliquidnitrogen.Allsamples
remainedstoredat–80°CuntilRNAextraction.
4.2.2.Processing
ForRNAextraction,amodifiedvariantofthesinglestepmethodaccordingto
ChomczynskiandSacchiusingTRIzol®reagent(ThermoFisherScientific,Carlsbad,CA,
USA)wasperformed.Tothisend,frozentissuesamplesweremechanicallypulverized
bypestlinginaliquidnitrogen‐cooledmortar.50–100mgofgroundtissuewere
Int.J.Mol.Sci.2021,22,323317of25
immediatelyplacedin2mLRNAse‐freetubesand1mLofTRIzol®reagentwasadded.
Aftera5minincubationstepatroomtemperature,0.2mLofchloroform(Sigma‐Aldrich,
Taufkirchen,Germany)wereaddedfora2–3minincubationatroomtemperature.
Subsequenttoacentrifugationstepat12000gat4°Cfor15min,theRNAcontaining
aqueousphasewascarefullytransferredinafresh2mLRNAse‐freetubeand0.5mL
isopropanol(Sigma‐Aldrich,Taufkirchen,Germany)wereaddedforanother10min‐
incubationatroomtemperature.Acentrifugationstepat12000gat4°Cfor10minwas
followedbyresuspendingtheRNAprecipitatein1mLof75%ethanol(Sigma‐Aldrich,
Taufkirchen,Germany).Afteralastcentrifugationstepat7500gat4°Cfor5minthe
remainingRNApelletwasairdriedandafterwardsdissolvedin100μLofRNAsefree
DEPC‐treatedwater(ThermoFisherScientific,Carlsbad,CA,USA)byheatblock
incubationat57°Cfor15min.RNAquantificationwasperformedbyphotometric
analysis(BioPhotometerPlus,Eppendorf,Hamburg,Germany).Reversetranscriptionof
alldifferentRNAsamples(2μgperreaction)tocDNAwasperformedbytheusageof
High‐CapacitycDNAReverseTranscriptionKit(50U/μl;ThermoFisherScientific,
Carlsbad,CA,USA)accordingtomanufacturer’sinstructions.Forgene‐specificPCRs12.5
μLofRedMastermix(2×)TaqPCR‐Mastermix(GENAXXONbioscience,Ulm,Germany)
werecombinedwith4μLofa1μMstocksolutionofeachforwardandreverseprimer,1
μLtemplateDNA,and3.5μLRNAsefreeDEPC‐treatedwater.PCRreactionwas
performedinProFlexPCRSystemthermalcycler(ThermoFisherScientific,Carlsbad,CA,
USA).AllprimersweresynthesizedatInvitrogen(ThermoFisherScientific,Carlsbad,CA,
USA).AlistofthedifferentprimersequencesandthecycleconditionsisprovidedinTable
3.
Table3.OverviewofusedprimerpairsforRT‐PCRincludingcycleconditions.
Gene
ForwardPrimer
ReversePrimer
Primerreference
CycleConditions
β‐Actin
(154bp)
(F)5′‐GGCTGTATTCCCCTCCATCG‐3′
(R)5′‐CCAGTTGGTAACAATGCCATGT‐3′
Self‐Designed
InitialDenaturation10min95°C
35
Cycles
Denaturation45s95°C
Annealing30s57°C
Extension45s72°C
Finalextension10min72°C
GFAP
(199bp)
(F)5′‐CAACGTTAAGCTAGCCCTGGACAT‐3′
(R)5′‐CTCACCATCCCGCATCTCCACAGT‐3′
Shietal.[77]
Initialdenaturation10min95°C
35
Cycles
Denaturation45s95°C
Annealing30s60°C
Extension45s72°C
Finalextension10min72°C
PLP
(218bp)
(F)5′‐AGCGGGTGTGTCATTGTTTGGGAA‐3′
(R)5′‐ACCATACATTCTGGCATCAGCGCA‐3′
Chewetal.[78]
Initialdenaturation10min95°C
35
Cycles
Denaturation45s95°C
Annealing30s58°C
Extension45s72°C
Finalextension10min72°C
MBP
(342–642bp)
(F)5′‐ATGGCATCACAGAAGAGACC‐3′
(R)5′‐CATGGGAGATCCAGAGCGGC‐3′
Yeetal.[79]
Initialdenaturation10min95°C
35
Cycles
Denaturation45s95°C
Annealing30s56°C
Extension45s72°C
Finalextension10min72°C
MAG
(355–400bp)
(F)5′‐CTCTATGGCACCCAGAGCCT‐3′
(R)5′‐TGTCCTTGGTGGGTCGTTTT‐3′
Yeetal.[79]
Initialdenaturation10min95°C
35
Cycles
Denaturation45s95°C
Annealing30s56°C
Extension45s72°C
Finalextension10min72°C
OSP(F)5′‐GATTGGCATCATCGTCACAACG‐3′ Initialdenaturation10min95°C
Int.J.Mol.Sci.2021,22,323318of25
(339bp)(R)5′‐AGCCAGCAGAATAAGGAGCACC‐3′
Hellanietal.[80]35
Cycles
Denaturation45s95°C
Annealing30s50°C
Extension45s72°C
Finalextension10min72°C
MOG
(841bp)
(F)5′‐GACCTCAGCTTGGCCTGACCC‐3′
(R)5′‐TGCTGGGCTCTCCTTCCGC‐3′
Delarasseetal.[81]
Initialdenaturation5min94°C
35
Cycles
Denaturation1min95°C
Annealing1min66°C
Extension3min72°C
Finalextension5min72°C
Agarosegelelectrophoresisforallamplifiedproductswascarriedoutona2%
agarosegelinTris/acetate/EDTA(TAE)‐buffer(pH8.0)containingGelRed®nucleicacid
gelstain(GENAXXONbioscience,Ulm,Germany).ReversetranscriptasePCRproducts
werevisualizedunderultravioletlight.Thehousekeepinggeneforβ‐actinwasusedas
loadingcontrol.
4.3.Immunohistochemistry
Forabetteroverview,theprimaryandsecondaryantibodiesusedareshowninTable4.
Table4.Characterizationofantibodiesandtoxinsusedforimmunohistochemicalstaining.
PrimaryAntibodiesHostSpeciesDilutionSource(CatalogueNumber)
CalbindinD28kGuineapig1:100SynapticSystems
Göttingen,Germany(214004)
ChATGoat1:40Millipore
Temecula,CA,USA(AB144P–1ML)
DsRedRabbit1:1000TakaraBio
MountainView,CA,USA(632496)
GFAPRabbit1:800–1:2000Dako
Glostrup,Denmark(Z0334)
GFPChicken1:1000ThermoFisher
Waltham,MA,USA(A10262)
MBPRabbit1:200–1:500Abcam
Cambridge,UK(ab40390)
MBPChicken1:500–1:3000Abcam
Cambridge,UK(ab134018)
PGP9.5Guineapig1:500FitzgeraldInd.
Acton,MA,USA(20R–PG011)
PLP(PLP1)Rat1:1000 KindgiftfromWendyB.Macklin
SynaptophysinGuineapig1:1000SynapticSystems
Göttingen,Germany(101004)
βIII‐TubulinRabbit1:500–1:4000Abcam
Cambridge,UK(ab18207)
SecondaryAntibodiesandToxinsDilutionSource(CatalogueNumber)
DonkeyAnti‐ChickenAlexa6471:1000JacksonImmunoResearch
WestGrove,PA,USA(703–605–155)
GoatAnti‐ChickenAlexa6471:1000JacksonImmunoResearch
WestGrove,PA,USA(103–605–155)
DonkeyAnti‐GoatAlexa4881:1000MolecularProbes
Eugene,OR,USA(A11055)
DonkeyAnti‐GoatAlexa6471:1000MolecularProbes
Eugene,OR,USA(A21447)
DonkeyAnti‐GuineaPigDYE4051:200
J
acksonImmunoResearch
WestGrove,PA,USA(706–475–148)
GoatAnti‐GuineaPigAlexa5551:1000MolecularProbes
Int.J.Mol.Sci.2021,22,323319of25
Eugene,OR,USA(A–21435)
DonkeyAnti‐GuineaPigAlexa6471:1000JacksonImmunoResearch
WestGrove,PA,USA(706–605–148)
DonkeyAnti‐RabbitDYE4051:200
J
acksonImmunoResearch
WestGrove,PA,USA(711–475–152)
DonkeyAnti‐RabbitAlexa4881:1000ThermoFisher
Waltham,MA,USA(A–21206)
DonkeyAnti‐RabbitAlexa6471:1000MolecularProbes
Eugene,OR,USA(A31573)
DonkeyAnti‐RatAlexa4881:1000ThermoFisher
Waltham,MA,USA(A–21208)
α‐BungarotoxinAlexa5551:1000MolecularProbes
Eugene,OR,USA(B35451)
Hoechst1:1000Sigma‐Aldrich
St.Louis,MO,USA(H6024)
4.3.1.TissuePreparationandFixation
Followingeuthanasia,micewereperfusedtranscardiallywith20mLofRinger
solution(B.Braun,Melsungen,Germany)prewash,followedby100mL4%phosphate‐
bufferedformaldehyde(pH7.4).
Forfrozensectionsthecerebellum,thecervical,thoracic,andabdominalportionsof
theesophagusandbothanteriortibialmuscleswereprepared.Forequalfull‐length
divisionoftheesophagus,thethoracicportion,whichisapproximatelytwiceaslongas
theothertwoparts,wasdividedintoanupperandalowerhalf.Tissueswerethen
postfixedin4%phosphate‐bufferedformaldehyde(pH7.4)foranother5hat4°Cand
rinsedinphosphatebuffer(pH7.4)at4°Covernight.Forcryoprotection,tissueswere
immersedin12%phosphate‐bufferedsucrosesolutionfor24hat4°C.Forlongtime
storageandfurtherprocessing,tissuesweremountedinOCTEmbeddingMatrix(Carl
Roth,Karlsruhe,Germany)andfrozeninliquidnitrogen‐cooledisopentane.
Inthosemice,whichwereintendedforwholemountpreparations,priorto
perfusion,aplastictubingwitha2mmouterdiameterwasinsertedintheesophagusin
ordertodistendtheorgan.Afterperfusion,theentireesophagustogetherwiththeplastic
tubingwasdissectedandpostfixedasdescribedabove,beforerinsinginphosphatebuffer
(pH7.4)at4°Covernight.Incontrasttotheothertissues,wholemountsoftheesophagus
werenotfrozenbutfreshlyusedforimmunohistologicalstaining.Therefore,theplastic
tubingwasremoved,theorganwasopenedlongitudinally,andthemucosa,including
submucosa,wasgentlypeeledoff.
4.3.2.FrozenSections
Forallfrozensectionstainings,12‐μm‐thickcryostatsectionswerecutusingaLeica
CM1900cryostat(Leica,Wetzlar,Germany).Sliceswerethenmountedonpoly‐L‐lysine
coatedslidesandair‐driedforatleastonehouratRT.Ahydrophobicbarrieraroundthe
specimensontheslideswasprovidedbydrawnlineswithImmEdge™Hydrophobic
BarrierPen(VectorLaboratories,Burlingame,CA,USA).Thepreincubationsolution
containedamixtureof1%bovineserumalbumin(BSA;CarlRoth,Karlsruhe,Germany),
0.5%Triton®×100(CarlRoth,Karlsruhe,Germany),Tris‐bufferedsaline(TBS;0.05M,pH
7.3),and,dependingonthehostspeciesoftheusedsecondaryantibody,5%normal
donkeyserum(JacksonImmunoResearch,WestGrove,PA,USA)and/or5%normalgoat
serum(JacksonImmunoResearch,WestGrove,PA,USA).Beforeandafterpreincubation,
whichwasperformedforonehouratroomtemperature,slideswerewashedinTBS.In
protocolswherethePLP‐antibodywasapplied,anextrapreincubationwithamixtureof
TBSand10%Triton®X100(CarlRoth,Karlsruhe,Germany)wasprependedfor15minto
thecommonpreincubation.Primaryantibodyincubationwasperformedovernightat
roomtemperature,followedbyanotherrinsingstepinTBSfor15min.Secondary
Int.J.Mol.Sci.2021,22,323320of25
antibodyincubationwassubsequentlyperformedforonehouratroomtemperature,
completedwitha15minwashingstepinTBS.Formusculartissues,i.e.,esophagus‐and
anteriortibialmuscle‐sections,a20minincubationwithfluorochrome‐taggedα‐BTwas
added,inordertolabelmotorendplates.Ifnecessary,anuclearstainingwithHoechst
wasappendedintheendfor10min.AfterafinalwashinTBSfor15min,allsectionswere
coverslippedwitha1:1mixtureofTBS‐glycerol(pH8.6).Theexactsettingofprimary
andsecondaryantibodiesforeachstainingisshowninTable1.
4.3.3.WholeMounts
Aftertheremovalofthemucosaltissue,whole‐mountpreparationswere
preincubatedatroomtemperaturefortwohoursonashakerwithapreincubation
solutionconsistingoutof1%BSA(CarlRoth,Karlsruhe,Germany),2.5%Triton®X100
(CarlRoth,Karlsruhe,Germany),TBS(0.05M,pH7.3),0.05%Thimerosal(CarlRoth,
Karlsruhe,Germany),and,dependingonthehostspeciesoftheusedsecondaryantibody,
5%normaldonkeyserum(JacksonImmunoResearch,WestGrove,PA,USA)and/or5%
normalgoatserum(JacksonImmunoResearch,WestGrove,PA,USA).Inprotocolswhere
thePLP‐antibodywasapplied,anextrapreincubationwithamixtureofTBSand10%
Triton®X100(CarlRoth,Karlsruhe,Germany)wasprependedfor15mintothecommon
preincubation.Whole‐mounttissueswerethenrinsedinTBSfor10minandsubsequently
putintothespecificprimaryantibodyincubationforthreedaysat4°Conashaker.
Afterwards,awashingstepinTBSforonedayat4°Conashakerwasperformed,before
thesecondaryantibodyincubationwasstartedforfourhoursatroomtemperatureona
shaker,followedbyanadditionalrinsingstepinTBSforonedayat4°Conashaker.In
thecaseofmotorendplate‐labellingwithfluorochrome‐taggedα‐BT,incubationtimewas
prolongedtoonehouratroomtemperature.Fornuclearstaining,anincubationwith
Hoechstwasappendedintheendfor10min.AfterafinalwashinTBSfor15min,all
sectionswerecoverslippedwitha1:1mixtureofTBS‐glycerol(pH8.6).Theexactsetting
ofprimaryandsecondaryantibodiesforeachstainingisshowninTable2.
4.3.4.ControlExperiments
Thespecificityoftheimmunohistochemicalreactionswasassessedbyreplacingthe
primaryantibodywithTBSortherespectivehostserumasnegativecontrolsorbypre‐
absorbingtheantibodyagainstCalbindinD28k,ChAT,PGP9.5andSynaptophysinwith
itsrespectiveantigen(CalbindinD28k:SynapticSystems,Göttingen,Germany;ChAT:
Millipore,Billerica,MA,USA;PGP9.5:FitzgeraldInd.,Acton,MA,USA;Synaptophysin:
SynapticSystems,Göttingen,Germany).ForspecificitycontrolofGFAP,PLP,andMBP,
weusedcryosectionsofthecerebellumaspositivecontrolsasdescribedabove.Specificity
ofDsRedandGFPantibodieswasconfirmedbycombinedcell‐typespecificexpressionof
PLP(redfluorescenceintransgenicPLP‐CreERT2×tdTmice)andGFAP(green
fluorescenceintransgenicGFAP‐EGFPxPLP‐DsRed1mice).
4.4.ImageAquisition
Allstainingswereevaluatedusingafluorescencemicroscopewithaconfocallaser
scanningsystem(NikonEclipseE1000‐M;NikonDigitalEclipseC1withsoftwareEZ‐C1
3.91;Tokyo,Japan)equippedwiththedigitalcamerasystemNikonDigitalSightDS‐
2MBWc.Thesystemprovidedaquadruplelaserconfiguration,consistingofa405nm
Diode‐Laser(Coherent:CUBE405‐100C),a488nmanda543nmSolid‐State‐Laser(both
fromCoherent,SantaClara,CA,USA)anda642nmDiode‐Laser(Melles‐Griot,Carlsbad,
CA,USA).Inordertoreduceunspecificbackgroundfluorescence,aBIO1‐Filterset
(DAPI/Cy5forC1‐Detector;AHFAnalysentechnik,Tübingen,Germany)wasadditionally
installed.
Dryobjectivelenseswith20×and40×magnificationandanumericalapertureof0.75
and0.95,respectively,wereusedincombinationwithanelectronicalzoomfactorfrom
Int.J.Mol.Sci.2021,22,323321of25
1.0to4.0.Toobtainall‐in‐focusimages,upto18opticalsectionsweretakenatintervals
of0.5–1μminthez‐axisandelectronicallysuperimposed.Imageprocessingwas
performedwithNikonFreeViewersoftware(EZ‐C13.91)andVolocityDemo6.1.1
(PerkinElmer,Waltham,MA,USA),brightnessandcontrastwereadjustedbytheusage
ofAdobePhotoshopCS6(AdobeSystems,SanJosè,CA,USA)andlayoutwasconfigured
withCorelDRAWX7(Ottawa,ON,Canada).
5.Conclusions
Thepresentstudyprovidesdetaileddataofmyelinandglialstructuresinthemurine
esophagus,whicharepossibletargetsforautoimmuneprocessesinthisorgan.Besides
PLP‐,GFAP‐andMBP‐positiveSchwanncellssurroundingvagalnervefibersandPLP‐
andGFAP‐positiveglialcellssurroundingmyentericneurons,weobservednumerous
PLP‐positiveglialcellsforminganetworkintheesophagealwall.Itistemptingto
speculate,thatsubtypesoftheseentericglialcells,whicharecloselyassociatedwith
striatedmusclefibers,areinvolvedintheentericco‐innervationofesophagealmotor
endplatesattheperipherallevel.Itwillbeworthwhile,tostudyalterationsofthesePLP‐
positiveentericglialcellsinmousemodelsofMSandotherdemyelinatingdiseasesand
todelineatetheirpossibleparticipationinthepathogenesisofdysphagia.
SupplementaryMaterials:Supplementarymaterialscanbefoundatwww.mdpi.com/1422‐
0067/22/6/3233/s1.SupplementaryFigureS1:ControlstainingsforPLP,MBPandGFAPinthe
murinecerebellum,SupplementaryFigureS2:ExpressionofGFAP,MBPandPLPintheNMJofthe
tibialisanteriormuscle,SupplementaryFigureS3:ExpressionofβIII‐tubulinandMBPinnervefiber
bundles,aroundbloodveselsandintheNMJofthetibialisanteriormuscle,SupplementaryFigure
S4:PositivecontrolstainingsofChATandcomparisonofChATandsynaptophysindistributionin
thetibialisanteriormuscle.
AuthorContributions:Conceptualization,C.K.andJ.W.;methodology,C.K.,J.W.,R.C.,S.K.,
W.L.N.,A.S.,K.S.G.,andW.B.M.;investigation,C.K.andJ.W.;resources,J.W.,A.S.,K.S.G.,W.B.M.,
andM.E.;writing—originaldraftpreparation,C.K.;writing—reviewandediting,C.K.,J.W.,R.C.,
M.E.,S.K.,W.L.N.,andA.S.;supervision,J.W.;projectadministration,J.W.;fundingacquisition,
J.W.,S.K.,andW.L.N.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:Theauthorsdisclosedreceiptofthefollowingfinancialsupportfortheresearch,
authorship,andpublicationofthisarticle:Thisresearchwasfundedby“Universitätsbund
Erlangen‐Nürnberge.V.”,grantnumberWö/2020and“KuratoriumdesSonderfondsfür
wissenschaftlichesArbeitenderFriedrich‐Alexander‐UniversitätErlangen‐Nürnberg“,grant
numberWö/2020/13.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decision
topublishorpreparationofthemanuscript.
InstitutionalReviewBoardStatement:Thestudywasconductedaccordingtotheethicalapproval
bythelocalveterinaryinspectionofficesoftheUniversityofErlangen‐NürnbergandtheUniversity
ofSaarland(filereference:TS‐99/20‐AnatomieI(15.12.1999)and36/2016(08.11.2016)).Fortheuse
ofallanimalstheEuropeanandGermanCommunitiesDirectiveandanimalwelfareprotocols,
endorsedbythelocalgovernment,andthe“ARRIVEguidelinesforreportinganimalresearch”were
followed.Themaximaleffortwasdoneinrespectingthe3Rrule.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Thedatapresentedinthisstudyareavailableonrequestfromthe
correspondingauthor.
Acknowledgments:WewouldliketoacknowledgetheexcellenttechnicalassistanceofAnita
Hecht,AndreaHilpert,StephanieLink,KarinLöschner,FrankRhode,DanielSchauenburgand
HedwigSymowski.WealsowouldliketothankVerenaSchroppandSabineTackefortechnical
supportanddiscussion.ThepresentworkwasperformedinfulfillmentoftherequirementsoftheFriedrich‐
Alexander‐UniversitätErlangen‐Nürnberg(FAU)forobtainingthedegree“Dr.med.“.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterests.
Int.J.Mol.Sci.2021,22,323322of25
Abbreviations
BSABovineserumalbumin
CALBCalbindin(D28k)
cDNAComplementaryDNA
ChATCholineAcetyltransferase
CNSCentralnervoussystem
DEPCDiethylpyrocarbonate
DsRedDiscosomasp.redfluorescentprotein
EAEExperimentalautoimmuneencephalomyelitis
EDTAEthylenediaminetetraaceticacid
EGCEntericglialcell
EGFPEnhancedgreenfluorescentprotein
ENSEntericnervoussystem
GFAPGlialfibrillaryacidicprotein
GFPGreenfluorescentprotein
MAGMyelin‐associatedglycoprotein(also:Siglec‐4)
MBPMyelinbasicprotein
MOGMyelinoligodendrocyteglycoprotein
MSMultiplesclerosis
NMJNeuromuscularjunction
OSPOligodendrocyte‐specificprotein(also:Claudin‐11)
PETZPräklinischesExperimentellesTierzentrum;animalfacilityoftheUniversityErlangen‐Nürnberg
PGP9.5Proteingeneproduct9.5
PLP/PLP1Proteolipidprotein1(also:lipohilin)
PNSPeripheralnervoussystem
PSCPerisynapticSchwanncell
RT‐PCRReversetranscriptasepolymerasechainreaction
TAETris/acetate/EDTA‐buffer
TBSTris‐bufferedsaline
tdTtdTomato
α‐BTα‐Bungarotoxin
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... The esophagus is innervated by many inputs, including vagal motor neurons, sensory neurons, and local enteric neurons [94,95]. Imbalance of inhibitory and excitatory neural activity [96] as well as sensitisation of esophageal afferent neurons by inflammatory mediators and endogenous substances (hydrogen, potassium ions, 5-HT, bradykinins, prostaglandins, etc.) [97], leads to various esophageal-related disorders. ...
... A subset of esophageal tumour cells could express both NGF and NGF receptors [104], suggesting an autocrine signalling loop. Antigenic characterization combined with the genetic tracing of peripheral glia in murine esophageal tissue revealed the presence of myelinating and non-myelinating SCs of motor processes, a network of non-myelinating perisynaptic SCs, several types of enteric glial cells, and some glial cells along the blood vessels [95]. To what extent this rich variety of cells is involved in esophageal pathology remains to be demonstrated, but p75 expression is a hallmark of activated SCs, and glia-cancer cell interaction via p75 signalling has been reported in other gastrointestinal tumours [51]. ...
Schwann Cells in Digestive System Disorders
Article
Full-text available
  • Feb 2022
Proper functioning of the digestive system is ensured by coordinated action of the central and peripheral nervous systems (PNS). Peripheral innervation of the digestive system can be viewed as intrinsic and extrinsic. The intrinsic portion is mainly composed of the neurons and glia of the enteric nervous system (ENS), while the extrinsic part is formed by sympathetic, parasympathetic, and sensory branches of the PNS. Glial cells are a crucial component of digestive tract innervation, and a great deal of research evidence highlights the important status of ENS glia in health and disease. In this review, we shift the focus a bit and discuss the functions of Schwann cells (SCs), the glial cells of the extrinsic innervation of the digestive system. For more context, we also provide information on the basic findings regarding the function of innervation in disorders of the digestive organs. We find diverse SC roles described particularly in the mouth, the pancreas, and the intestine. We note that most of the scientific evidence concerns the involvement of SCs in cancer progression and pain, but some research identifies stem cell functions and potential for regenerative medicine.
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... In the present study, TA was revealed to have no impact on MO3.13 cell proliferation. However, TA significantly increased the expression of MBP, a specific marker of mature OPCs (Kapitza et al., 2021), at mRNA and protein levels within the cells. Consistently, TA also enhanced MBP expression and suppressed myelin loss in CPZ-induced mice, indicating that TA could boost OPCs maturation and remyelination in vivo. ...
Total astragalosides promote oligodendrocyte precursor cell differentiation and enhance remyelination in cuprizone-induced mice through suppression of Wnt/β-catenin signaling pathway
Article
  • Aug 2022
  • J ETHNOPHARMACOL
Ethnopharmacological relevance Radix Astragali, a traditional Chinese medicine with various pharmacological effects. Total astragalosides (TA), the main effective ingredients in Radix Astragali, exert properties including anti-oxidative stress, anti-neuroinflammation, and neuroprotection. We previously found that TA alleviated experimental autoimmune encephalomyelitis (EAE) progression, a widely used animal model of multiple sclerosis (MS). As a chronic demyelination disease, MS generally manifests myelin loss and fails to myelin regeneration. Regulation of oligodendrocyte progenitor cells (OPCs) differentiation and remyelination is the fundamental strategy for MS treatment. However, whether TA could directly promote OPCs differentiation and remyelination is still unknown. Aims of the study This study was aimed to investigate pro-differentiation and myelin regeneration effects of TA on OPCs and CPZ-induced demyelination mice, an animal model of MS, and to explore mechanism underlying from regulation of OPCs differentiation and maturation. Materials and methods Mice were orally given CPZ (400 mg/kg) daily for 4 weeks to induce myelin loss, and then treated with TA (25 and 50 mg/kg) daily for 1 week. Cell proliferation assay, Western blot, RT-PCR, immunocytochemistry and immunohistochemistry were performed to explore the mechanisms. The role of TA in oligodendrocyte differentiation and maturation was evaluated using MO3.13, a human oligodendrocytic hybrid cell line. Results TA was shown to mitigate behavioral impairment in CPZ-induced mice. It markedly ameliorated myelin loss and enhanced remyelination in the corpus callosum of mice, evidenced by increased expression of myelin basic protein (MBP) and the number of CC1⁺ newly generated oligodendrocytes (OLs). TA also enhanced the expression of MBP at both mRNA and protein levels in MO3.13 cells. In CPZ-induced mice and MO3.13 cells, TA remarkably promoted the activation of GSK3β, repressed the phosphorylation of β-catenin, reduced the expression of transcription factor 4 and inhibitor of DNA binding 2. The agonist of β-catenin, SKL2001, partially abolished the pro-differentiation effect of TA in MO3.13 cells. Conclusions Taken together, we clarified that TA could effectively enhance the differentiation and maturation of OPCs and accelerate remyelination in CPZ-induced mice through inhibition of Wnt/β-catenin signaling pathway. This study provides new insight into the beneficial effect of TA in the treatment of MS.
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... Since this classification into four enteric glia subtypes mostly stems from studies on the murine ileum and colon, it will be important to investigate how it holds for other gut regions and whether similar enteric glia subtypes are present in other species, including human. Confirming the current classification, a recent study reported the presence of similar morphological subtypes in the murine oesophagus (Kapitza et al., 2021). Furthermore, both mucosal, intramuscular and myenteric enteric glia have been described in the human colon Graham et al., 2020). ...
Development, Diversity, and Neurogenic Capacity of Enteric Glia
Article
Full-text available
  • Jan 2022
Enteric glia are a fascinating population of cells. Initially identified in the gut wall as the “support” cells of the enteric nervous system, studies over the past 20 years have unveiled a vast array of functions carried out by enteric glia. They mediate enteric nervous system signalling and play a vital role in the local regulation of gut functions. Enteric glial cells interact with other gastrointestinal cell types such as those of the epithelium and immune system to preserve homeostasis, and are perceptive to luminal content. Their functional versatility and phenotypic heterogeneity are mirrored by an extensive level of plasticity, illustrated by their reactivity in conditions associated with enteric nervous system dysfunction and disease. As one of the hallmarks of their plasticity and extending their operative relationship with enteric neurons, enteric glia also display neurogenic potential. In this review, we focus on the development of enteric glial cells, and the mechanisms behind their heterogeneity in the adult gut. In addition, we discuss what is currently known about the role of enteric glia as neural precursors in the enteric nervous system.
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... A recent study used genetic reporters and immunohistochemistry to characterize expression of PLP1, GFAP, and myelin basic protein in the mouse esophagus in detail. It found that all four morphological types of glia were detectable in the esophagus and that they shared similarities with their counterparts in the intestines, such as presence of GFAP expression in myenteric glia and absence of GFAP in Type IV intramuscular glia (Kapitza et al., 2021). Many esophageal glia expressed both PLP1 and GFAP, but overall, PLP1 expression was much more widespread. ...
Enteric Glia in Homeostasis and Disease: From Fundamental Biology to Human Pathology
Article
Full-text available
  • Jul 2021
Glia, the non-neuronal cells of the nervous system, were long considered secondary cells only necessary for supporting the functions of their more important neuronal neighbors. Work by many groups over the past two decades has completely overturned this notion, revealing the myriad and vital functions of glia in nervous system development, plasticity, and health (Allen and Lyons, 2018; Barres, 2008). The largest population of glia outside the brain is in the enteric nervous system (ENS), a division of the autonomic nervous system that constitutes a key node of the gut-brain axis. Here we review the latest in the understanding of these enteric glia in mammals with a focus on their putative roles in human health and disease.
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Häufiger als gedacht – ein Update zu Dysphagie bei Multipler Sklerose
Article
  • Mar 2024
In Deutschland sind schätzungsweise mehr als 280 000 Menschen an Multipler Sklerose (MS) erkrankt. Neben sensiblen, motorischen, vegetativen und neuropsychologischen Funktionsstörungen ist die Dysphagie ein hochrelevantes, stark beeinträchtigendes und zugleich bislang nicht gut untersuchtes Symptom der Erkrankung. Der Artikel bietet eine Übersicht über die aktuelle Studienlage zur Dysphagie bei MS und soll die Wahrnehmung für das Symptom Schluckstörung im Kontext der MS-Erkrankung schärfen. Techniken zur weiterführenden Diagnostik und Therapie werden vorgestellt mit dem langfristigen Ziel, betroffene Menschen mit MS künftig zuverlässiger zu diagnostizieren, ihnen optimierte Hilfe zu bieten und ihre Lebensqualität zu verbessern.
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Mini-review: Enteric glial cell heterogeneity: Is it all about the niche?
Article
  • Jul 2023
  • NEUROSCI LETT
Enteric glial cells represent the enteric population of peripheral neuroglia. According to their 'glial' nature, their principal function is to support enteric neurons in both structural and functional ways. Mounting evidence however demonstrates that enteric glial cells crucially contribute to the majority of enteric nervous system functions, thus acting as pivotal players in the maintenance of gut homeostasis. Various types of enteric glia are present within the gut wall, creating an intricate interaction network with other gastrointestinal cell types. Their distribution throughout the different layers of the gut wall translates in characteristic phenotypes that are tailored to the local tissue requirements of the digestive tract. This heterogeneity is assumed to be mirrored by functional specialization, but the extensive plasticity and versatility of enteric glial cells complicates a one on one phenotype/function definition. Moreover, the relative contribution of niche-specific signals versus lineage determinants for driving enteric glial heterogeneity is still uncertain. In this review we focus on the current understanding of phenotypic and functional enteric glial cell heterogeneity, from a microenvironmental and developmental perspective.
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Lymphopenia and local antigen presentation combine to induce colitis in a transgenic mouse model of uveitis
Preprint
Full-text available
  • Jun 2022
Undifferentiated uveitis (intraocular inflammation, IOI) is an idiopathic sight-threatening, presumed autoimmune disease, accountable for ~ 10% of all blindness in the developed world. We have investigated the association of uveitis with inflammatory bowel disease (IBD) using a mouse model of spontaneous experimental autoimmune uveoretinitis (EAU). Mice expressing the transgene (Tg) hen egg lysozyme (HEL) in the retina were crossed with 3A9 mice expressing a transgenic HEL-specific TCR. Double transgenic (dTg TCR/HEL) mice with EAU also spontaneously develop clinical signs of colitis at ~ P30 (post-partum day) with diarrhoea, bowel shortening, oedema and lamina propria (LP) inflammatory cell infiltration. Single (s)Tg TCR (3A9) mice also show histological LP cell infiltration but no clinical signs. dTg TCR/HEL mice are profoundly lymphopenic at weaning. In addition, dTg TCR/HEL mice contain myeloid cells which express MHC Class II-HEL peptide (MHCII-HEL) complexes, not only in the inflamed retina but also in the colon. In this model the lymphopenia and reduction in the absolute Treg numbers in dTg TCR/HEL mice is sufficient to initiate eye disease. We suggest that cell-associated antigen released from the inflamed eye, activates colonic HEL-specific T cells which, in a microbial micro-environment, not only cause colitis but feed-back to amplify IOI.
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Colitis in a transgenic mouse model of autoimmune uveitis may be induced by neoantigen presentation in the bowel
Article
Full-text available
  • Jan 2023
Undifferentiated uveitis (intraocular inflammation, IOI) is an idiopathic sight-threatening, presumed autoimmune disease, accountable for ~ 10% of all blindness in the developed world. We have investigated the association of uveitis with inflammatory bowel disease (IBD) using a mouse model of spontaneous experimental autoimmune uveoretinitis (EAU). Mice expressing the transgene (Tg) hen egg lysozyme (HEL) in the retina crossed with 3A9 mice expressing a transgenic HEL-specific TCR spontaneously develop uveoretinitis at post-partum day (P)20/21. Double transgenic (dTg TCR/HEL) mice also spontaneously develop clinical signs of colitis at ~ P30 with diarrhoea, bowel shortening, oedema and lamina propria (LP) inflammatory cell infiltration. Single (s)Tg TCR (3A9) mice also show increased histological LP cell infiltration but no bowel shortening and diarrhoea. dTg TCR/HEL mice are profoundly lymphopenic at weaning. In addition, dTg TCR/HEL mice contain myeloid cells which express MHC Class II-HEL peptide complexes (MHCII-HEL), not only in the inflamed retina but also in the colon and have the potential for antigen presentation. In this model the lymphopenia and reduction in the absolute Treg numbers in dTg TCR/HEL mice is sufficient to initiate eye disease. We suggest that cell-associated antigen released from the inflamed eye can activate colonic HEL-specific T cells which, in a microbial micro-environment, not only cause colitis but feedback to amplify IOI.
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Dysphagia in Neuroinflammatory Diseases of the Central Nervous System
Chapter
Full-text available
  • Jan 2022
Neuroinflammatory disorders of the central nervous system (CNS) consist of a relatively heterogeneous group of diseases that share the autoimmune activity against different parts of the system. Swallowing problems could happen in many of these cases. Its effect on the patients’ quality of life is undeniable. It could be an important cause of morbidity and mortality. Detailed medical history and physical exam are important. Several questionnaires could help monitor dysphagia. Radiographic and endoscopic evaluations may be necessary to detect overlooked swallowing problems. The main treatment appears to be treating the underlying disease, besides general supplementary options like rehabilitation and speech therapy.
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Demyelinating Diseases of the CNS (Brain and Spine)
Chapter
Full-text available
  • Feb 2020
Multiple sclerosis (MS) is the most important idiopathic inflammatory disorders that affects both the brain and spine. Dissemination in space and time on MRI is not limited to MS and can occur in neuromyelitis optica (NMO) and a series of other inflammatory disorders. Spinal cord imaging is an important element of MS (differential) diagnosis and especially relevant in case of possible age-related vasculo-ischemic brain white matter lesions; a negative scan will help to rule out MS. Increasingly, MRI is used to monitor treatment and their complications such as progressive multifocal leukoencephalopathy (PML).
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Clinical and laboratory features of anti-MAG neuropathy without monoclonal gammopathy
Article
Full-text available
  • Apr 2019
Antibodies against myelin-associated glycoprotein (MAG) almost invariably appear in the context of an IgM monoclonal gammopathy associated neuropathy. Very few cases of anti-MAG neuropathy lacking IgM-monoclonal gammopathy have been reported. We investigated the presence of anti-MAG antibodies in 69 patients fulfilling diagnostic criteria for CIDP. Anti-MAG antibodies were tested by ELISA and confirmed by immunohistochemistry. We identified four (5.8%) anti-MAG positive patients without detectable IgM-monoclonal gammopathy. In two of them, IgM-monoclonal gammopathy was detected at 3 and 4-year follow-up coinciding with an increase in anti-MAG antibodies titers. In conclusion, anti-MAG antibody testing should be considered in chronic demyelinating neuropathies, even if IgM-monoclonal gammopathy is not detectable.
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Enteric Glia: S100, GFAP, and Beyond
Article
Full-text available
Since several years, the enteric nervous system (ENS) is getting more and more in the focus of gastrointestinal research. While the main interest was credited for years to the enteric neurons and their functional properties, less attention has been paid on the enteric glial cells (EGCs). Although the similarity of EGCs to central nervous system (CNS) astrocytes has been demonstrated a long time ago, EGCs were investigated in more detail only recently. Similar to the CNS, there is not “the” EGC, but also a broad range of diversity. Based on morphology and protein expression, such as glial fibrillary acidic protein (GFAP), S100, or Proteolipid‐protein‐1 (PLP1), several distinct glial types can be differentiated. Their heterogeneity in morphology, localization, and transcription as well as interaction with surrounding cells indicate versatile functional properties of these cells for gut function in health and disease. Although NG2 is found in a subset of CNS glial cells, it did not colocalize with the glial marker S100 or GFAP in the ENS. Instead, it in part colocalize with PDGFRα, as it does in the CNS, which do stain fibroblast‐like cells in the gastrointestinal tract. Moreover, there seem to be species dependent differences. While GFAP is always found in the rodent ENS, this is completely different for the human gut. Only the compromised human ENS shows a significant amount of GFAP‐positive glial cells. So, in general we can conclude that the EGC population is species specific and as complex as CNS glia. Anat Rec, 302:1333–1344, 2019. © 2019 Wiley Periodicals, Inc.
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Autoimmune glial fibrillary acidic protein astrocytopathy
Article
Full-text available
  • Feb 2019
Purpose of review: To describe a recently characterized autoimmune, inflammatory central nervous system (CNS) disorder known as autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy. Recent findings: Affected patients present with symptoms of one or more of meningitis (headache and neck ache), encephalitis (delirium, tremor, seizures, or psychiatric symptoms), and myelitis (sensory symptoms and weakness). Optic disc papillitis (blurred vision) is common. CNS inflammation is evident in characteristic T1 postgadolinium enhancement of GFAP-enriched CNS regions, and lymphocytic cerebrospinal fluid (CSF) white cell count elevation. CSF is more reliable than serum for GFAP-immunoglobulin G (IgG) testing. Ovarian teratoma commonly coexists, particularly among patients with accompanying N-methyl-D-aspartate receptor or aquaporin-4 autoimmunity. Parainfectious autoimmunity is suspected in some other patients, though the culprit organism is rarely verified. Pathophysiologic relevance of T cells is underscored by neuropathology and cases of dysregulated T-cell function (HIV or checkpoint inhibitor cancer therapy). Corticosteroid-responsiveness is a hallmark of the disease. Relapses occur in approximately 20% of patients, necessitating transition to a steroid-sparing drug. Reported outcomes vary, though in the authors' experience, early and sustained intervention usually portends recovery. Summary: Autoimmune GFAP astrocytopathy is a treatable autoimmune CNS disease diagnosable by GFAP-IgG testing in CSF. This disease presents opportunities to explore novel mechanisms of CNS autoimmunity and inflammation.
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Serum GFAP as a biomarker for disease severity in multiple sclerosis
Article
Full-text available
  • Oct 2018
While neurofilament light chain (NfL) measurement in serum is a well-established marker of neuroaxonal damage in multiple sclerosis (MS), data on astroglial markers in serum are missing. In our study, glial fibrillary acid protein (GFAP) and NfL were measured in cerebrospinal fluid (CSF) and serum of MS patients and patients with other non-inflammatory neurological diseases (OND) using the Simoa technology. Clinical data like age, gender, expanded disability status scale (EDSS) and MRI findings were correlated to neurochemical markers. We included 80 MS patients: 42 relapsing-remitting MS (RRMS), 38 progressive MS (PMS), as well as 20 OND. Serum GFAP levels were higher in PMS compared to RRMS and OND (p < 0.001, p = 0.02 respectively). Serum GFAP levels correlated with disease severity in the whole MS group and PMS (Spearman-rho = 0.5, p < 0.001 in both groups). Serum GFAP correlated with serum NfL in PMS patients (Spearman-rho = 0.4, p = 0.01). Levels of serum GFAP were higher with increasing MRI-lesion count (p = 0.01). in summary, we report elevated levels of GFAP in the serum of MS patients. Since serum levels of GFAP correlate with the clinical severity scores and MRI lesion count, especially in PMS patients, it might be a suitable disease progression marker.
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Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β
Article
Full-text available
  • May 2018
Multiple sclerosis (MS) is a neurodegenerative autoimmune disease, where chronic inflammation plays an essential role in its pathology. A feature of MS is the production of autoantibodies stimulated by an altered-peptide-ligand response and epitope spreading, resulting in loss of tolerance for self-proteins. The involvement of autoantibodies in MS pathogenesis has been suggested to initiate and drive progression of inflammation; however, the etiology of MS remains unknown. The effect of etomoxir and interferon-β (IFN-β) was examined in an experimental-autoimmune-encephalomyelitis (EAE) model of MS. Moreover, the impact of etomoxir and IFN-β on recognition of brain proteins in serum from EAE rats was examined with the purpose of identifying the autoantibody reactivities involved in MS. Animals treated with etomoxir on day 1 exhibited a statistically significantly lower disease score than animals treated with IFN-β (on day 1 or 5) or placebo. Etomoxir treatment on day 5 resulted in a significantly lower disease score than IFN-β treatment on day 1. After disease induction antibodies was induced to a broad pallet of antigens in the brain. Surprisingly, by blocking CPT1 and therewith lipid metabolism several alterations in the antibody response was observed suggesting that autoantibodies play a role in the EAE animal model.
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Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia
Article
Full-text available
  • Apr 2018
Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-CreERT2/loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.
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Dysphagia in Multiple Sclerosis: Evaluation and Validation of the DYMUS Questionnaire
Article
Full-text available
  • Jun 2018
  • Dysphagia
Objective The 10-item Dysphagia in Multiple Sclerosis (DYMUS) questionnaire is a self-administered tool used to identify swallowing problems in adults with MS. The questionnaire was not validated against other existing questionnaires to assess its convergent validity. Moreover, its test–retest reliability was not measured previously. Therefore, the purpose of this study was to assess the factor analysis, internal consistency and test–retest reliability of the DYMUS, as well as its convergent validity against an established and validated questionnaire, the EAT-10. Method English-speaking adults with MS in New South Wales, Australia who were seen for routine medical check-ups were invited to complete two questionnaires across two phases. One hundred participants completed phase 1, while 55 completed phase 2. Statistical analyses were performed to investigate the psychometric properties of the DYMUS questionnaire. ResultsInternal consistency (Cronbach’s Alpha) reduced the DYMUS questionnaire from ten to five items. The shortened version of the DYMUS showed high internal consistency (alpha = 0.904). It also showed satisfactory reproducibility, and adequate correlation with the 10-item Eating Assessment Tool (EAT-10). Conclusion Evaluation of the DYMUS resulted in a shortened version of the questionnaire with five questions related to dysphagia. This shortened version is considered an easy and useful tool in identifying patients with MS-related dysphagia.
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Regional variations in the number distribution of intrinsic myenteric neurons and coinnervated motor endplates on the striated muscles in the rat esophagus
Article
  • Jul 2019
  • AUTON NEUROSCI-BASIC
The roles of intrinsic neurons and the significance of the coinnervated striated muscles in the esophagus are unclear. We examined the number distribution of intrinsic neurons and coinnervated motor endplates on the striated muscles in the rat esophagus using immunohistochemistry to investigate whether these neurons and coinnervated striated muscles may be relevant to the local control of esophageal motility. The number of PGP9.5-positive neurons was higher in the cervical esophagus (segment 1) and gradually decreased toward the aboral, with a moderate increase in the abdominal (segment 5). This pattern was similar to that of NOS-positive neurons, while the number of ChAT-positive neurons decreased toward the aboral, but it was not significantly different among segments 3 to 5. The number of ChAT-positive motor endplates increased toward the aboral, with the highest number in segment 5. The proportion of coinnervated motor endplates was approximately 80% in segments 1 to 4, but approximately 66% in segment 5. NPY-IR was localized in some nerve terminals among the smooth muscles of the muscularis mucosa and some NOS- or ChAT-positive esophageal intrinsic neurons. ENK-8-IR was found in some NOS- or ChAT-positive intrinsic neurons, and nerve terminals surrounding intrinsic neurons in the esophagus, but not in motor neurons at the NA or DMV. This study suggests that regional variations in the number of intrinsic neurons and coinnervated striated muscles in the rat esophagus may be involved in local regulations of esophageal motility, and that the rat esophageal intrinsic neurons may contain, at least, motor neurons and interneurons.
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Altered gastrointestinal motility involving autoantibodies in the experimental autoimmune encephalomyelitis model of multiple sclerosis
Article
  • Feb 2018
Background Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that, in addition to motor, sensory, and cognitive symptoms, also causes constipation, which is poorly understood. Here, we characterize gastrointestinal (GI) dysmotility in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and evaluate whether autoantibodies target the enteric nervous system (ENS) and cause dysmotility. Methods EAE was induced in male SJL and B6 mice. GI motility was assessed in vivo and ex vivo in wild type (WT) and B cell‐deficient mice. MS and EAE serum was used to survey potential targets in the ENS and changes in the ENS structure were characterized using immunohistochemistry. Key Results EAE mice developed accelerated gastric emptying and delayed whole GI transit with reduced colonic motility. Fecal water content was reduced, and colonic migrating myoelectrical complexes (CMMC) and slow waves were less frequent. Colons from EAE mice exhibited decreased GFAP levels in glia. Sera from MS patients and from EAE mice targeted ENS neurons and glia. B‐cell deficiency in EAE protected against colonic dysmotility. Conclusions & Inferences Consistent with symptoms experienced in MS, we demonstrate that EAE mice widely exhibit features of GI dysmotility that persisted in the absence of extrinsic innervation, suggesting direct involvement of ENS neurocircuitry. The absence of GI dysmotility in B cell‐deficient mice with EAE together with EAE and MS serum immunoreactivity against ENS targets suggests that MS could be classified among other diseases known to induce autoimmune GI dysmotility.
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