教科書-hanyuan麻醉學(xué)筆記

SectionI–1–HistoryofAnestheticPracticeMerlinD.LarsonKeyMethodstosafelyalleviateseverepainarerelativelyrecentdiscoveries,asviewedwithinthetimespanofhumanhistory.ThepublicdemonstrationofetheranesthesiaonOctober16,1846,ranksasoneofthemostsignificanteventsinthehistoryofmedicine.NosingleindividualcanbesaidtohavediscoveredThespecialtyofanesthesiarestsondiscoveriesmadefromseveralscientificMajordiscoverieswereoftenmadebysmallgroupsofcuriousindividualswithdiverseTechniquesincommonuseatanyonetimeoftenseemdangeroustosubsequentgenerationsofanesthesiologists.MajorinnovationsweresometimesignoreduntiltheirrediscoveryseveraldecadesDevelopmentsinanesthesiaoftenarosetomeettheneedsofpatientswithseverecomorbidconditionsthatrequiredcomplexsurgicalprocedures.Consequently,advanceswithinthespecialtiesofsurgeryandanesthesiaarecloselyintegrated.SectionII–Anesthetic11–Sleep,Memory,andConsciousnessMaxB.Kelz,GeorgeA.Mashour,TedG.Abel,MervynMazeKeySleepisanactiveprocessgeneratedintheStructuresinthebrainstem,diencephalon,andbasalforebraincontrolthesleep-wakecycleandaredirectlymodulatedbygeneralanesthetics.Sleepandanesthesiaaresimilarstateswithdistincttraits,witheachsatisfyingneurobiologicfeaturesoftheother.DistinctmemoryfunctionsaresubservedbydistinctneuralLimbicsystemstructuressuchasthehippocampusandamygdalaarecriticalformemoryandplayaroleinanesthetic-inducedamnesia.Althoughbrainstem,diencephalon,andbasalforebrainstructuresgeneratewakefulness,thecontentsofconsciousnessarethoughttobegeneratedbythecortex.MultipleneuralcorrelatesofconsciousnessarethoughttobethetargetsofgeneralConsciousnessandsubsequentexplicitrecallofintraoperativeevents—knownas“awarenessduringgeneralanesthesia”—occurin1to2casesper1000.Monitoringanestheticdepthhasevolvedtoelectroencephalographicmethods,althoughlimitationsstillexist.【140731ethernitrousoxide Anesthetizedstate,includingamnesia,hypnosis,gesia,immobility,andbluntingofautonomicreflexes.Thebeneficialeffectsofsleepincluderestorationoftheneuronalhomeostasisessentialforsynapticfunction,consolidationofmemoriesandinitiationandexpressionofneuronalplasticity.Theneuronalunderpinningsforcontrolofarousallieinsubcorticalstructuresdeepwithinthebrainstemthalamus,andhypothalamusinregionswheretheyareconservedacrosstheanimalkingdom.的控制基于皮質(zhì)下結(jié)構(gòu)，以及腦干、丘腦和下丘腦。睡眠的學(xué)Bremer實(shí)驗(yàn)（cerveauisolecat），橫切延髓尾端，需要機(jī)械通氣，但睡眠-周期正常；Underthispassiveview,sleepwasnothingmorethanadefaultstateproducedbycessationoftheactivestate—waking. BremerMoruzziascendingreticularactivatingsystem（網(wǎng)狀結(jié)構(gòu)上行激活系Sleepisgeneratedwhenspecificneuronalsystemsincreasetheirfiringratesandthusinhibittheoutputofotherneuronalstructuresrequiredforwakefulness. 【140807睡眠和的生理模式由EEG和EMG（肌電圖）記Wakefulnessisdefinedbyadesynchronized,low-amplitudefast-frequencyelectroencephalogram(EEG)withprominentmuscleactivity.REMsleepshowssimilarsignsofcorticalactivationwithadesynchronized,low-amplitude,fast-frequencyEEGinwhichθrhythmsof4to8HzdominatethepowerspectrumHowever,unlikewakefulness,motoractivityisminimalinthisstate.NREMsleephasanEEGappearancethatismarkedlydifferentfromtheothertwostates.DuringNREMsleep,theEEGdisplaysslow-frequencylarge-amplitudeoscillations.MotortoneduringNREMisdramaticallyreduced. 是去同步化的、低幅、高頻的EEG，以及顯著的肌肉運(yùn)動。EEG，0.5-4Hz的δnoradrenergicneuronsofthelocusceruleus(LC),histaminergicneuronsofthetuberomammillarynucleus(TMN),serotonergicneuronsofthedorsalandmedianraphenuclei(RN),andthenewlyrecognizedpopulationofdopaminergicneuronsintheventralperiaqueductalgray(vPAG)matterareallmonoaminergiccentersthatdisplayarousalstate–dependentfiringpatterns. 斑LC、組胺能神經(jīng)元結(jié)節(jié)核TMN、胺能神經(jīng)元中縫核RN的背側(cè)核和正中vPAG。Neuronscontainingthewake-promotingandwake-sustainingneuropeptideorexinalsoknownashypocretin)sharesimilaritieswithothermonoaminergicsystems.Theseneuronsexhibitalactivityduringwakefulness,reducetheirfiringduringNREMsleep,and equiescentduring 促和喚醒維持的神經(jīng)肽食欲素orexin與其它單胺能系統(tǒng)相似，在時表現(xiàn)出最大活性，NREM時放電頻率減少，REM期間為靜態(tài)。Orexin正性刺激單胺能系統(tǒng)，維持。破壞orexin信號傳遞導(dǎo)致發(fā)作性嗜睡病。對于而言，沒有任何一種單胺類神LDTPPT投射至前腦。單胺類神經(jīng)元彌散投射至前腦，并直接調(diào)節(jié)下丘腦核團(tuán)，為活性區(qū)域。睡眠促進(jìn)神經(jīng)元腹外側(cè)視前區(qū)核VLPO，含有抑制性神經(jīng)γ-氨基丁酸和甘丙肽，VLPO抑制性拮抗上行網(wǎng)狀激活結(jié)構(gòu)。Orexin能神經(jīng)元與Althoughbrainstemandhypothalamicactivitymodulateswakefulness,thecerebralcortexitselfcontributestoself-awakeningthroughitsefferentprojectionstothethalamusandreticular 【140808Neuronsthatareactiveduringwakefulness(W)includethosewithascendingprojectionstothecortexthatstimulateafast-frequencydesynchronizedEEG(gamma+),alongwithdescendingprojectionstothespinalcordthatstimulatetheposturalmuscletonerequiredforwakingbehavior.Wake-activeneuronsdisplayalactivityduringwakefulness,dramaticallyslowtheirfiringduringnon–rapideyemovement(NREM)sleep,and evirtuallyquiescentduringrapideyemovement(REM)sleep. 活性神經(jīng)元，上行投射至皮質(zhì)刺激形成高頻EEG（γ波），下行至脊髓刺激形成肌張力，這些神經(jīng)元在時顯示出最大活性，而在NREM時放電頻率大幅下降，REMNAH、食欲素能Orx、谷氨GluACh、谷氨酰胺能Gluγ-氨基丁酸能GABA等神經(jīng)元REM時也激活。Sleep-activeneuronsincludecellswhosecorticalascendingprojectionsdampenfastcorticalactivityandthosewithdescendingprojectionstothespinalcordandbrainstem,whichdiminishbehavioralarousalandmuscletoneThesleep-activeneuronsdischargeinassociationwithslowEEGactivity(gamma-/delta+)duringNREMsleep. 和下行消除行為和肌張力的細(xì)胞，在NREM時呈慢波（Δ波）。NREMWithanotableexceptionoftheventrolateralpreoptic(VLPO)nucleus,overallelectricalactivityinmostregionsofbrainisdecreasedduringNREMsleep.DuringNREMsleep,monoaminergic,orexinergic,andcholinergicgroupsareinhibitedbyefferentsignalsemanatingfromthepreopticanteriorhypothalamus,specifically,aclusterofneuronslocalizedtotheVLPOnucleusthatusetheinhibitoryneurotransmittersGABAandgalanin.VLPOneuronsaresleepactiveanddisplayincreasedfiringratesandc-Fosimmediateearlygeneexpressionduringsleep.Sleep-activeVLPOneuronshaveanantagonisticrelationshipwithwake-activecenterssuchthatVLPOactivationinhibitsfiringinwake-activecenters.Conversely,rapidfiringofwake-activeregionsinhibitstheVLPOnucleus.Thisnetworkdesignleadstoabistablebehavioralstateofarousalfavoringeithersleeporwakefulnessbutnotrapidtransitionsbetweenthetwo.NotsurprisinglydestructionoftheVLPOnucleuswithanexcitatoryaminoacidlesionthatdestroyscellbodieswhileleavingfibersofpassageintactcausesinsomnia. VLPO之外，NREM時腦電活性全面減弱。VLPO釋放抑制性神經(jīng)遞質(zhì)GABA和甘丙肽抑制單胺能、食欲素能、膽堿能等神經(jīng)元。VLPO在睡眠時激活，放電頻率增加，c-Fos即刻早期表達(dá)。睡眠活性神經(jīng)元VLPO與活VLPO后引起失眠。Burstfiringofthalamocorticalneuronstransientlycausesdeafferentationofthecortexbyreversiblydisconnectingitfromsensorystimulinormallyconveyedtothecortexfromthe NREM時改變腦電活動，形成睡眠REMAlthoughseveralneuroanatomiccentersparticipateinregulationandcoordinationofREMonsetandoffset,themaineffectorresponsibleforthegenerationofREMsleepresidesinthepontinereticularformation.TheponstothenucleuspontisoralisPnOwhichappearstobenecessaryforREMexpression.DirectinjectionofcholinergicagonistsintothePnOproducesastatethatmimicsnaturalREMsleep.TheendogenouscholinergictoneinthebrainstemarisesfromtheLDTandPPT,twonucleilocatedatthejunctionbetweentheponsandmidbrain.ExcitotoxiclesionsthatablatetheLDTandPPTdramaticallyimpairREMsleep.LDTandPPTcholinergicneuronalactivitymayinitiateREMsleep.NeuronsoftheextendedVLPO(eVLPO)nucleusalsoexhibitpreferentialactivationduringREMsleep. REM的產(chǎn)生中具有重要作用，REMREM睡眠的啟動者。延伸的eVLPO核在REM中也具有特異性的重要作用。ThecardinalsignsofREMsleepincluderapideyemovement,atoniaofallmotorgroupsexceptforthediaphragm,andactivationofalow-voltage,fast-frequencyEEGrhythm.Subcorticalrecordingsdemonstrateponto-geniculo-occipital(PGO)waves.ThischaracteristicyEEGpatternofREMsleeporiginatesinthepons,istransmittedtothethalamiclateralgeniculate,andterminatesintheoccipitalcortex.TheREMandwake-activepopulationofLDTandPPTneuronswithrostralprojectionsisimportantforproductionofthedesynchronizedfast-frequency,low-amplitudeEEGfoundinbothwakefulnessandREMsleep.REMatoniaisinitiatedbyagroupofpontinereticularneuronsthatsynapseinthebulbarreticularformationbeforeterminatingtheirsignalonspinalcordmotorneurons. REM的主要特征斑塊快速眼球運(yùn)動、膈肌外的其他肌EEGPGO波，這種高尖的波產(chǎn)生于腦橋，發(fā)送到丘腦外側(cè)膝狀體，并終止于枕葉皮質(zhì)。LDT和PPT是產(chǎn)生和REM中快頻低幅EEG的關(guān)鍵。REM肌肉麻痹由一組延髓網(wǎng)狀神經(jīng)元形成，終止于脊髓運(yùn)動神經(jīng)元。ExitfromREMsleeptransitionsintoeitherNREMsleeporwakefulnessandistriggeredby“REM-off”groups.TheobservationthatnoradrenergicLCneuronsdecreasetheirfiringrateduringNREMsleepand evirtuallyquiescentduringREMsleep,togetherwithpharmacologicandlesionstudies,hadsuggestedthatinhibitionoftheLCwasarequirementforentryintoREMsleepandthatLCneuronsmightserveasREM-offcells.However,geneticstudiesinnoradrenergic-deficientmicehaveconclusivelydemonstratedthecontinuedexistenceofnormal,ornearlynormal,REMsleepdespitetheabsenceofnorepinephrine.Thus,theadrenergicneuronsoftheLCcannotbeanexclusiveREM-offpopulation.Neuronsoftheventrolateralperiaqueductalgray(vlPAG)matteralsoservetoterminateREMepisodes,asprovedbypharmacologicstudiesduringwhichmuscimolinhibitionofthisregionincreasesREMsleepandalsobyelegantimmunohistochemicalmapcombinedwithvlPAGlesions.vlPAGneuronsformamutuallyantagonisticinhibitoryloopwiththoseoftheSLDnucleustoefficientlygenerateorinhibitREMsleep. 從REM進(jìn)入到NREM或狀態(tài)是由REM-off群組觸發(fā)的。LC神NREMREMLCREM的關(guān)鍵，故其可REM-off細(xì)胞。然而，膽堿能缺陷小鼠盡管缺少去甲腎上腺素，仍表現(xiàn)正?；蚪咏！lPAGREMSLDREM睡眠?！?20809Intrathecalinfusionofcerebrospinalfluid(CSF)harvestedfromsleep-depriveddogsintorestednormaldogscausedtherecipientdogstopromptlyfallasleep.Thisresultsuggestedtheexistenceofanendogenoussomnogen,ahormonecirculatingintheCSFwhoseaccumulationcouldcausetheonsetofsleep. 正常狗鞘內(nèi)注射來自失眠狗的CSF后立即入睡。睡眠或狀態(tài)的進(jìn)入或脫離機(jī)制尚不明確，體液理論認(rèn)為存在一種內(nèi)源Somnogen，在CSF中積聚引起InfusionoffemtomolarconcentrationsofPGD2intothethirdventricleinducesbothNREMandREMsleepinratsthatisindistinguishablefromnaturalsleep.PGD2levelsfluctuateintheCSF,withcircadianfrequencyparallelingsleep-wakecycles.SleepdeprivationproportionallyelevatesCSFPGD2levels,thusalsosupportingaroleforPGD2asanendogenoussomnogen.PGD2issynthesizedbytheenzymeprostaglandinDsynthetase,whichislocalizedinthearachnoidmembraneandchoroidsplexus,andissecreteddirectlyintoCSF,whereitisthesecondmostabundantprotein.However,thissomnogenicactivityispresentonlywhenPGD2isinfusedinthevicinityofthepreopticareaofthehypothalamus.ThemostpronouncedactivityofPGD2isobservedwhenitisinfusedbeneaththeVLPOnucleus.InfusionofaPGD2antagonistintothethirdventriclereversiblyanddose-dependentlyinhibitsbothREMandNREM 與睡眠-周期相似，失眠者CSF中PGD2水平增高。PGD2由位于蛛網(wǎng)膜和脈絡(luò)膜的前DCSFCSF中第二豐富的蛋白。Somnogen效應(yīng)僅在PGD2VLPO核附近效應(yīng)最強(qiáng)，而注射其拮抗劑可REM和NREM。OnbindingtotheD-typeprostanoidreceptor(DPR),whichislocalizedtothearachnoidmembraneliningtheventralsurfaceofthebrain,thesomnogenicsignalofPGD2appearstobetransducedindirectlybyactivationoftheVLPOnucleus.ThemechanismforVLPOactivationaftersubarachnoidinfusionofPGD2appearstorequireadenosinebecausecoadministrationofanadenosinereceptorA2aantagonistblocksthesomnogenicactivityofPGD2.Conversely,administrationofanadenosineA2aagonistmimicsthesomnogenicactivityofPGD2.Asadenosinelevelsaccumulate,theyactivateA2areceptor–expressingneuronstodirectlyorindirectlyactivatetheVLPOnucleus.Hence,itappearsthatadenosinemayfunctionastheneurotransmitterthatcouplesthehumoraltotheneuralmechanismsdrivingsleep-wakeregulation. 側(cè)蛛網(wǎng)膜的D型素受體DPR，PGD2的somnogenic信號可能是由活化的VLPO核間PGD2VLPO還需要腺苷，因?yàn)楹嫌孟佘帐荏wA2a拮抗劑阻斷PGD2somnogenic活動。而A2a受體激動劑能夠模擬的PGD2somnogenic活性。腺苷積聚激活A(yù)2a受體直接或間接激活VLPO核。因此，腺苷作為神經(jīng)遞質(zhì)，可能Duringsleepandgeneralanesthesia,thereisreducedresponsivenesstoexternalstimuli.Functionalbrainimagingduringanesthetic-inducedunconsciousnesshasbeenshowntoinhibitthalamicandmidbrainreticularformationnuclei.Anestheticblockadeofthalamicinformationtransfer,whichdisruptssomatosensoryinputfromreachinghighercorticalcenters,hasalsobeenconfirmedwithmoredirectmicroelectroderecordings.Inbothinstances,theseanestheticeffectsonthethalamusresemblethenaturallyoccurringthalamocorticalinhibitioncharacteristicofNREM NREMInfusionofadenosineinlowdosespotentiatesthehypnoticactionsofintravenousandvolatileanesthetics,therebyreducingtheamountofanestheticrequiredtoachieveagivendepthofanesthesia.Mechanistically,thesedatafitwellwithaneffectofadenosineonactivationofthehypothalamicsleepcenter,theVLPOnucleus.Meanwhile,exposuretoanestheticssuchasisofluraneaffectsthelevelsofendogenoussomnogens,withisofluranealteringthebalancebetweenprostaglandinE2,awake-promotingprostaglandin,andPGD2,asleep-inducingprostaglandin,inthehypothalamus. 度的藥物劑量。于異氟烷影響內(nèi)源性somnogens，改變prostaglandinE2（促）和【140812AnestheticsappeartoactonNREMsleepcircuits,therebyleadingtosharedmechanismsofaction.Withinthethalamus,thereisasimplearchitectureofcelltypesconsistingofreticularneuronsandthalamocorticalneuronsthatcommunicatewiththecortexwhilealsointegratingperipheralinput.ActivationofthereticularneuronsduringNREMsleepandanesthesiacauseshyperpolarizationofthalamocorticalrelayneurons,whichinturnblockspropagationoftheactionpotentialthroughthalamocorticalrelayneurons.Asaresult,thalamocorticalneuronsarepreventedfromrelayingperipheralinputtohighercorticalcenters.Midlinethalamicnucleiarethoughttoplayacriticalroleingeneratingconsciousawarenessandappropriayreceivingafferentinputfrommostreticularactivatingarousal-promotingcenters.Imagingstudiesconfirmaregionallyselectivereductioninmidlinethalamicbloodflow,metabolism,andbyextension,activity.Supportforathalamocorticalconsciousnessswitchhasrecentlybeenstrengthenedbythefindingthatmicroinjectionofnicotineintothecentromediannucleusofthethalamusreversessevoflurane-inducedhypnosis.Theseconclusionsaremitigatedbythefactthatadministrationofnicotineintothecentromediannucleusresultsinseizures.However,supportforthecentralthalamusasanarousalcenterthatiscapableofreversingunconsciousnessalsocomesfromliteratureonthepersistentvegetativestate.High-frequencystimulationofthecentralthalamusintherathasbeenassociatedwithwidespreadcorticalactivationandenhancedcognitivefunction.Furthermore,deepbrainstimulationofthecentralthalamushasbeenshowntoreversesomeofbehavioraldeficitsinapatientsufferingtraumaticbraininjury. NREM睡眠相似，其機(jī)NREM和麻醉時網(wǎng)狀神經(jīng)元激活引起丘腦皮質(zhì)傳遞神經(jīng)元超極化，阻斷丘腦皮質(zhì)神經(jīng)元動Becausenicotinicacetylcholinereceptorsareheavilyexpressedinthethalamusandbecausemanyanestheticsinhibitsignalingvianicotinicacetylcholinereceptors,suppressionofthecholinergicarousalsystemmaybeonemechanismthroughwhichmanyanestheticsproduceunconsciousness.ProcessedEEGmeasuresofanestheticdepthalsorevealanimportantroleforthecholinergicarousalsysteminasmuchasintracerebroventricularinfusionsofneostigmineorthemuscarinicagonistoxotremorinearouseisoflurane-anesthetizedrats. 【140813Thalamicnucleireceiveinputfromtheascendingbrainstemreticularactivatingsystemviaadorsalpathwayandalsoreceivehypothalamicinputfromwake-activecenterssuchashistaminergicandorexinergicneurons.Asdiscussedearlier,thethalamicgatescloseduringNREMsleepandexposuretoseveralanestheticsandthisclosureisfacilitatedbydecreasedinputofmonoaminergiccholinergicandorexinergicsignalsduringanesthesia.GABAergicanestheticssuchaspropofolandbarbituratesexerttheirhypnoticeffectsbyinactivatinghistaminergicneuronsoftheTMN.ThisactionmaybeexplainedatthemolecularlevelbypotentiationofinhibitoryGABAergicprojectionfromthesleepcenter,theVLPOnucleus.DisinhibitionoftheVLPOnucleus,inturn,shutsdownotherwake-activegroupsandfurtherreinforcesVLPOactivity.Thisfeed-forwardmechanismstabilizesthehypnoticstate.Blockadeofthewake-promotinghistaminergicsignalisalsothemechanismbywhichtheantihistaminergicdrugdiphenhydramineprecipitatessleep.Recoveryoremergencefromanesthetichypnosisisfacilitatedbywake-promotingorexinergicneurons,whichareinhibitedbyvolatileanestheticssuchasisofluraneandsevoflurane. 息，并接收來自下丘腦喚醒活性中心如組胺能和orexinergic神經(jīng)元的傳入信息。NREM眠和麻醉時，丘腦關(guān)閉，而這種封閉通過減少單胺能、膽堿能、orexinergic的輸入。GABA能丙泊酚和巴比妥類藥物通過滅活TMN的組胺能神經(jīng)元發(fā)揮作用。VLPO核的去抑制，阻斷其他喚醒活性基團(tuán)，并進(jìn)一步增強(qiáng)了VLPO活性。這種前饋機(jī)制穩(wěn)定了狀態(tài)?？菇M胺能藥物苯海拉明阻斷促組胺能信號。促orexinergic神經(jīng)Onefindingthathasemergedfromstudyingthehypnoticpropertiesofdifferentanestheticagentsisthatthereisneitheraunitarymoleculartargetnoraninvariantneuronalsiteofactioncommontoallanesthetics.Thispointisillustratedbydexmedetomidine,anα2-adrenergicagonist.Thebehavioralhypnosisofdexmedetomidineresultsfromthedrug'sabilitytoinactivatenoradrenergicneuronsoftheLC.ThiseventdisinhibitstheVLPOnucleus,whichsubsequentlyinactivatesotherarousalcentersviatheVLPO'sGABAergicandgalaninergicinhibitorysignaling.AswithpropofolandbarbituratesactingontheTMN,thecommonconsequenceofVLPOdisinhibitionisstabilizationofthehypnoticstate.Pharmacologicandlesionexperimentsthatalterbothmonoaminergicreticularactivatingfunctionandanestheticsensitivitycannowbereinterpretedintheframeworkofintegratedarousalnetworkactivity.DepletionofCNScatecholamines,includingnorepinephrine,serotonin,dopamine,andhistamine,produceshypersensitivitytoanesthetics.Conversely,pretreatmentwithamonamineoxidaseinhibitororacuteexposuretoamphetamine,bothofwhichincreasecatecholaminelevelsinthebrain,producespartialtoanesthetics.FocusingonceagainonnoradrenergicneuronsoftheLC,chemicaldepletionofnorepinephrinewith6-hydroxydopamineandelectrolyticdestructionofLCneuronsbothproducehypersensitivitytoanesthetics,probablybyremovinganinhibitorysignaltotheVLPOnucleus.DestructionofserotonergicRNneuronswiththetoxin5,6-dihydroxytryptamineordirectelectrolyticlesionsofserotonergicRNneuronsalsocausehypersensitivitytoanesthetics.不同物的特性并腺素能受體激動劑）LC的去甲腎上腺素能神經(jīng)元滅活，VLPO核去抑制，繼而滅活其它經(jīng)由VLPO的GABA能的中心和甘丙肽能抑制性信號。如丙泊酚和巴比妥類作用在TMN，VLPO去抑制穩(wěn)定的狀態(tài)。藥理和病損實(shí)驗(yàn)改變單胺能網(wǎng)狀激活功能和麻醉敏性于安非他明，都增加大腦中兒茶酚胺水平，對麻醉產(chǎn)生部分抵抗。6羥基多巴胺化學(xué)性消耗或電解破壞去甲腎上腺素，都對產(chǎn)生過敏，可能由于去除VLPO核的抑制性信號。用毒素5,6雙羥色胺或直接電解破壞素神經(jīng)元RN也會引起對的過Thediscoverythatpentobarbitalandmuscimol,aGABAAagonist,causebehavioralandEEGsignsofhypnosiswhenmicroinjectedintoad