Distinctive Hu-like Receptors Gene Expression And Glial Response in Different Brain Regions Of Natural Scrapie Part 3
Jun 13, 2024
Peb tau tshawb xyuas qhov kev sib raug zoo ntawm cov kab mob neuropathological ntawm tus kab mob prion thiab TLR gene qhia nyob rau hauv plaub cheeb tsam sib txawv ntawm lub paj hlwb ntawm yaj ib txwm muaj kab mob nrog scrapie. Hauv tag nrho plaub lub paj hlwb, peb tau pom qhov sib txawv tseem ceeb hauv PrPScdeposition, spongiosis, astrogliosis, thiab microgliosis nyob rau hauv cov kab mob tshwm sim tiv thaiv cov yaj.
Tus kab mob Prion, tseem hu ua kab mob ncaj ncees hauv tuam tsev, kab mob noj quav ciab, thiab lwm yam, yog tus kab mob kis tau los ntawm prions. Tus kab mob no kis tau los ntawm kev noj cov zaub mov uas tsis muaj prions. Thaum kis tus kab mob prions, nws yuav ua rau mob hlwb thiab lub paj hlwb puas, thiab cov tsos mob xws li mob taub hau, ntuav, thiab kub taub hau tuaj yeem tshwm sim.
Txawm li cas los xij, thaum ntsib qhov xwm txheej zoo li no, peb tsis tuaj yeem poob kev ntseeg siab hauv peb lub cim xeeb vim tias muaj tus kab mob prion. Cov neeg mob feem ntau tuaj yeem rov qab tau thiab rov qab nco tau tom qab kho.
Tau kawg, los ntawm kev tiv thaiv kev xav, peb tuaj yeem sim noj zaub mov ntau dua kom tsis txhob muaj kab mob prion. Nyob rau tib lub sijhawm, yog tias koj hnov cov tsos mob, koj yuav tsum tau mus ntsib kws kho mob sai li sai tau txhawm rau tiv thaiv tus kab mob tsis zoo.
Hauv lub neej niaj hnub, peb tuaj yeem sim qee qhov kev qhia nco yooj yim, xws li nyeem ntawv, sau cov npe, teeb tsa cov xwm txheej thiab cov ntaub ntawv, thiab lwm yam los txhim kho peb lub cim xeeb. Cov kev cob qhia no tsis xav tau ntau lub sijhawm thiab lub zog, tab sis lawv tseem ceeb heev rau kev tswj xyuas lub hlwb noj qab haus huv thiab kev nco ua haujlwm zoo.
Nyob rau hauv luv luv, thaum ntsib cov kab mob xws li kab mob prion, peb yuav tsum fim nws zoo, nrhiav kev kho mob thiab kev tiv thaiv kev tiv thaiv, thiab xyuam xim rau kev nco txhua hnub kom peb lub cev noj qab haus huv thiab ua hauj lwm zoo. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche tuaj yeem txhim kho kev nco zoo vim tias nws tuaj yeem tswj hwm qhov sib npaug ntawm cov neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob, uas tseem ceeb heev rau kev nco thiab kev kawm. Tsis tas li ntawd, Cistanche tseem tuaj yeem txhim kho cov ntshav khiav thiab txhawb nqa cov pa oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov khoom noj txaus thiab lub zog, yog li txhim kho lub hlwb tseem ceeb thiab kev ua siab ntev.
Nyem paub txoj hauv kev los txhim kho lub hlwb
Cov kev sib txawv no tau pom tseeb tshaj plaws hauv medulla oblongata, feem ntau caudalarea. Los ntawm qhov sib txawv, qhov mob me tshaj plaws tau pom nyob rau hauv frontal cortex, feem ntau rostralarea. Cov qauv sib txuas no tau pom zoo nrog txoj hauv kev ntawm prion neuroinvasion thiab kev tshaj tawm thoob plaws hauv CNS, pib ntawm qhov chaw nkag hauv tus txha caj qaum thiab obex thiab thaum kawg mus txog lub frontal cortex [1,5,51].
Kev npaj txhij txog, txiav txim siab txog lawv qhov kev sib thooj, peb tau pom qhov txo qis hauv PrPSc deposition thiab vacuolization, thiab qhov poob qis hauv astrogliosis, thaum txav los ntawm thalamus mus rau hippocampus, tsis ua raws li kev maj mam caudo-rostral kev loj hlob ntawm pathology.
Ib qho tshwj xeeb microgliamorphology hu ua qws microglia tau pom nyob rau hauv qhov sib thooj ntawm pyramidal cellneurons ntawm CA3 tab sis tsis tuaj ntawm tag nrho lwm thaj chaw neuroanatomic kawm.
Toour kev paub, qhov microglial profile no tsis tau cuam tshuam nrog neuroinflamation tshwm sim los ntawm prions rau hnub tim, txawm hais tias kev tshawb fawb tsis ntev los no tau tshaj tawm tias muaj tus pas nrig-shapedmicroglia hauv cerebellum ntawm tib neeg cov neeg mob nrog CJD [52].
Txawm hais tias me ntsis tau kawm txij li thawj cov lus piav qhia hauv xyoo 1900s, rod microglia tau raug tshaj tawm tsis ntev los no hauv cov kab mob neurological xws li qaug dab peg, Lewy lub cev dementia, Huntington's kab mob, thiab AD, tshwj xeeb hauv cov cheeb tsam me me ntawm lub paj hlwb cortex thiab hippocampus [40,41,53]. Thephenotypic qhia thiab kev ua haujlwm ntawm pas nrig microglia tseem tsis tau meej meej.
Txawm li cas los xij, qhov tseeb tias qhov no morphological feem ntau coincides nrog lub xub ntiag ntawm neuronal cov ntsiab lus uas puas los yog tsis yooj yim rau kev puas tsuaj qhia lub luag hauj lwm neuroprotective [42,54,55].
Qws microglia tsis xav tias yuav cuam tshuam nrog cov kab mob hnyav, vim tias qhov kev loj hlob mus rau ameboid morphology yuav tsum tau nyob hauv cov xwm txheej no [56]. Lub xub ntiag ntawm rodmicroglia nyob rau hauv lub hippocampus ntawm scrapie-mob yaj yaj, tab sis tsis nyob rau hauv lwm lub paj hlwb cheeb tsam, tej zaum yuav qhia tau hais tias ib tug neuroprotective ib puag ncig, nyob rau hauv kev pom zoo nrog lub limitedneuronal poob nyob rau hauv lub cheeb tsam no.
Peb qhov kev soj ntsuam ntawm TLR qhia qhia txog kev sib raug zoo ntawm qhov mob hnyav thiab TLR gene overexpression uas coincides nrog theaforementioned caudo-rostral kev loj hlob ntawm prion neuropathology.
Hloov pauv, TLR4 zoo ib yam dhau los ntawm txhua qhov chaw, tsis hais qhov mob hnyav npaum li cas. TLR4 overexpression nyob rau hauv cov cheeb tsam ntawm me me neuronal puas (piv txwv li, lub hippocampus thiab frontal cortex) tej zaum yuav muaj kev cuam tshuam lub luag hauj lwm aneuroprotective, raws li yav tas los piav rau phagocytic hlwb xws li macrophages andmicroglia [23,57].Qhov upregulation ntawm TLR7 tau yav tas los qhia nyob rau hauv nas qauv ntawm priondisease thiab tib neeg cov neeg mob [22,26,31].
Peb tsuas yog pom qhov kev tswj hwm ntawm TLR7 nyob rau hauv medulla oblongata, qhov chaw puas tsuaj tshaj plaws nyob rau hauv qhov siab tshaj plaws ntawm spongiosis thiab PrPSc deposition tau kuaj pom.
Neuronal kev tuag ntawm apoptosis tau yav dhau los txuam nrog TLR7 stimulation [58,59], thiab nws overexpression nyob rau hauv lub medulla oblongatamay muaj feem xyuam rau cov txheej txheem no [60]. Txawm hais tias lub luag haujlwm ntawm TLR1 hauv cov kab mob neurodegenerative tseem tsis tau meej meej, TLR2 kev koom tes hauv microglial activation tau nce ntxiv hauv amyotrophic lateral sclerosis, MS, thiab AD [61,62].
Thaum lub luag hauj lwm ntawm TLR2in prion kab mob tsis tau nkag siab tag nrho, cov txiaj ntsig tau raug npaj: lub sijhawm muaj sia nyob tau txo qis hauv cov nas uas tsis qhia TLR2 tom qab intracerebral inoculation withscrapie [22].
TLR2 thiab MyD88 overexpression kuj tseem yog qhov qhia tau hais tias muaj tus kab mob proinflammatorymicroglia phenotype, uas tuaj yeem piav qhia txog qhov nce hauv TNF- thiab IL-6 uas peb tau pom thalamus [8,63].
Interestingly, thwmsim siv EOC 13.31 hlwb, ib tug immortalizedmicroglia-zoo li nas cell kab, tau qhia ib tug dysregulation ntawm tus kab mob inflammatory teb nyob rau hauv teb rau TLR2 activation thiab qhia ib tug kev sib txuas ntawm TLR2 qhia thiab tsub zuj zuj ntawm microglia nyob rau hauv lub xeev tsis zoo rau phagocytosis [26] .
Yog li ntawd, TLR2 overexpression nyob rau hauv lub medulla oblongata thiab lub thalamus, lub feem ntau puas cheeb tsam nyob rau hauv uas ntau heev theem ntawm phagocytic microglia kuj tau kuaj pom, qhia hais tias lub dysfunctional clearance ntawm PrPSc yuav ua rau kom sustained tsub zuj zuj ntawm PrPSc thiab neuronaldamage [9,17].
Peb qhov kev tshawb pom nyob rau hauv lub hippocampus ntawm scrapie-mob yaj yaj qhia ib qho piv txwv piv nrog rau lwm qhov chaw ntawm lub hlwb soj ntsuam, nrog rau downregulation ntawm TLR1, TLR2, thiab MyD88 thiab tsis muaj cytokine alterations.
Qhov no yuav qhia tau tias microglia teb txawv ntawm hippocampus; Tseeb tiag, nws tau pom tias TLR2 tsis muaj peev xwm hauv cov kab lis kev cai hauv cov kab lis kev cai ntawm cov kab mob hauv lub cev, los ntawm cov nas me me (0-3 hnub qub) thiab txhawb nqa nrog neurotoxicpeptide PrP106-126, hloov microglial activation los ntawm neurotoxic mus rau neuroprotectivephenotype [63 ].
Txawm li cas los xij, qhov tseeb ntawm PrP106-126 peptide hauv prion pathology tau raug nug [64]. CD36 yog ib qho txawv ntawm cov qauv kev lees paub receptor, muaj peev xwm paub txog cov ligands uas tau txais los ntawm cov ligands xws li amyloid-forming peptides, uas tau tsim lub luag haujlwm hauv endocytic uptake ntawm cov khoom no [65,66]. Cov receptor no tau cuam tshuam nrog cov txheej txheem proinflammatory microglial [67].
Hauv vitro, stimulation ntawm BV-2 hlwb, ib hom ntawm immortalized microglial cell, nrog PrP106-126 ua rau CD36 upregulation, nce proinflammatory cytokines thiab iNOS thiab TSIS MUAJ ntau lawm [68,69].
Tsis tas li ntawd, kev lees paub ntawm -amyloid peptide los ntawm CD36 ua rau muaj kev sib dhos ntawm ib qho tshiab heterotrimeric complexCD36-TLR4-TLR6 uas ua rau lub cev tsis muaj zog teb [70,71].
Hauv peb txoj kev tshawb fawb, tag nrho cov cheeb tsam tau pom muaj kev txhim kho tseem ceeb ntawm CD36 tshwj tsis yog hippocampus. Lub upregulation ntawm CD36, TLR4, thiab TLR6 nyob rau hauv lub feem ntau puas cheeb tsam, lub medulla oblongata thiab obex, qhia txog kev koom tes ntawm no triad nyob rau hauv ua rau ib tug pro-inflammatory microglial statusin teb rau prion kab mob.
Conversely, qhov tsis muaj CD36 thiab TLR6 overexpression nyob rau hauv lub hippocampus qhia tias qhov no heterotrimer tsis yog tsim, qhia dua ntawm aneuroprotective ib puag ncig nyob rau hauv lub hlwb cheeb tsam.
Nws tseem yuav tsum tau piav qhia tias vim li cas microglia teb txawv ntawm thaj tsam ntawm lub hlwb no.Thaum prion strain-specific cell tropism tuaj yeem txiav txim siab tus qauv ntawm microgliosis thiab astrogliosis, kev tshawb pom tsis ntev los no qhia tias ob qho kev cuam tshuam feem ntau cuam tshuam los ntawm lub hlwb [18,72].
Tsis yog microglia lossis astroglia teb tsis sib xws thoob plaws CNS, thiab cov lus teb tshwj xeeb hauv cheeb tsam no tuaj yeem ua rau muaj kev xaiv tsis zoo ntawm qee lub hlwb hauv cov kab mob prion [16,18].
Nyob rau hauv no hais txog, nws tau raug postulated tias inflammatory teb ntawm microglia rau prion kab mob yog tswj los ntawm sialylation ntawm PrPSc [14,73–75] thiab hais tias PrPSc sialylation yog lub paj hlwb nyob rau hauv cheeb tsam [18]. Tshwj xeeb, qib siab dua ntawm sialylation ntawm PrPSc pom nyob rau hauv hippocampus thiab cortex dua li hauv thalamus thiab lub hlwb, qhia txog lub luag haujlwm tseem ceeb hauv kev xaiv qhov tsis zoo ntawm cov paj hlwb no [18,73,75].
Cov theem siab ntawm PrPSc sialylation nyob rau hauv hippocampus tej zaum yuav txuam nrog deceleratedprion replication, ua rau qhov txawv prion-induced microglial activation nyob rau hauv cheeb tsam no, raws li qhov txo qis ntawm cov cheeb tsam no implied los ntawm peb qhov kev tshawb pom.
Qhov zoo siab, qhov kev tshawb pom yav dhau los qhia tias hippocampus tuaj yeem tiv thaiv los ntawm cov kab mob neurotoxicity hauv cov kab mob CJD ntuj [76,77], thiab cov ncauj lus kom ntxaws txog kev tshawb fawb neuropathological ntawm CJD cov neeg mob tau tshaj tawm cov kab mob me me hauv hippocampus dua li hauv lwm lub hlwb [76].
Tshwj xeeb, cov archicortex, uas feem ntau suav nrog hippocampus, zoo li yuav tsis tshua muaj txiaj ntsig piv nrog lwm thaj chaw cortical hauv CJD [77]. Qhov kev koom tes ntawm hippocampal me me piav qhia hauv ntuj CJD yog pom zoo nrog cov txiaj ntsig tam sim no hauv yaj ib txwm muaj kab mob nrog scrapie.
Interestingly, qhov no ib feem tiv thaiv tsawg kawg yog ob yam kab mob prion tshwm sim nyob rau hauv lub hippocampus, uas yog phylogenetically lub qub cheeb tsam ntawm lub paj hlwb cortex thiab muaj cov feem ntau yooj yim hom ntawm corticaltissue.
Cov kev tshawb fawb ntxiv yuav tsum tau tshawb nrhiav qhov tseeb ntawm qhov kev sib raug zoo no.Nyob rau hauv cov ntsiab lus, peb cov txiaj ntsig tau qhia tshwj xeeb tshaj yog mob neuropathology nyob rau hauv lub hippocampus ntawm ntuj scrapie-infected yaj, tus cwj pwm los ntawm qib qis ntawm spongiosis, PrPScdeposition, thiab astrogliosis tshaj qhov xav tau muab cov caudal-rau. - rostral kis ntawm scrapielesions.
Ntxiv mus, lub xub ntiag nyob rau hauv lub hippocampus ntawm ib tug tshwj xeeb microglial morphology, qws microglia, uas yuav ua tau ib tug neuroprotective lub luag hauj lwm [54], ua ke nrog ib tug txawv patternrecognition receptor (TLR noob thiab CD36) noob qhia profile, ntxiv txawv lub hlwb cheeb tsam los ntawm lwm yam neuroanatomic. thaj chaw nyob rau hauv ntuj scrapie-mob yaj.
Cov kev tshawb pom no qhia txog qib ntawm kev tiv thaiv kab mob tiv thaiv kab mob prion nyob rau hauv lub hippocampus uas tsim nyog rau kev tshawb nrhiav ntxiv.Neurodegenerative kab mob tshwm sim los ntawm cov protein misfolding cuam tshuam nrog lub voj voog tsis zoo ntawm cov kab mob uas muaj xws li misfolded protein ntau, glial activation, thiab tso tawm ntawm glial inflammatory mediators, uas exacerbate protein deposition thiab deposition. neuroinflamation.
Kev cuam tshuam lub voj voog tsis zoo no los ntawm kev tsom mus rau microglia ua kom siv cov tshuaj tshwj xeeb TLR inhibitors ntawm cov kab mob tshwj xeeb yuav ua rau muaj kev cia siab rau kev txwv tsis pub muaj kab mob neuroinflammation ntxiv. Peb qhov kev tshawb pom tau hais txog TLR2 downregulation raws li lub hom phiaj rau txoj hauv kev zoo li no, vim qhov no yuav ua rau muaj kev hloov pauv hauv microglia los ntawm neurotoxic rau aneuroprotective phenotype [63].
Thaum kawg, thaum muaj kev vam meej tau ua tiav hauv kev qhia txog ntau haiv neeg ntawm glialphenotypes siv cov qauv nas ntawm cov kab mob neurodegenerative, txawm tias cov qauv nas ncaj ncees qhia txog qhov tseem ceeb ntawm cov kab mob prion yog qhov teeb meem ntawm qee qhov kev sib cav [78,79].
Peb cov txiaj ntsig tau los ntawm tus qauv tg338 transgenic rov tsim dua cov cim kab mob ntawm cov kab mob prion, suav nrog cov cim PrPSc deposition, neuropil spongiosis, astrogliosis, thiab microgliosis.
Txawm li cas los xij, cov nas kis tau pom qhov tseem ceeb tshaj tawm ntawm TLR1 thiab TLR2 thiab muaj kev cuam tshuam rau TLR7 overexpression. Thaum qhov kev hloov pauv ntawm cov noob no tau piav qhia yav dhau los hauv lwm tus qauv nas kis nrog scrapie [22,26], qhov qauv no sib txawv nrog peb qhov kev tshawb pom hauv lub hlwb ovine. Remarkably, nyob rau hauv nas hlwb, peb pom tsis muaj kev hloov nyob rau hauv qhov kev qhia ntawm TLR4, cov noob uas qhov loj tshaj plaws kev hloov nyob rau hauv qhia tau pom nyob rau hauv cov yaj cov qauv.
Cov kev tshawb pom tsis sib haum no yuav yog qhov tshwm sim ntawm txoj kev sib txawv ntawm kev kis kab mob thiab / lossis prion protein qhia theem hauv tg338 nas [80,81]. Txawm li cas los xij, peb cov txiaj ntsig thaum kawg qhia tias qhov intracerebralinoculation ntawm scrapie nyob rau hauv cov nas loj tg338 tsis rov tsim dua lub cev tiv thaiv kab mob uas tau pom nyob rau hauv cov kab mob scrapie.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Scrapie-Infected thiab Tswj Yaj
Nees nkaum ib tug poj niam Rasa Aragonesa yaj (hnub nyoog 2-6 xyoo) tau suav nrog hauv qhov kev tshawb fawb tam sim no. Txhua tus tau genotyped rau PRNP polymorphisms, raws li yav dhau los tau tshaj tawm [82], thiab tau pom los tso saib ARQ / ARQ genotype. Tswj tsiaj (n=8) tau raug xaiv los ntawm ib pab yaj uas tsis tau tshaj tawm cov xwm txheej.
Scrapie-infected tsiaj (n=13) tau txais los ntawm cov tsiaj uas cuam tshuam nrog scrapie thiab tau kuaj pom los ntawm immunohistochemistry (IHC) ntawm qhov quav mucosa biopsies. Kev kis kab mob tau lees paub los ntawm kev tiv thaiv kab mob tom qab tuag ntawm PrPSc hauv obex raws li cov txheej txheem luam tawm [83].
Cov tsiaj txhu tau raug kho los ntawm kev txhaj tshuaj ntau dhau ntawm barbiturates thiab exsanguination.
Thaum lub sij hawm ntawm euthanasia, tag nrho cov yaj uas muaj kab mob scrapie pom cov tsos mob ntawm ntau yam mob hnyav: qee cov tsiaj txhu pom cov tsos mob tshwm sim xws li pruritus ntawm nraub qaum thiab flanks tom qab digitalstimulation thiab txo qis ntawm lub cev mob, thaum lwm tus pom cov tsos mob zoo xws li kev khawb ntawm lub cev. cov hauv paus hniav, lumbar cheeb tsam, thiab ceg ceg, cov tsos mob ntawm lub paj hlwb nrog rau ataxia thiab lub taub hau tshee, thiab cov hniav sib tsoo, wool poob, thiab hnyav poob [84].
4.2. Kab mob ntawm Tg338 nas
Txhawm rau ntsuas TLR gene qhia hauv lub hlwb ntawm tus qauv murine ntawm scrapie, kaum-lub lim tiam qub tg338 nas (n=8) (overexpressing lub ovine VRQ/VRQ PrPC 8- rau 10-fold [ 85]) wereinoculated nrog lub hlwb lub pas dej los ntawm ib tug tej yam ntuj tso scrapie-infected yaj Rasa Aragonesa txidat lub chaw kho mob theem.
Cov nas tau inoculated nrog 20 uL ntawm scrapie inoculum (diluted2% w/v hauv PBS) rau hauv txoj cai cerebral hemisphere nyob rau hauv isoflurane tshuaj loog. Intracerebralinjections tau ua nrog 50 µL koob txhaj tshuaj thiab koob 25G. Tom qab inoculation, lawv tau txhaj tshuaj subcutaneous ntawm buprenorphine (0.3 mg / kg) rau induceanalgesia.
Raws li kev tswj hwm, tg338 nas (n=8) tau inoculated nrog lub hlwb homogenate los ntawm cov yaj scrapie-negative raws li cov txheej txheem uas tau piav saum toj no. Cov nas raug saib xyuas rau kev txhim kho ntawm cov tsos mob thiab euthanized los ntawm lub ncauj tsev menyuam dislocation thaum cov tsos mob ntawm tus kab mob xws li mob hnyav ataxia thiab tsis muaj peev xwm noj tau tshwm sim.
Cov nas kis nrog cov kab mob zoo-zoo inoculum pom lub sijhawm muaj sia nyob ntawm 187 ± 26 dpi.
4.3. Cov ntaub so ntswg
Cov qauv ntawm CNS tau sau thiab muab faib ua ob ntu; ib qho tau kho nyob rau hauv 10% nruab nrab-buffered formalin rau histopathological thiab immunohistochemicalanalysis, thiab lwm yam tau ncaj qha khov thiab khaws cia ntawm −80 ◦C rau kev tsom xam cov protein orstabilized hauv RNAlaterTM Solution (InvitrogenTM, Waltham, MA, USA) rau RNA extractionand ces khov thiab khov. ntawm -80 ◦C.
4.4. PRNP Sequence
DNA tau muab rho tawm los ntawm cov ntshav kuaj nrog Speedtools Tissue DNA Extraction kit (Biotools, Madrid, Spain) raws li cov chaw tsim khoom cov lus qhia. PCR amplification thiab sequencing tau ua raws li tau piav ua ntej [82].
4.5. Immunohistochemistry
Formalin-taw cov ntaub so ntswg tau ua tiav raws li cov txheej txheem histopathological. Cov ntaub so ntswg tau muab tso rau hauv paraffin, txiav rau hauv 4 µm tuab seem, thiab stained nrog hematoxylin-eosin (HE) rau kev ntsuam xyuas ntawm vacuolation thiab neuropil spongiosis.
IHC rau PrPSc kev kuaj pom tau ua tiav siv nas monoclonal thawj antibodyL42 hauv yaj (1:500 dilution ntawm chav sov rau 30 min) (R-Biopharm, Darmstadt, Lub Tebchaws Yelemees) thiab luav polyclonal antibody R486 hauv cov nas (1: 8000 dilution, hmo ntuj ntawm 4 ◦C)(R. Jackman, unpublished) raws li yav tas los piav [86,87].
Cov tshooj kuj tau txais kev tiv thaiv kab mob rau cov astrocyte marker glial fibrillary acidic protein (GFAP) (1: 500; Dako, Glostrup, Denmark), thiab microglia marker ionized calcium-bindingadaptor molecule 1 (Iba1) (1: 1000; Wako, Richmond. , VA, USA), raws li cov txheej txheem luam tawm [88] .Tag nrho cov kev ntsuam xyuas histological thiab IHC tau ua los ntawm ob tus kws kho tsiaj cov kws kho mob dig muag rau cov ntaub ntawv kho mob.
Kev soj ntsuam ntawm spongiosis thiab PrPSc staining siv tau yog ib nrab-quantitatively ua thiab yoog raws li cov txheej txheem tau piav qhia hauv cov kev tshawb fawb yav dhau los: vacuolation ntawm neuropil thiab perikarya tau qhab nia los ntawm 0 (tsis tuaj) mus rau 5 (ntau heev thiab confluent) [51], Lub teeb liab PrPSc tau ntsuas raws li qhov extentof immunostaining los ntawm 0 (tsis muaj daim ntawv lo) mus rau 5 (cov ntawv sau hnyav hnyav) raws li tau tshaj tawm yav dhau los [89], thiab qhov twg ntawm GFAP thiab Iba1 immunolabelling tau qhab nia ntawm qhov ntsuas xws li {{ 8}} mus rau 5 (0=qaug zog staining; 5=ntau yam tshuaj tiv thaiv kab mob thoob plaws hauv cheeb tsam) raws li tau piav qhia [88]. Plaub cheeb tsam ntawm lub paj hlwb raug soj ntsuam: frontal cortex (Fc) thalamus (Th), hippocampal tsim (Hc), thiab medulla oblongata (Mo), thiab txhua cheeb tsam tau raug soj ntsuam thoob ntiaj teb rau qhov qhab nia thiab cov duab sawv cev raws li qhov ntsuas ± tus qauv yuam kev.
4.6. Western Blot
100 mg ntawm cov ntaub so ntswg ntawm lub hlwb ntawm txhua cheeb tsam ntawm lub hlwb (Fc, Th, Hc, thiab Mo) los ntawm 13 scrapie-infected yaj, 8 nrog cov tsos mob siab heev thiab 5 nrog cov tsos mob tshwm sim, yog homogenized hauv 1 mL ntawm lysis tsis.
Hemiencephalons ntawm 8 tus kab mob thiab 8 controlmice tau homogenized ntawm 10% (w/v) hauv lysis tsis. Cov qauv ntaub so ntswg yog homogenized ingrinding hlab (Bio-Rad, Hercules, CA, USA) siv TeSeEPrecess 48 TM homogenizer (Bio-Rad, Hercules, CA, USA), thiab cov protein ntau tau ntsuas siv cov khoom siv PierceTM BCA Protein Assay (ThermoScientificTM, Waltham, MA, USA) raws li cov chaw tsim khoom cov lus qhia.
Rau kev soj ntsuam PrPres, sib npaug ntawm cov protein ntau los ntawm cov ntaub so ntswg homogenates tau incubated rau 10 min ntawm 37 ◦C nrog cov proteinase K tov, raws li tau piav qhia yav dhau los [90].
Muaj cov qauv kuaj tau raug rau electrophoresis hauv 12% CriterionTM XT Bis-Tris Protein Gel (Bio-Rad, Hercules, CA, USA) thiab pauv mus rau PVDF daim nyias nyias uas raug thaiv rau 1 h nrog 2% tsis muaj roj qhuav mis hauv TBST (Tris- buffered saline nrog 0.1% Tween20).
Rau kev txhaj tshuaj tiv thaiv kab mob, daim nyias nyias tau muab tso rau ib hmos ntawm 4 ◦C nrog Sha31primary antibody (SPI-Bio, Montigny-le-Bretonneux, Fabkis) ntawm qhov concentration ntawm 1 µg / mLfollowed los ntawm 1 h incubation ntawm chav tsev kub (RT) nrog horseradish peroxidase- conjugated anti-nas IgG Secondary antibody (1:5000) (Santa Cruz Biotechnology, Dallas, TX, USA).
Immunoreactivity tau kuaj pom siv cov chemiluminescent substrate ImmobilonCrescendo Western HRP (Merck, Darmstadt, Lub teb chaws Yelemees).Lub TLR4 protein qhia tau soj ntsuam los ntawm cov ntaub so ntswg homogenates tov nrog 2 × Laemmli Sample buffer (Bio-Rad, Hercules, CA, USA) raws li cov chaw tsim tshuaj paus cov lus qhia. .
Plaub caug micrograms ntawm tag nrho cov protein tau loaded rau ib qhov dej, khiav hauv 7.5% CriterionTM TGXTM Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA), thiab pauv mus rau PVDF daim nyias nyias, uas tau raug thaiv rau 2 h nrog 4% bovine serum albumin. (BSA)(Merck, Darmstadt, Lub teb chaws Yelemees) hauv TBST ntawm RT. Cov membranes tau incubated overnightat 4 ◦C nrog luav polyclonal anti-TLR4 antibody (Novus Biological, Minneapolis, MN, USA) ntawm ib tug concentration ntawm 0.5 µg/mL, thiab ces ntxuav thiab incubated nrog tshis antirabbit IgG ( H + L) HRP Secondary antibody (ThermoScientificTM, Waltham, MA, USA) ntawm 1:20,{13}} rau 1 teev ntawm RT.
Blots tau pom raws li tau piav qhia saum toj no. Tom ntej no, cov membraneswere stripped nrog RestoreTM Western Blot Stripping buffer (ThermoScientificTM, Waltham, MA, USA) rau 15 min ntawm 37 ◦C, ntxuav, thiab thaiv dua.
Tom qab ntawd, cov daim nyias nyias tau muab tso rau ib hmos ntawm 4 ◦C nrog nas monoclonal -actin thawj antibody (Santa CruzBiotechnology, Dallas, TX, USA) ntawm 1: 1000, ntxuav, thiab incubated nrog anti-nas m-IgGкBP-HRP theem nrab antibody (Santa Cruz Biotechnology, Dallas, TX, USA) 1 h. Tom qab ntxuav, blots tau tsim raws li tau piav saum toj no.
Densitometries tau ua tiav nrog ImageJ software thiab cov txiaj ntsig tau normalized siv -actin. Cov txiaj ntsig zoo li qub tau sawv cev nrog GraphPad Prism 6.0 (SanDiego, CA, USA).
Cov kev txheeb xyuas txheeb cais los sib piv cov kab mob thiab cov pab pawg tswj tau ua tiav nrog Cov Tub Kawm Ntawv t-test, thiab qhov sib npaug ntawm qhov sib txawv tau txiav txim siab los ntawm Levene'stest siv SPSS software (SPSS Statistics for Windows, Version 17.0, Chicago, IL, USA). Qhov sib txawv ntawm cov pab pawg tau suav tias yog qhov tseem ceeb ntawm * p<0.05.
4.7. RNA Extraction, cDNA Synthesis, thiab Gene Expression
Yaj tag nrho RNA tau muab rho tawm los ntawm 90 mg ntawm cov ntaub so ntswg kuaj los ntawm frontal cortex, thalamus, hippocampus, thiab medulla oblongata. Cov nas hlwb tau muab faib rau hauv nruab nrab thiab tag nrho RNA tau muab rho tawm los ntawm<90 mg obtained from the thalamic area.
Tissueswere homogenized siv TeSeEPrecess 48TM homogenizer (Bio-Rad) nrog RNeasy LipidTissue Mini Kit (Qiagen, Hilden, Lub teb chaws Yelemees) ua ke nrog TURBO DNase (Invitrogen TM, Waltham, MA, USA) kom tshem tawm cov kab mob DNA sib kis tau. RNA concentration tau txiav txim siab spectrophotometrically nrog NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), thiab rau txhua tus qauv, 260/280 thiab 260/230 ratioswere soj ntsuam xyuas cov qauv purity.
Ib microgram ntawm complementary DNA (cDNA) tau tsim los siv qScriptTM cDNA SuperMix (Quantabio BiosciencesTM, Beverly, MA, USA) raws li cov chaw tsim khoom cov lus qhia. Tsis tas li ntawd, qhov ua tau zoo ntawm kev kho DNase tau soj ntsuam hauv cov qauv RT-tsis zoo. Tom qab rov qab cov ntaub ntawv rov qab, tib pawg ntawm diluted cDNA raug rau qPCR los ua kom nrov TLRs.
Ob hom kev tu vaj tse (HK) feem ntau raug xaiv los ua kom qhov kev qhia ntawm cov noob hom phiaj: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) andactin-beta (ACT) [91]. Qhov ruaj khov ntawm HK noob no tau txheeb xyuas raws li peb qhov kev sim. Cov lus qhia RNA (mRNA) tau txiav txim siab los ntawm qPCR rau 1 txog 10 ovine TLR noob, 1 txog 9 murine TLR noob, thiab MyD88 noob rau ob hom.
Twoproinflammatory (TNF- thiab IL-6) thiab ob qho tshuaj tiv thaiv kab mob (IL-10 thiab TGF-) cytokines kuj tau kawm hauv yaj thalamus thiab hippocampus. Primer sequences thiab kev ua tau zoo tau luam tawm yav dhau los lossis tau tsim los siv cov cuab yeej Primer3Plus [92](Table 2).
Peb tau txheeb xyuas qhov ua tau zoo ntawm txhua tus noob tsim cov qauv nkhaus los ntawm kev nthuav dav 1: 2 serial dilutions ntawm kev tswj cDNA thiab tom qab ntawd kuaj xyuas cov kab nruab nrab ntawm qhov pib qauv concentration thiab lub voj voog pib (Ct) qhov tseem ceeb. Tag nrho cov noob tau pom qhov sib txheeb coefficient ntawm 0.9 thiab 0.99, nrog tus nqi nqes hav ntawm tus qauv nkhaus hauv thaj tsam ntawm −3.2 txog −3.5 thiab qPCR efficiency ntawm 90–110%.
Cov tshuaj tiv thaiv qPCR tau khiav siv Applied BiosystemsTM QuantStudioTM 5 RealTime PCR System, 96-zoo nrog rau kev nthuav dav dav dav: qhov pib ua kom zoo thiab cDNA denaturation kauj ruam ntawm 10 min ntawm 95 ◦C, ua raws li 40 lub voj voog ntawm 3 s ntawm 95 ◦C thiab 30 zaum 60 ◦C.
Txhawm rau txheeb xyuas qhov muaj ntawm PCR amplicons lossis qib siab ntawm primerdimers, peb tau ua ib qho kev sib txuas lus nkhaus tom qab txhua qhov qPCR cov tshuaj tiv thaiv. Txhua tus qauv raug txheeb xyuas hauv triplicate hauv tag nrho cov tshuaj tiv thaiv ntim ntawm 10 µL, suav nrog 15 ng ntawm cDNA, 5 µL Fast SYBR Green Master Mix (2X) (ThermoFisher Scientific, Waltham, MA, USA), thiab qhov yuav tsum tau muaj ntawm kev xa mus thiab thim rov qab. primers (Table 2).
Nuclease-dawb dej tau ntxiv rau qhov kawg ntim ntawm 10 µL. Cov theem ntawm cov noob qhia tau txiav txim siab siv txoj kev sib piv Ct. Cov txiaj ntsig tau sawv cev raws li kev hloov pauv thiab qhov sib txawv ntawm cov noob caj noob ces txheeb ze rau qib nruab nrab ntawm pawg tswj hwm tau ntsuas rau 1.
4.8. Cov Ntaub Ntawv Kev Tshawb Fawb thiab Kev Tshawb Fawb
Tag nrho cov ntaub ntawv khaws tseg tau raug sim rau qhov qub nrog Shapiro-Wilk Wtest. Histological thiab immunohistochemical qhov sib txawv ntawm cov kab mob thiab cov pab pawg tswj tau raug soj ntsuam los ntawm Cov Tub Ntxhais Kawm T-test los yog Mann-Whitney U xeem nyob ntawm cov ntaub ntawv parametric lossis nonparametric.
Kev sib txawv ntawm plaub thaj tsam ntawm lub hlwb sib txawv hauv scrapie kab mob-cov yaj tau txiav txim siab siv ib txoj kev ntsuas qhov sib txawv (ANOVA) ua raws li Bonferroni post hoc test lossis Kruskal-Wallis test, nyob ntawm seb cov ntaub ntawv parametric lossis nonparametric faib. Kev txheeb xyuas ntawm cov ntaub ntawv qPCR tau ua los ntawm qhov nruab nrab ∆Ct qhov tseem ceeb rau txhua tus noob.
Rau qhov kev sib piv ntawm cov kab mob thiab cov pab pawg tswj hwm, Cov Tub Ntxhais Kawm T-test lossis Mann-Whitney U test tau ua nyob ntawm qhov kev faib tawm ntawm txhua tus noob, thiab qhov sib npaug ntawm qhov sib txawv tau txiav txim los ntawm Levene qhov kev sim. Qhov sib txawv ntawm kev qhia tau suav tias yog qhov tseem ceeb ntawm p < 0.05.
Cov ntawv sau hauv qab no tau siv los txheeb xyuas p-tus nqi hauv cov duab: * p < 0 05; ** p Tsawg dua lossis sib npaug rau 0.01; # p < 0.1. SPSS (SPSS Statistics for Windows, Version 17.0, Chicago, IL, USA) software tau siv rau kev txheeb xyuas kev txheeb xyuas. Graphs raug generated nrog GraphPad Prism 6.0 (San Diego, CA, USA) thiab cov ntaub ntawv qhia nyob rau hauv cov duab sawv cev qhov txhais thiab tus qauv yuam kev ntawm qhov nruab nrab (txhais tau tias ± SEM).
5. Cov lus xaus
Rau peb txoj kev paub, txoj kev tshawb fawb tam sim no yog thawj zaug piav qhia txog qib ntawm TLRgenes hauv thaj chaw sib txawv ntawm lub hlwb ntawm cov yaj uas muaj kab mob thiab cov kab mob loj tshaj tg338 miceexperimentally kis nrog scrapie.
Peb txoj kev tshawb fawb qhia tau meej meej tias TLRs, thiab tshwj xeeb tshaj yog TLR4 hauv yaj thiab TLR1 thiab TLR2 hauv cov nas, koom nrog cov kab mob ntawm scrapie.Ntxiv rau, sib piv rau lwm thaj chaw kawm, qhov txawv ntawm TLR geneexpression, ua ke nrog cov cim microglial morphology. thiab mob me me neuropathology pom nyob rau hauv hippocampus, qhia txog lub hlwb thaj tsam ntawm lub cev tiv thaiv kab mob hauv cov kab mob naturalscrapie.
Txawm li cas los xij, kev tshawb fawb ntxiv yuav tsim nyog los ua tus cwj pwm TLR kev qhia hauv microglia, astroglia, thiab cov paj hlwb hauv cov kab mob scrapie kom nkag siab txog qhov tseeb ntawm txhua hom cell rau neuroinflammation. Tsis tas li ntawd, nws tseem tsis tau txiav txim siab seb qhov kev ua kom TLR yog ib qho lus teb ncaj qha rau prion toxicity los yog tshwm sim rau lwm yam kev mob.
TLRs ua ib lub hom phiaj zoo rau kev kho cov kab mob toprion, thiab yog li kev nkag siab zoo ntawm TLR-tswj cov tshuaj neuroinflammatory teb yuav xav tau los xyuas kom muaj kev nce qib ntxiv hauv cheeb tsam no.
Cov Khoom Siv Ntxiv: Cov hauv qab no muaj nyob online ntawmhttps://www.mdpi.com/article/10.3390/ijms23073579/s1.
Sau Kev Pabcuam: Kev xav, kev tshawb fawb, thiab kev kho cov ntaub ntawv, MCG thiab LMC; kev sau ntawv-kev npaj ua ntej, MG-M., MCG, thiab LMC; Kev sim txheej txheem, MG-M.; sau- tshuaj xyuas thiab kho, MG-M., MCG, LMC, thiab AO; hauv vivo methodology, MB; kev nrhiav nyiaj txiag, RB, thiab JJB; Molecular Methodology, BS-P.; Kev saib xyuas, MCG, thiab JJB Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.
Cov Nyiaj Txiag: Qhov kev tshawb fawb no tau txais nyiaj los ntawm "Departamento de Ciencia, Universidad y Sociedad delConocimiento" (Aragon Government) los ntawm txoj haujlwm "A05_20R: Enfermedades Priónicas, Vectoriales y Zoonosis Emergentes".
Institutional Review Board Statement: Tag nrho cov txheej txheem uas muaj cov tsiaj ua raws li cov lus qhia suav nrog hauv Spanish Txoj Cai Lij Choj rau Kev Tiv Thaiv Tsiaj RD53/2013 thiab European Union Cov Lus Qhia 2010/63 txog kev tiv thaiv tsiaj txhu siv rau kev sim. Cov txheej txheem tau txais kev pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm University of Zaragoza (Tshaj Ntawv Tso Cai: PI38/159 Lub Kaum Hli 2015 thiab PI19/14 11 Lub Plaub Hlis 2014).
Cov Lus Qhia Txog Kev Pom Zoo: Tsis siv tau.
Cov Lus Qhia Txog Kev Muaj Peev Xwm: Cov ntaub ntawv qhia hauv qhov kev tshawb fawb no muaj nyob rau hauv cov ntawv nyeem, cov duab, thiab cov khoom siv ntxiv.
Kev lees paub: Peb ua tsaug Olivier Andreoletti (UMR INRAEENVT 1225-IHAP) thiab VincentBeringue (UMR VirologieImmunologieMoleculaires (VIM-UR892), INRAE, Universite Paris-Saclay) rau siab zoo muab tg338 nas siv rau cov kev sim no.
Kev tsis sib haum xeeb ntawm kev txaus siab: Cov neeg sau ntawv tshaj tawm tsis muaj teeb meem ntawm kev txaus siab. Cov nyiaj txiag tsis muaj lub luag haujlwm hauv kev tsim qauv ntawm kev kawm; hauv kev sau, txheeb xyuas, lossis txhais cov ntaub ntawv; nyob rau hauv kev sau ntawv ntawm cov ntawv sau, lossis hauv kev txiav txim siab tshaj tawm cov txiaj ntsig. Cov duab thiab cov duab hauv phau ntawv no yog cov ntaub ntawv qub.
Cov ntaub ntawv
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