ANeurodegenerative DiseaseoftheRetina andBeyond-PartA
Editedby
GiacintoBagetta
PreclinicalandTranslationalPharmacology, DepartmentofPharmacy, HealthandNutritionalSciences, UniversityofCalabria,Rende,Italy
CarloNucci OphthalmologyUnit, DepartmentofExperimentalMedicine, UniversityofRomeTorVergata,Rome,Italy
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Contributors
AnnagraziaAdornetto
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
MarcelinoAviles-Trigueros
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
GiacintoBagetta
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
AnnaMariaBassi
DepartmentofExperimentalMedicine(DIMES),UniversityofGenoa,Genoa; Inter-UniversityCenterforthePromotionofthe3RsPrinciplesinTeaching& Research(Centro3R),Pisa,Italy
JavierBenı´tez-del-Castillo
ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022,Madrid;Departmentof OphthalmologyattheHospitalofJerez,JerezdelaFrontera,Ca ´ diz,Spain
JoseManuelBernal-Garro
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
DongFengChen
SchepensEyeResearchInstituteofMassachusettsEyeandEar,Departmentof Ophthalmology,HarvardMedicalSchool,Boston,MA,UnitedStates
MariaTizianaCorasaniti
SchoolofHospitalPharmacy,University"MagnaGraecia"ofCatanzaroand DepartmentofHealthSciences,University“MagnaGraecia”ofCatanzaro, Catanzaro,Italy
RosadeHoz
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo;Facultadde O ´ pticayOptometrı´a,DepartamentodeInmunologı´a,Oftalmologı´ayORL, UniversidadComplutensedeMadrid,Madrid,Spain
JuanAntonioMirallesdeImperial-Ollero DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
PedrodelaVillaPolo
DepartmentofSystemsBiology,UniversityofAlcala ´ ;InstitutoRamo ´ nyCajalde Investigacio ´ nSanitaria,Madrid,Spain
JoseA.Ferna ´ ndez-Albarral
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo,Universidad ComplutensedeMadrid,Madrid,Spain
AlejandroGallego-Ortega
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
StefanoGandolfi
OphthalmologyUnit,DepartmentofBiological,Biotechnologicaland TranslationalSciences,UniversityofParma,Parma,Italy
JoseJ.Garcı´a-Medina
OphthalmicResearchUnit“SantiagoGrisolı´a”/FISABIOandCellularand MolecularOphthalmo-biologyGroupoftheUniversityofValencia,Valencia; ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022,Madrid;Departmentof OphthalmologyattheUniversityHospital“MoralesMeseguer”andDepartmentof OphthalmologyattheFacultyofMedicine,UniversityofMurcia,Murcia,Spain
RafaelGimenez-Go ´ mez
ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022,Madrid;Departmentof OphthalmologyattheUniversityHospital“ReinaSofia”,Co ´ rdoba,Spain
EugenioLuigiIorio
InternationalObservatoryofOxidativeStress,Salerno,Italy
AlbertoIzzotti
DepartmentofExperimentalMedicine(DIMES),UniversityofGenoa; MutagenesisUnit,IRCCSOspedalePoliclinicoSanMartino,Genoa,Italy
KenjiKashiwagi
DepartmentofOphthalmology,FacultyofMedicine,UniversityofYamanashi, Kofu,Japan
HanspeterEsrielKiller
DepartmentofOphthalmology,KantonsspitalAarau,Aarau;Centerfor BiomedicineUniversityofBasel,Basel,Switzerland
MariaLuisaLagana `
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
EsterLicastro
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
InesLo ´ pez-Cuenca
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo,Universidad ComplutensedeMadrid,Madrid,Spain
FumihikoMabuchi
DepartmentofOphthalmology,FacultyofMedicine,UniversityofYamanashi, Kofu,Japan
LuigiAntonioMorrone
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
FranciscoJ.Mun ˜ oz-Negrete
ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022;OphthalmologyDepartment attheUniversityHospital“Ramo ´ nyCajal”(IRYCIS)andSurgeryDepartmentat theFacultyofMedicine,UniversityAlcaladeHenares,Madrid,Spain
Marı´aNorte-Mun ˜ oz
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
CarloNucci
OphthalmologyUnit,DepartmentofExperimentalMedicine,UniversityofRome TorVergata,Rome,Italy
MariaD.Pinazo-Dura ´ n
OphthalmicResearchUnit“SantiagoGrisolı´a”/FISABIOandCellularand MolecularOphthalmo-biologyGroupoftheUniversityofValencia,Valencia; ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022,Madrid,Spain
AnaI.Ramı ´ rez
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo;Facultadde O ´ pticayOptometrı´a,DepartamentodeInmunologı´a,Oftalmologı´ayORL, UniversidadComplutensedeMadrid,Madrid,Spain
JoseM.Ramı ´ rez
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo,Universidad ComplutensedeMadrid,Madrid;FacultaddeMedicina,Departamentode Inmunologı´a,Oftalmologı´ayORL,UniversidadComplutensedeMadrid,Spain
PilarRojas
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo,Universidad ComplutensedeMadrid;HospitalGeneralUniversitarioGregorioMaran ˜ o ´ n, InstitutoOfta ´ lmicodeMadrid,Madrid,Spain
RossellaRusso
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
SergioC.Sacca `
PoliclinicoSanMartinoUniversityHospital,DepartmentofNeuroscienceand senseorgans,OphthalmologyUnit,Genoa,Italy
YoichiSakurada
DepartmentofOphthalmology,FacultyofMedicine,UniversityofYamanashi, Kofu,Japan
JuanJ.Salazar
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo;Facultadde
O ´ pticayOptometrı´a,DepartamentodeInmunologı´a,Oftalmologı´ayORL, UniversidadComplutensedeMadrid,Madrid,Spain
ElenaSalobrar-Garcı ´ a
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo;Facultadde
O ´ pticayOptometrı´a,DepartamentodeInmunologı´a,Oftalmologı´ayORL, UniversidadComplutensedeMadrid,Madrid,Spain
SilviaM.Sanz-Gonza ´ lez
OphthalmicResearchUnit“SantiagoGrisolı´a”/FISABIOandCellularand MolecularOphthalmo-biologyGroupoftheUniversityofValencia,Valencia; ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022,Madrid,Spain
AndreaSatriano
PreclinicalandTranslationalPharmacology,DepartmentofPharmacy,Health andNutritionalSciences,UniversityofCalabria,Rende,Italy
JingTang
DepartmentofOphthalmology,WestChinaHospital,SichuanUniversity, Sichuan,China;SchepensEyeResearchInstituteofMassachusettsEyeandEar, DepartmentofOphthalmology,HarvardMedicalSchool,Boston,MA, UnitedStates
YizhenTang
DepartmentofOphthalmologyandVisionScience,Eye&ENTHospital,Shanghai MedicalCollege,FudanUniversity,Shanghai,China;SchepensEyeResearch InstituteofMassachusettsEyeandEar,DepartmentofOphthalmology,Harvard MedicalSchool,Boston,MA,UnitedStates
G€ ulg€ unTezel
DepartmentofOphthalmology,VagelosCollegeofPhysiciansandSurgeons, ColumbiaUniversity,EdwardS.HarknessEyeInstitute,NewYork,NY, UnitedStates
SaraTirendi
DepartmentofExperimentalMedicine(DIMES),UniversityofGenoa,Genoa; Inter-UniversityCenterforthePromotionofthe3RsPrinciplesinTeaching& Research(Centro3R),Pisa,Italy
PaoloTonin
RegionalCenterforSeriousBrainInjuries,S.AnnaInstitute,Crotone,Italy
AlbertoTrivin ˜ o
InstitutodeInvestigacionesOftalmolo ´ gicasRamo ´ nCastroviejo,Universidad ComplutensedeMadrid,Madrid;FacultaddeMedicina,Departamentode Inmunologı´a,Oftalmologı´ayORL,UniversidadComplutensedeMadrid,Spain
MarValero-Vello ´
OphthalmicResearchUnit“SantiagoGrisolı´a”/FISABIOandCellularand MolecularOphthalmo-biologyGroupoftheUniversityofValencia,Valencia,Spain
FranciscoJavierValiente-Soriano
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
StefaniaVernazza
IRCCS,FondazioneG.B.Bietti,Rome,Italy
ManuelVidal-Sanz
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
Marı´aPazVillegas-Perez
DepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianode Investigacio ´ nBiosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain
IrvinYi
SchepensEyeResearchInstituteofMassachusettsEyeandEar,Departmentof Ophthalmology,HarvardMedicalSchool,Boston,MA,UnitedStates
VicenteZano ´ n-Moreno
OphthalmicResearchUnit“SantiagoGrisolı´a”/FISABIOandCellularand MolecularOphthalmo-biologyGroupoftheUniversityofValencia,Valencia; ResearchersoftheSpanishNetofOphthalmicResearch“OFTARED”ofthe InstituteofHealthCarlosIII,NetRD16/0008/0022,Madrid;International UniversityofValencia,Valencia,Spain
CHAPTER1Functionalandmorphologicalalterationsina glaucomamodelofacuteocularhypertension ............. 1 AlejandroGallego-Ortega,Marı´aNorte-Mun ˜ oz, JuanAntonioMirallesdeImperial-Ollero, JoseManuelBernal-Garro, FranciscoJavierValiente-Soriano,PedrodelaVillaPolo, MarcelinoAviles-Trigueros,Marı´aPazVillegas-Perez,and ManuelVidal-Sanz
1. Introduction.......................................................................................2
2. Methods............................................................................................4
2.1Animalhandling......................................................................4
2.2Experimentaldesign................................................................4
2.3Acuteocularhypertensioninduction.......................................5
2.4Spectraldomainopticalcoherencetomography (SD-OCT)................................................................................5
2.5Electroretinography(ERG)......................................................5
2.6Tissueprocessing.....................................................................7
2.7Immunohistofluorescence........................................................7
2.8Imageacquisition.....................................................................7
2.9Quantificationandspatialdistribution....................................8
2.10Statisticalanalysis....................................................................8
3. Results...............................................................................................8
3.1Earlythinningoftheinnerretinaandadelayedthinning oftheexternalretina..................................................................8
3.2Dramaticandpermanentalterationsofallretinalwaves recorded.....................................................................................9
3.3DeathofCBCsbutnot RBCs...................................................9
3.4LossofRGCsandconesbutnotHZs.....................................13
4. Discussion.......................................................................................19
4.1AOHTreducesthethicknessoftheinnerretinaandover timetheouterretina................................................................19
4.2AOHTresultsinearlypermanentretinaldisfunction.............20
4.3AOHTcausesthelossofganglioncellsandconesbutnot HZs...........................................................................................21
4.4AOHTresultsinthedeathofconebipolarcellsbutnot rodbipolarcells.......................................................................21
CHAPTER2Geneticsofprimaryopen-angleglaucomaandits
2. Genome-wideassociationstudiesonPOAGendophenotypes......32
3. Discareaandverticalcup-to-discratio(VCDR)...... ....................32 4. Intraocularpressure(IOP)..............................................................38
CHAPTER3Abroadperspectiveonthemolecularregulationof retinalganglioncelldegenerationinglaucoma .... .....
G€ ulg€ unTezel
1. ComplexityofRGCdegenerationinglaucoma.............................49
2. InjuryofRGCaxonsattheopticnerve head................................53
3. MolecularlydistinctcompartmentalizedprocessesforRGC degeneration....................................................................................55
4. Mitochondrialdysfunction,akeypathogeniceventinRGC degenerationatdifferentcompartments.........................................57
5. Glia-drivenneuroinflammation,awidespreadoutcome promotingRGCdegeneration.........................................................58
6. MolecularsignalingfordegenerationofRGCaxons....................60
7. MolecularsignalingforRGCsomadeath.....................................62
8. LossofRGCsynapsesatdendritesand axonterminals................67
9. Conclusions.....................................................................................69
CHAPTER4Theroleofcommensalmicroflora-inducedTcell responsesinglaucomaneurodegeneration ................ 79 JingTang,YizhenTang,IrvinYi,andDongFengChen
1. Introduction.....................................................................................80
2. Microbiotainneuroinflammation.... ..............................................80
2.1Commensalmicrobiotaandimmuneregulation.....................80
2.2MicrobiotaandtheautoimmunediseasesintheCNS.... ........81
2.3Microbiotaandtheblood-retinabarrier ..................................82
2.4Microbiotaandmicrogliadevelopment,maturationand function....................................................................................82
2.5Microgliaandneurodegenerationinglaucoma.......................84
3. Heatshockproteinsinglaucomatous neurodegeneration..............85
3.1HSPsandtheirupregulationinglaucomatous neurodegeneration....................................................................85
3.2TheroleofHSPsinautoimmuneconditionsand neurodegenerativediseases,includingglaucoma....................87
4. InteractionsbetweenTcellsandmicroglia...................................88
4.1CNSTcellinfiltrationandactivatedmicroglia......................88
4.2CNS/retina:Animmuneprivilegedsite..................................90
5. Conclusion......................................................................................90
Acknowledgment.................................................................................90 References............................................................................................91
CHAPTER5Theroleofneuroinflammationinthepathogenesis ofglaucomaneurodegeneration ................ .................. 99
MariaD.Pinazo-Dura ´ n,FranciscoJ.Mun ˜ oz-Negrete, SilviaM.Sanz-Gonza ´ lez,JavierBenı´tez-del-Castillo, RafaelGimenez-Go ´ mez,MarValero-Vello ´ , VicenteZano ´ n-Moreno,andJoseJ.Garcı´a-Medina
1. Introduction...................................................................................100
2. Thelinkbetweenneuroinflammationandoxidativestressin glaucomaneurodegeneration........................................................100
2.1Neuroinflammation................................................................100
2.2Oxidativestressinglaucomaneurodegeneration..................103
3. Geneticsandomicsinglaucomaneuroinflammat ion..................104
3.1Polymorphismsofcytokinegenesinglaucoma neuroinflammation.................................................................104
3.2Omicsinglaucomaneuroinflammation ................................106
4. Updateonuveiticglaucoma.........................................................107
4.1Uveiticglaucomapathophysiology.......................................107
4.2Uveiticglaucomatreatment...................................................109
5. Imagingmethodsforocularinflammationandglaucoma...........111
5.1Anteriorsegmentopticalcoherencetomography...... ...........112
5.2Innovationinimagingtechniques .........................................113
6. Cluesinglaucomaneuroprotection..............................................115
6.1Potentialglaucomaneuroprotectants.....................................115
7. Closingremarks............................................................................116
Acknowledgments..............................................................................117 References..........................................................................................117
CHAPTER6Microglialchangesintheearlyagingstageina healthyretinaandanexperimentalglaucoma model .......................................................................... 125 AnaI.Ramı´rez,JoseA.Ferna ´ ndez-Albarral,RosadeHoz, InesLo ´ pez-Cuenca,ElenaSalobrar-Garcı´a,PilarRojas, FranciscoJavierValiente-Soriano, MarcelinoAviles-Trigueros,Marı´aPazVillegas-Perez, ManuelVidal-Sanz,AlbertoTrivin ˜ o,JuanJ.Salazar,and JoseM.Ramı ´ rez
1. Introduction...................................................................................126
2. Materialandmethods...................................................................127
2.1Ethicsstatement.....................................................................127
2.2Animalsandanesthetics....
2.3Experimentalgroups..............................................................128
2.4LasertreatmentandIOPmeasurement .................................128
2.5Immunohistochemistry..........................................................129
2.6Quantitativeretinaanalysis...................................................130
2.7Statisticalanalysis..................................................................132
3. Results...........................................................................................132
3.1Laser-inducedocularhypertension........................................132
3.2GeneralmorphologicalcharacteristicsofIba-1+retinal cells........................................................................................132
3.3MorphometriccharacteristicsoftheIba-1+cells.................133
3.4MHCIIexpression.... ........................137
3.5CD68expression....................................................................137
3.6P2RY12expression...............................................................137
4.
CHAPTER7Molecularchangesinglaucomatoustrabecular meshwork.Correlationswithretinalganglioncell deathandnovelstrategiesforneuroprotection ........ 151 SergioC.Sacca ` ,StefaniaVernazza,EugenioLuigiIorio, SaraTirendi,AnnaMariaBassi,StefanoGandolfi,and AlbertoIzzotti
1. Introduction...................................................................................152
2. Oxidativestress.............................................................................154
3. TheNrf2andNF-κBsystems......................................................159
4. Aging............................................................................................161
4.1AgingandTM.......................................................................164
5. Theaqueoushumorproteome:Itsimportanceinglaucoma.......165
6. Neuroprotectivestrategies............................................................169
6.1Polyphenols............................................................................170
6.2Omega-3.................................................................................171
7. Conclusions...................................................................................173
Acknowledgments..............................................................................174 References..........................................................................................174
CHAPTER8Effectsofcaloricrestrictiononretinalagingand neurodegeneration ...................................................... 189
AnnagraziaAdornetto,LuigiAntonioMorrone, AndreaSatriano,MariaLuisaLagana ` ,EsterLicastro, CarloNucci,MariaTizianaCorasaniti,PaoloTonin, GiacintoBagetta,andRossellaRusso
1. Introduction...................................................................................189
2. Glaucoma:Anocularneurodegenerativedisease........................190
3. Caloricrestrictionandfastinginbrief .........................................192
4. Theeffectofcaloricrestrictioninretinalagingand neurodegeneration.........................................................................192
5. Autophagy:Mechanismandfunction..........................................195
6. Autophagyandglaucoma.............................................................196
7. Theeffectofcaloricrestrictiononautophagy.............................197
8. Conclusion....................................................................................199 References..........................................................................................199
CHAPTER9Isstagnantcerebrospinalfluidinvolvedinthe pathophysiologyofnormaltensionglaucoma ........... 209 HanspeterEsrielKiller
1. Summary.......................................................................................216 References..........................................................................................217
Preface
Glaucomaisaneurodegenerativediseaseofthevisualsystemcharacterizedbythe progressivedeathofretinalganglioncells(RGCs)andthelossoftheiraxonsthat formtheopticnerve.Increasedintraocularpressure(IOP)hasbeendemonstrated tobethemainriskfactorassociatedwiththeonsetandprogressionofthedisease. DespitemedicalandsurgicalapproachestoreduceIOPareavailable,actually,this diseaseisoneofthemajorcauseofblindnessintheworld.
Overthelastdecade,dissectionundernormalandpathologicalconditionsof molecularmechanismsimplicatedinthecontrolofcellsurvivalanddemiseand interplayamongretinalcellsofdiverseembryologicoriginhavewidenedthe perspectivefordevelopingneuroprotectivetherapeuticapproachesotherthanIOP controlinglaucoma.
Accordingly,aconsiderableinteresthasbeenrecentlydirectedtowardthe identificationofnoveltherapeuticstrategiesbaseduponneuroprotection.
InthisissueofProgressinBrainResearchdedicatedtoglaucoma,leadersinthis scientificfieldhavediscussedtheiroriginalandinnovativedataaswellasdata originatedfromtheclinic.Inviewofthelargecollectionyielded,thislattereffort hasbeenreportedintwoseparatedvolumes.
Thefirstvolume(256)isdedicatedtothewealthofmechanismsunderlyingthe neuronaldamageinglaucoma.Inparticular,thereaderisintroducedtothepotential contributionofalteredfundamentalcellularmechanisms(seeautophagy)aswellas microbialdysbiosisthroughthemodulationofadaptiveimmunity.Themore extensivelydissectedroleofinnateimmunityviamicrogliaactivationandsoluble neuroinflammatorymediatorsreleaseinretinalganglioncellsaswellasthe contributionofoxidativestressaredeeplyexplored.Theselattermaycontribute totheobservedalterationsofthecerebrospinalfluiddynamicand/orcomposition thatmayplayaroleinthepathogenesisofglaucoma.
Particularlyinterestingarethedatareportedinthesecondvolume(257)starting withachapterdevotedtotheuseofartificialintelligenceinthediagnosisandtherapy ofglaucoma.Therearemanyvariablesthataffecttheearlydiagnosisofthedisease andthemonitoringofitsprogression;theintroductionofartificialintelligencein clinicalpracticecouldgreatlysupporttheworkofophthalmologists.
Datasupportingthehypothesisthatglaucomamaybeinfluencedbyormayshare commonpathogenicmechanismswithdiseasesofthecentralnervoussystem(CNS) havebeenaccumulated.Inagreementwiththelatter,evidenceexistssuggestingthat thecentralnervouspathwaysdamagedinglaucomamayplayaroleinsustaining functionalanddailylivingdisabilitycausedbythedisease.
Thissecondvolumeincorporatesameta-analysisontheclinicalevidenceofthe neuroprotectivepropertiesofbrimonidineandcurrentscrutinyofnaturallyoccurring potentialneuroprotectans.Anoutlooktofuturestrategiesforglaucomaneuroprotectionisalsoproposed.Infact,achapteroverviewsthecurrentprogressontheregenerationofpluripotentstemcell-derivedRGCsandoutlookstheperspectiveinthis field.
Wewouldliketoacknowledgetheoutstandingcontributionofalltheauthorsto thesuccessofthesevolumesofProgressinBrainResearchdedicatedtoglaucoma andtheexcellentcollaborationofthehighlyprofessionalstaffofElsevier.Inparticular,wewouldliketoacknowledgetheconstantandskillfulsupportofMr.Hilal Johnsonalongthewholeeditorialprocess.Finally,wewouldalsothanktheReferees whohavecontributedagreatdealtoimproveoureditorialwork.
TheEditors
GiacintoBagetta CarloNucci
Functionaland morphologicalalterations inaglaucomamodelof acuteocularhypertension
AlejandroGallego-Ortegaa,Marı´aNorte-Mun ˜ oza, JuanAntonioMirallesdeImperial-Olleroa,JoseManuelBernal-Garroa, FranciscoJavierValiente-Sorianoa,PedrodelaVillaPolob,c, MarcelinoAviles-Triguerosa,Marı´aPazVillegas-Pereza,andManuelVidal-Sanza,* aDepartmentofOphthalmology,UniversityofMurciaandInstitutoMurcianodeInvestigacio´n Biosanitaria-VirgendelaArrixaca(IMIB-Arrixaca),Murcia,Spain bDepartmentofSystemsBiology,UniversityofAlcala´,Madrid,Spain cInstitutoRamo´nyCajaldeInvestigacio´nSanitaria,Madrid,Spain *Correspondingauthor:Tel.:+34868884330,e-mailaddress:manuel.vidal@um.es
Abstract
Tostudyshortandlong-termeffectsofacuteocularhypertension(AOHT)oninnerandouter retinallayers,inadultSprague-DawleyratsAOHT(87mmHg)wasinducedfor90minandthe retinaswereexaminedlongitudinally invivo withelectroretinogram(ERG)recordingsand opticalcoherenttomography(OCT)from1to90days(d). Exvivo,theretinaswereanalyzed forrod(RBC)andcone(CBC)bipolarcells,withantibodiesagainstproteinkinaseCα and recoverin,respectivelyincrosssections,andforcones,horizontal(HZ)andganglion (RGC)cellswithantibodiesagainstarrestin,calbindinandBrn3a,respectivelyinwholemounts.Theinnerretinathinnedprogressivelyupto7dwithnofurtherchanges,whilethe externalretinahadanormalthicknessuntil30d,witha20%thinningbetween30and90d. Functionally,thea-waveshowedaninitialreductionby24handafurtherreductionfrom 30to90d.AllothermainERGwavesweresignificantlyreducedby1dwithoutsignificant recoveryby90d.RadialsectionsshowedanormalpopulationofRBCsbuttheirterminals werereduced.TheCBCsshowedaprogressivedecreasewithalossof56%by30d.In wholemountretinas,RGCsdiminishedto40%by3dandto16%by30dwithoutfurtherloss. Conesdiminishedto58%and35%by3and7d,respectivelyandfurtherdecreasedbetween 30and90d.HZsshowednormalvaluesthroughoutthestudy.Inconclusion,AOHTaffects boththeinnerandouterretina,withamorepronounceddegenerationoftheconethanthe rodpathway.
Keywords
Acuteocularhypertension,Glaucoma,Ratretina,Bipolarcells,Horizontalcells,Cones, Retinalganglioncells,Transientretinalischemia
1 Introduction
Glaucomaisanopticneuropathycharacterizedbyprogressiveretinalganglioncell (RGC)losswithconcomitantvisualfielddeficitsthatmayprogresssilentlyuntiladvancedstagesofthedisease(Leiteetal.,2011)andconstitutesthemaincauseof irreversibleblindness(Quigley,2006).Additionalhallmarkcharacteristicsofglaucomaincludestructuralabnormalitiesofthenervefiberlayerandopticdisc (Chauhanetal.,2014; Weinrebetal.,2014).Independentresearchfromanumber oflaboratorieshasshownthatglaucomaisnotonlyadiseaseoftheinnerretina, butalsoofthemainretinofugalpathway.Ithasbecomeclearinhumanglaucoma studiesandexperimentalmodelsofocularhypertensionthatthisdiseasealsoaffects themainthalamicandmidbrainrelaynuclei(Dekeysteretal.,2015; ValienteSorianoetal.,2015a; Y€ ucel,2003; Y€ ucelandGupta,2008),aswellastheprimary (Nuccietal.,2013)andassociated(Frezzottietal.,2014)areasofthevisualcortex. Moreover,increasingevidenceindicatesthatthepre-ganglioncellcircuitofthe visualpathwayisalsoaffectedbothfunctionallyandstructurally(Mittagetal., 2000; Norketal.,2000; Ortı´n-Martı´nezetal.,2015).Thus,glaucomamaybeconsiderednowadaysadiseaseofthewholeprimaryvisualpathwaythatstarts somewhereneartheopticnervehead,fromwhereitprogressestowardsthecortical areasandbackwardsinamoreprotractedfashiontowardsthephotoreceptors(VidalSanzetal.,2012,2015a,2017).
Althoughthepathologyofglaucomaisnotfullyunderstood,itisgenerally acceptedthatisamultifactorialneurodegenerativediseasewhereaxonalcompression,vasculardysfunction,oxidativestress,immune-relatedneuroinflammation andischemia(Burgoyne,2015; Flammeretal.,2002; Tezeletal.,2010)mayplay arole.Accordingly,anumberofstudieshaveinvestigatedtheeffectsofaxotomy, excitotoxicity,oxidativestress,immune-relatedinflammationandischemiainan efforttounderstandthepathophysiologyofglaucoma.
Intraorbitalopticnerveinjuryisaclassicmodelthatallowstostudyplasticity, thatis,axonalregeneration(Aguayoetal.,1987; Brayetal.,1987),targetreinnervation(Aviles-Triguerosetal.,2000; Vidal-Sanzetal.,1987,2002)synapseformation (Keirsteadetal.,1989; Vidal-Sanzetal.,1987,1991; Whiteleyetal.,1998)aswellas neuroprotection(Lindqvistetal.,2004; L€ onngrenetal.,2006; Vidal-Sanzetal., 2007)oftheadultmammalianvisualsystem.Moreover,opticnerve(ON)injury hasalloweddetailcharacterizationoftheeffectsofaxotomyinadultratsandmice followingcompletecrushortransection(Nadal-Nicola ´ setal.,2015,2017; Sa ´ nchezMigallo ´ netal.,2018; Villegas-Perezetal.,1993)oftheintraorbitalON.ThesestudieshaveshownthatfollowingaxotomythereisarapidlossofRGCsfollowedbya
moreprotractedRGCdeaththatoccursoverthefollowingmonthswithaverysmall proportionofRGCsurvivinglongafterinjury.
TheeffectsofretinalexcitotoxicityhavealsobeenstudiedbymeansofintraocularadministrationofNMDAinanattempttolearnabouttheeffectsofthisinjury (Blancoetal.,2017; Calvoetal.,2020; Vidal-Villegasetal.,2019).Excitotoxicity resultedinrapidandmassivelossofthegeneralpopulationofBrn3a+ RGCs,while theentiremelanopsin+ RGCpopulationwaspreserved.Interestingly,theseretinas showedwithtimeanimportantthinningoftheoverallretinalwidthindicatingaprotracteddelayedouterretinaldegeneration.Transientischemiaoftheretinaisanother commonmodeltoinvestigatetheshortandlongtermeffectsofthisinjuryonthe survivalandfunctionoftheretina(Selles-Navarroetal.,1996).Thismaybe achievedusingamethodthatinvolvesselectiveligatureoftheophthalmicvessels withoutdirectdamagetotheON(Vidal-Sanzetal.,2007).Thesestudiesindicated thatRGClossisprogressiveanddependsnotonlyontheischemicintervalbutalso onthesurvivalperiod,andthatfollowingatransientperiodofretinalischemia,there wasadelayedprotracteddegenerationoftheouterretinaaswellasoftheprimary visualpathway(Mayor-Torroglosaetal.,2005).Morerecently,theearlyinvolvementofimmune-relatedneuroinflammationfollowingacuteocularhypertension hasbeenstudiedinadultrodents(deHozetal.,2013,2018; Ramı´rezetal., 2010,2020).
Becauseelevatedintraocularpressureremainsthemainriskfactoranditscontrol constitutesthemaintargettoslowdowntheprogressionofthedisease,anumberof experimentalanimalmodelshaveinvestigatedthephysiopathologyanddeteriorationoftheretinafollowingchronicocularhypertension(AOHT)(forreview,see (Morrisonetal.,2005; Vidal-Sanzetal.,2012,2015a,2017).TheseincludespontaneousgeneticmicemodelssuchastheDBA/2Jmice(PerezdeLaraetal.,2014, 2019).Inaddition,anumberofanimalmodelshavebeendevelopedtoinduceAOHT including,episcleralveincauterization(Garcia-Valenzuelaetal.,1995; Vecino etal.,2018),injectionintoepiscleralveinsofhypertonicsaline(Morrisonetal., 1997)orsclerosantagents(Blancoetal.,2019),injectionofmicrobeadsorviscoelasticsintotheanteriorchamber(Itoetal.,2016)orlaserphotocoagulationofthe episcleralandperilimbalveins(Salinas-Navarroetal.,2010).Thelaterhasbeen quiteapopularmodelusedtocharacterizetheeffectsofAOHTinadultalbino (Ouetal.,2016; Salinas-Navarroetal.,2010; Valiente-Sorianoetal.,2015a)rats aswellasinalbino(Salinas-Navarroetal.,2009)andpigmentedmice(ValienteSorianoetal.,2015b).
Anothermodeltostudyocularhypertensionconsistsoftheinductionofanacute elevationoftheintraocularpressure(AOHT)abovebasallevels.Thismodelmimics somehowtheeffectsofanacuteangle-closureglaucoma,andhasbeenwidely employed(QuigleyandAnderson,1976).RecentstudiesfromourLaboratoryin adultalbinoratsindicatethatAOHTresultsinprogressiveRGCloss,thatmaybe preventedwithintravitrealadministrationofbrainderivedneurotrophicfactor (BDNF)(Rovereetal.,2016).Furthermore,thegeneralpopulationofBrn3a+ RGCs orthepopulationmelanopsin+ RGCsresponddifferentlynoonlytotheinsultbutalso
toneuroprotection.AOHTalsoresultedin significantincreaseofthemicroglial cellswithintheRGClayer,withimportantchangesintheexpressionofmiRNAs associatedwithneuroinflammation(Wangetal.,2017).Inaddition,severalstudieshaveinvestigatedtheeffectsofAOHTintheinnerandouterretina,butthese wereanalyzedatshortersurvivalintervalsof3 – 8weeks( Palmhofetal.,2019 ; Schmidetal.,2014 ).Thepresentstudiesweredesignedtofurtherinvestigate inadultalbinoratstheshort-andlong-termeffectsofAOHTontheretina.Using functionalandmorphologicaltechniqueswestudiedlongitudinally invivo upto 90daystheeffectsofAOHTonthearchitecture,andfunctionofthemainERG wave-generatingretinalneurons.Moreover,usingimmunohistochemistrywe assessedneuronalsurvivalfromtheinnermost(RGCs),inner(rodandconebipolars,andhorizontalcells)andouter(conephotoreceptors)nuclearlayersof theretina.
2 Methods
2.1 Animalhandling
Sprague-Dawley(SD)rats( 180–210g)(CharlesRiverLaboratories;L’Arbresle, France)werehousedinanimalfacilitiesofMurciaUniversityintemperatureand lightcontrolledrooms(12hlight/darkcycle)withfoodandwater“adlibitum.” ExperimentalprotocolswereapprovedbytheEthicalandAnimalStudiesCommitteeofMurciaUniversity,andwefollowedSpanishandEuropeanUnionregulations forAnimalCare,aswellasthestatementfortheUseofAnimalsinOphthalmicand VisionResearchapprovedbytheAssociationforResearchinVisionand Ophthalmology.
Surgicalmanipulationswereperformedundergeneralanesthesia;intraperitoneal(i.p.)injectionofamixtureofketamine(70mg/kg,Ketolar®,Parke-Davies, S.L.,Barcelona,Spain)andxylazine(10mg/kg,Rompu ´ n ®,Bayer,S.A.,Barcelona,Spain).Tominimizeanydiscomfortanalgesiawasprovidedpostoperatively withsubcutaneousbuprenorphine(0.1mg/kg;Buprex,Buprenorphine0.3mg/mL; Schering-Plow,Madrid,Spain).Animalsweresacrificedwithani.p.overdoseof pentobarbital(DolethalVetoquinol®,EspecialidadesVeterinarias,S.A.,Alcobendas, Madrid,Spain).
2.2 Experimentaldesign
Toinvestigatetheshort-andlong-timeeffectsofAOHTweusedtwogroups.One groupwasemployedforthelongitudinal invivo morphologicalSpectralDomain OpticalCoherenceTomography(SD-OCT)andfunctionalrecordedelectroretinogram(ERG)responsesaswellasforthe exvivo whole-mountretinalstudiesat1, 3,7,15,30or90d(n ¼ 5–7ratspertimepoint).Asecondgroupwasanalyzedin crosssectionsat3,7,15,30or90dafterAOHT(30experimental; n ¼ 6pertime point,and4naıverats).
2.3 Acuteocularhypertensioninduction
AOHTwasinducedasdescribed(Rovereetal.,2016; Wangetal.,2017).Briefly, anesthetizedratswereplacedoveraheatingpadtomaintainnormalbodytemperature.A30-gaugeinfusionneedleplacedintheanteriorchamberofthelefteyewas connectedtoa500-mlcontainerof0.9%NaCl1.5mabovetheeye.Intra-ocularpressure(IOP)raisedfrombaseline(10 2mmHg)to87 4mmHg,asmonitoredwith Tono-Pen(Tono-Pen;MedtronicCo.,Dublin,Ireland)(Ortı´n-Martı´nezetal.,2015; Valiente-Sorianoetal.,2015a,2015b).Following90minofAOHT,theneedlewas removed,IOPreturnedtobasalvaluesandthecorneaswerecoveredwithanointment(Tobrex;AlconS.A.,Barcelona,Spain)topreventcornealdesiccation.Retinal bloodflowwasexaminedbydirectfunduscopywithoperatingmicroscope(Spot OPMI11,CarlZeiss,Oberkochen,Germany)previously,duringandafteracute OHT.WhileAOHTresultedinlackofretinalperfusion,therewascompleteblood flowreperfusionafterneedleremoval.
2.4 Spectraldomainopticalcoherencetomography(SD-OCT)
TheeffectsofAOHTwerecharacterizedlongitudinally invivo inthesameretinasat 1,3,7,15,30(n ¼ 12)and90(n ¼ 7)days,usingSD-OCT(Spectralis;Heidelberg Engineering,Heidelberg,Germany)asdescribed(Ortı´n-Martı´nezetal.,2014b; Rovereetal.,2015; Valiente-Sorianoetal.,2019).Asshownin Fig.1,totalretinal (fromnervefiberlayertotheretinalpigmentepithelium),innerretinal(fromthe fiberlayertotheouterendoftheinnernuclearlayer)andouterretinal(fromtheinner endoftheouterplexiformlayertotheretinalpigmentepithelium)thicknesswere measuredusingtheaverageofthreemeasurementsofcalipersprovideddirectly bythesoftwareofthedevicefrom3differentsectionspereye(onesectioncontaining theON,one1500 μmaboveandone1500 μmbelow).
2.5 Electroretinography(ERG)
ERGrecordingswereobtainedlongitudinallyfrombotheyesofthesame7ratsat1,3, 7,15,30and90dafterAOHTasdescribed(Alarco ´ n-Martı´nezetal.,2009,2010; Valiente-Sorianoetal.,2019).Inbrief,initiallyscotopicERGwaveswererecorded binocularlyfromanesthetizedratsunderdarkadaptation,inresponseto 4.4 (ScotopicThresholdResponse), 2.5(RodResponseandScotopicFlicker Response)and0.5(MixedResponse)logcd s/m2 providedbyaGanzfelddome. Animalswerethenlightadapted(30cd/m2)for5minandConeResponseandPhotopic FlickerResponses(0.5logcd s/m2)werefurtherrecorded.Retinalresponseswere recordedbyBurian-Allenbipolarelectrodesplacedonbothcorneasoftheanimal; adropofmethylcellulose(Methocel2%®;NovartisLaboratoriesCIBAVision,Annonay,France)wasplacedbetweenthecorneaandtheelectrodetoincreaseelectrical conductivity;onereferenceelectrodewasplacedinthemouthandaneedleplacedsubcutaneouslyatthebaseofthetailwasconnectedtothegroundelectrode.Theelectrical signalsweredigitizedat20KHzusingaPowerLabdataacquisitionboard(AD Instruments,Chalgrove,UK).StandardERGwaveswereanalyzedaccordingtothe InternationalSocietyforClinicalElectrophysiologyofVision(ISCEV).
SD-OCTimagesfromarepresentativeretinaanalyzedbeforeandat1,3,7,15,30and90d afterAOHT.SD-OCTrepresentativeimagesofaretinaanalyzedbefore(A)andat1(B),3(C), 7(D),15(E),30(F)and90(G)daysaftertheinductionofAOHT.ImageAshowsthe delineatedretina;thebluelinesdepictthefibberlayer;thegreenlineoutlinestheIPLandONL limitandthegreenlineoutlinestheRPE.(H)Graphshowingquantitativestudyoftotalretinal thickness(entirecolumn),innerretinalthickness(lightgraycolumn)andouterretinal thickness(darkgraycolumn)acrossalltimeintervalsstudied.Graphsshowprogressofinner retina(I)andouterretina(J)thickness. *Statisticallysignificantdifferencewithrespecttothe control; #Statisticallysignificantdifferencewiththe3-daygroup; ΦStatisticallysignificant differencewiththepreviousgroup.
FIG.1
2.6 Tissueprocessing
Ratsweredeeplyanesthetized,perfusedtranscardiallywith0.9%salinefollowedby 4%paraformaldehyde(PF)in0.1Mphosphatebufferandeyeswereenucleatedand postfixedfor1hin4%PF.Forcrosssectionanalysis,theeyecupswereimmersedin increasingsucroseconcentrations(15–30%),orientedandfrozenintissuetackand 16 μmthickcrosssectionscontainingtheONheadwereobtainedinthecryostatas described(Ortı´n-Martı´nezetal.,2014a; Valiente-Sorianoetal.,2014).Forwholemountanalysis,retinaswerepreparedasflattenedretinasmaintainingretinalorientationbymakingfourradialcutsintheretina(thedeepestonesignalsthesuperior poleoftheeye)asdescribed(Vidal-Sanzetal.,2015b,2017).
2.7 Immunohistofluorescence
Immunodetectionwascarriedoutfollowingpreviousdescribedprotocolsforcross sections(Ortı´n-Martı´nezetal.,2014b)orwhole-mountedretinas(Vidal-Sanzetal., 2017).Primaryandsecondaryantibodiesanddilutionsusedinthisstudyaredetailed in Table1.Allcross-sectionswerecounterstainedwithDAPI.
2.8 Imageacquisition
Crosssectionsandwhole-mountswereexaminedandphotographedusingepifluorescencemicroscopes(Axioscop,ZeissETCandLeicaDM6-B,LeicaMicrosytems, Wetzlar,Germany)controlledbyimageanalysissoftwareasdescribed (DiPierdomenicoetal.,2020; Ortı´n-Martı´nezetal.,2014a; Valiente-Sorianoetal., 2020).Fortheanalysisoftheretinalsections,threenon-consecutivesagitalretinal
Table1 Antibodiesusedinthiswork. Primary
Cross sections
Retinal Wholemounts
Mouse α-PKCα b
Rabbit αRecoverinc
Mouse α-Brn3ad
Guinea αcalbindine
Rabbit α-arrestinf
1:200ABCAM,ab11723,UKIgg1Goat α-mouse488
1:1000AB5585,Millipore,GermanyDonkey α-rabbit594
1:500MAB1585,Millipore,GermanyIgg1Goat α-mouse488
1:500214–004,SynapticSystems, Germany Goat α-guineapig 647
1:1000AB15282;Chemicon-Millipore Iberica,Madrid,Spain Donkey α-rabbit555
aAllsecondaryantibodieswerepurchasedfromMolecularProbes,ThermoFisher,Madrid,Spain.All wereusedina1:500dilution. bDetectsrodbipolarcells(Cuencaetal.,2010). cDetectsconebipolarcells(Cuencaetal.,2010). dDetectsretinalganglioncells(Nadal-Nicolasetal.,2014). eDetectsHorizontalcells(Pasteelsetal.,1990). fDetectsconeouter-segments(Palmhofetal.,2019).
