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1、and the elucidation of neuropsychiatric disease mechanisms.i12j Recent optogenetics developments have provided a potential treatment of epiIepsy,(3,4 Par- kinson,s disease,s,6 depression网 and other brain disorders,(9- nj where the cell type-specific modulation of neural circuits may address the path
2、ological symptoms.i21 To manipulate the activities of specific neurons or neural circuits in vivo using optogenetics, implantable optical waveguides are frequently used to deliver laser light into the virus-infected brain regions.”/ Primarily, these waveguides are silica optical fibers, with an aver
3、age Young,s modulus at least six orders of magnitudes larger than that of the neural tissues415-17 The elastic mismatch between the silica optical fibers and organisms may lead to the host tissue injuries, which subsequently induce neuronal death in the implant surroundings. To prevent this, stretch
4、able and flexible optoelectronic implantss- 21 and polymer integrated PrObeS【22- 25j have been developed, with a decreased tissue response and consistent performance duringCOMMUNICATIONwww.advopticalmat.deHydrogelOpticalFibersUltrasoftandHighlyStretchableHydrogelOpticalFibersforInVivoOptogeneticModu
5、lations1.uluWang,ChengZhong,DingningKe,FengmingYefJieTu,LipingWang/andYiLu*Optogeneticshasbeenwidelyappliedasacell-specifictechniquewithhightemporalresolutionforthemodulationofneuralcircuitryinvivo,offeringpotentialnoveltreatmentsforneuropsychiatricdiseases.However,todate,themostwidelyusedoptogeneti
6、cswaveguidesremainsilicaopticalfibers,whichmayleadtoamismatchinthemechanicalpropertiesbetweentheimplantsandneuraltissues.Toresolvethisissue,alginate-polyacrylamidehydrogelopticalfiberscanbefabricatedinasimplifiedone-stepprocess,andtheyshowsignificantlyimprovedcharacteristicsfortheinvivoOptogeneticap
7、plications,includinglowlight-propagationlossandYoung,smodulus,andhighstretchability.AftertheexpressionofAAV-CaMKIIigqpdatoflow-modulusandhigh-stretchablealgi1eQ(amideSi(PAAm)hydrogelopticalfibersandinvestigc3T15(pplica-tioninvivo.ThoOlGinat。PAAmprecursor?andcross-linkedinasimplifiedone-stepprocess,w
8、hileits三molecularstructurewasconfirmedusingtheRamanspectraganalysis(Figure1a).WiththeincreaseinthePAAmcontents,mainbandintensityincreasedaswell:1101cm1attheNH2itwisting,1322cm1attheCH2wagging,1429cm1atthe.CNvibration,1452cm1attheCH2bending,1621cm1JattheCOstretchingvibration,and1677cm1attheNH(1of6)20
9、18WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim(SeCQheTermsandcondn(httpsFigure 1. Chemical and physical properties OfaIginate-PAAm hydrogels, a) Raman spectra of prepared hydrogels, b) Linear expansion ratios of hydrogel W samples after swelling, determined from the cube root of the swelling ratio (n= 6). c
10、) Refractive index of swollen hydrogels (= 6). d,e) Represera- tive images of a dehydrated (d) and swollen (e) hydrogel optical fiber coupled with an optical ceramic ferrule, f) Blue light (L = 472 nm) propagationcloss of the swollen hydrogel optical fibers (= 12). g) Youngs modulus of swollen hydro
11、gel optical fibers n= 10). In (b), (c)f (f), and (g), dat arejpresented as mean standard errorof the mean.fited high optical conductivity even in the aqueous medium, suit-1bending,3o.3iSwellingofthehydrogeliscrucialforitsinvivoapplications,asitaffectstheinertphysicalcharacteristicsandinsertiontrauma
12、duringimplantation.Hydrogelsamplesweredehydratedandimmersedintoartificialcerebrospinalfluid(ACSF)for72h,andtheirweightsweredetermined.Theaveragelinearexpansionratiosofthefullyswollenalginate-PAAmhydrogelswere1.900-1.927,comparabletothoseinthefullydehydratedstates(Figure1b)andalmostindependentoftheAA
13、mconcentration.Therefractiveindexofthefullyswollenhydrogelslightlyincreasedfrom1.3454withtheAAmcontentandreachedtheplatformatH1.3533(Figure1c).Wefabricatedhydrogelopticalfiberswithvariousdiametersusingdifferenttubemolds(FigureS1,SupportingInformation).Tobalancethesizeandperformance,includingbothmech
14、anicalandopticalcharacteristics,ahydrogelopticalfiberobtainedbypolymerizingthealginate-PAAmprecursorinasiliconerubbertube(innerdiameter=300Xm)wasused.Hydrogelfiberwasdehydrated,threadedthroughanopticalceramicferrule(innerdiameter=300m),andimmersedinACSFfor2hbeforeuse.Thefullyswollenhydrogelfiberhold
15、sthestiffceramicferrulesteadily,formingasmoothopticalconnection(Figure1d).Bluelaserlight(L=472nm),frequentlyusedfortheexcitationofchannelrhodopsin-2(ChR2)-expressingneurons,wasconductedintothehydrogelfibersthroughsilicaopticalfibers(diameter=200OCm,NA=0.37)terminatedwithaceramicconnector(Figure1e).T
16、hepowerdensityofthelighttransmittedthroughthehydrogelopticalfibersfabricatedusingdifferentAAmconcentrationswasdetermined(FigureS2,SupportingInformation),andtheirpropagationloss(dBcm)wascalculated(Figure1f).Theresultindicatesthatthepropagationlossofthefullyswollenhydrogelopticalfiberssomewhatdecrease
17、dwithanincreaseinthePAAmcontent,reachingaminimumvalue(0.249dBcm1)inhydrogelfibersfabricatedwith40wt%oftheAAminprecursorsolution.However,furtherincreaseinPAAmledtoaosharpincreaseinthepropagationloss,probablyduetoamorecompactpolymernetworkofsemicross-linkedhydrogels.Addi-Xtionally,themechanicalpropert
18、iesofthefullyswollenhydrogel三fiberswerecharacterizedbyYoungsmodulus,whichslowlyincreasedfrom48.234to90.849kPawiththeAAmconcentra-stionincrease(Figure1g),showingthattheYoung,smodulusof三thehydrogelfibersissignificantlylowerthanthatofthecon-ventionalsilicaopticalfibers(H10GPa),andmorecompatiblewiththen
19、euraltissues(H1kPa).is-17,29)Therefore,duetoitsswellingratioandYoung,smodulus,aswellaslowpropagationloss,hydrogelopticalfibersfabricatedwith40%AAmconcentrationwereselectedforfurtherstudies.Thefeasibilityofthehydrogelopticalfiberuseforinvivooptogeneticmodulationswasexamined.Theswollenhydrogelopticalf
20、ibershowedexcellentelasticstretchability,anditsconductivitydroppedonly13.97and30.15%whenstretchedto120and140%ofitsinitiallength,respectively(FigureS3,SupportingInformation),sufficienttomeetbrainorneuraltissuedeformation.Furthermore,thefabricatedhydrogelfiberehib-ableforinvivoguidingofthelaserlight(F
21、igureS4,SupportingInformation).Todeterminewhetherthefabricatedhydrogelopticalfibersdeliverenoughlighttoexciteglutamatergicneurons,adeno-associatedvirus(AAV)-CaMK11(-ChR2-mCherrywasinjectedintoC57mousehippocampus(Figure2a).Acustom-madehydrogel-optrodearraycontainingfourstere-otrodeswasusedforoptogene
22、ticstimulationandelectricalrecordinginvivo(Figure2b;andFigureS5,SupportingInformation).ThehippocampalneuronsweretransducedwithAAV4weeksaftertheinjection,andbluelightpulses(20Hz,5ms)weredeliveredforoptogeneticactivation.Astheimplantationdepthwasonly2.0mm,thereforethediameter-to-lengthratioofthehydrog
23、elopticalfiberwasrelativelyhigh.Thisisbeneficialfordecreasingpropagationlossduringopticalstimulation.RepresentativeexamplesofrawspikedatashowedthateachFigure 2. Optogenetic modulations in vivo, a) Expression of CaMK-ChR2-mCherry (red) in hippocampal neurons 4 weeks after injection (bar =500m).b)Asch
24、ematicdiagramofahydrogelopticalfiber-coupledelectrode(hydrogel-optrode)array,c)日ectroPhySioIogiCaIreCOrdingSbeforeand?duringoptogeneticstimulation(bluebar)usinghydrogel-optrodearrayinvivo,d)Principal-componentanalysisoftwoseparableneuronalunitpre-Isentedin(c).Thepointsarecoloredaccordingtotheassigne
25、dcluster,andlow-amplitudespikesarenotshown,e)WaveformsoftherecordedUIipresentedin(c).f)ExpressionofCaMKII(-ChR2-mCherry(red)intheM14weeksafterinjection(bar=200m).g,h)Representativemovingtracesinaanopenfieldbefore(g)andduring(h)optogeneticmodulation,i)Totaldistancetravelledintheopenfield(n=6,*p0.005,
26、Mest).flashofthepulsetraingeneratedalargeelectrophysiologicalresponsebytheactivatedneurons(Figure2c).3Dviewsoftheunitclustersandaveragedspikewaveformsoftherecordedneuronswerealsoanalyzed.Twotypesofneuronsactivatedbyhydrogelopticalfiberwereclearlyobserved(Figure2d),andthepeak-to-peakamplitudesofthese
27、sortedwaveformswere150-200ocV(Figure2e),implyingthatthefabricatedhydrogelopticalfiberscanbeusedaswaveguidesforoptogeneticstimulationinvivo.Wefurtherinvestigatedthelong-termperformanceofhydrogelopticalfibersinvivo,byimplantingthemintotheprimarymotorcortex(M1)ofmiceafterinjectingAAV-CaMK11(-ChR2-mCher
28、ry(Figure2f;andFiguresS6andS7,SupportingInformation).Themicecouldfreelyexploreanopen-fieldarenafor10minwithoutopticalstimulation(Figure2g;andVideoS1fSupportingInformation).Afterward,weexaminedwhetherthebluelightpulses(10mW,20H乙5msduration)intheimplantedhydrogelopticalfiberscanactivatetheM1glutamater
29、gicneuronsandsubsequentlymodulateanimalbehavior.Thesemiceexhibitedincreasedright-turningandrotatingmovementsimmediatelyafterlightdelivery(Figure2h;andVideoS2,SupportingInformation),attributabletothecontralateralM1neuronactivation.Asignificantdecreaseinthetotalmovingdistanceduringoptogeneticmodulatio
30、nperiodwasObSerVed(Figure2i),inaccordancewiththebehavioralresultsobtainedonmiceimplantedwithconventionalsilicaopticalfibers(FigureS8,SupportingInformation).Furthermore,toexaminethelong-termfunctionalstabilityandtissue-compatibilityofthehydrogelopticalfibers,sampleswereimplantedintothedeepbrainofC57m
31、ice.Wefoundthatthestretchabilityandopticalconductivityofthehydrogelfiberswerenotsignificantlyaffected4weeksafterimplantation(FigureS9,SupportingInformation).Thetissueresponsetotheimplantswascharacterizedbyglialfibrillaryacidicprotein(GFAP)immunoreactivity(Figure3a),andreactivatedastrocytesoccupiedth
32、ezonearoundthesilicafiberimplant,whileamuchlighterGFAP-positivezonewasWshowntorepresenthydrogelsurroundings.Quantitativeanal-ysisofGFAPintensityatthesilicaandhydrogelopticalimplant电asafunctionofdistancefromtheinterfaceispresentedinFigure3b,indicatingthattheGFAPintensityinthehydrogel冷groupwassignific
33、antlylowerp0.005)thanthatofthesilicagroup,along175mtotheimplantinterface.Neuronalsur-Svivalaroundtheimplantswasassessedbyanalyzingneuronal三nucleus(NeuN)immunoreactivity.Severeneuronallosswas?observedadjacenttotheconventionalsilicaopticalfiber,butnextatthehydrogel/tissueinterface(Figure3c).Quantitati
34、veanalysidemonstratedthattheneuronaldensityinthehydrogelopticalfibergroupremainedalmostunchangedacross500OCmdistancf4weeksafterimplantation,significantlyhigher(p0.005)thanthatobservedinthesilicaopticalfibergroupinthetestzonewithin100mfromtheimplant/tissueinterface(Figure3d).Thissuggeststhatthehydrog
35、elopticalfibersaresuitableforchronicoptogeneticmodulationsinvivo,withadecreasedglialencapsulationandanimprovedneuronalviabilityaroundtheLimplants.However,itisworthmentioningthatthesizeofthefabricatedhydrogelfibersshouldbefurtherdecreasedatthisstagetomeettherequirementsofmultisiteopticalStimula-tiona
36、ndminimalimplantationtrauma.Someissues,suchasSnovelcoreandcladdingmaterials,andimprovedopticalimplantdesigns,stillrequirefurthersystematicinvestigation.IInconclusion,wedemonstratedthefeasibilityandadvan-1tagesofanalginate-PAAmhydrogelopticalfiberuseforchronioptogeneticbrainmodulationinfree-movingani
37、mals.ThisFfiberwaspolymerizedinaone-stepprocess,exhibitingalow-5modulusandhigh-stretchablepropertiescompatiblewithbio-?logicaltissues.Opticalceramicferrule-coupledhydrogelfiberEimplantswerefabricatedbyasimplifiedswollen-fixingstrategy,1whichfacilitatestheircombinationwithelectrodearraysand三invivoopt
38、ogeneticstimulationapplications.Owingtothelowwww.advopticalmat.deoHvdroeelFiber(GFAP)SHkaFiber(GFAP)SilicaFiber(NeuN)HydrogelFiber(NeuNjIFigure3.Inflammatoryresponseandneuronalsurvivalaroundopticalimplants,a,c)GFAP(a)andNeuN(c)immunostainingofconventional?silicaIandhydrogelopticalfibers(red:GFAP;gre
39、en:NeuN;blue:DAPI;bar=200m)at4weeksafterimplantation,b,d)QuantitativecomparisonsofGP(b)andNeuN(d)immunoreactivitybetweenconventionalsilicaandhydrogelopticalfibers;comparisonswereperformedusingintensityprofileasafunctionofdistancefromtheimplantinterface,shownasmeanvaluesstandarderrorofthemean(GFAPrn=
40、16;NeUN,n=12).Shaded嗓a,significantdifference(p0.005,f-test).I：一、i4r,三OJblue-lightpropagationlossofthefullyswollenhydrogelfiber,weobservedlight-evokedandfrequency-dependentresponsesofthehippocampalneuronsusingahydrogel-optrodearray.Furthermore,wedemonstratedthatlightdeliverythroughthechronicimplantedhydrogelopticalfiberscanactivatetheAAV-epressingneuronsinM1,modulatinganimalbehavior.Hydrogelopticalfiberssignificantlyalleviatedtissueresponseandimprovedneuronalsurvivalattheimplant/tissueinterface,incontrasttothoseoftheconventionalsilicaopticalfibers.Althoughwepresentedonlysomeoft
