Kev nqus pa luv luv ntawm Sevoflurane tuaj yeem txo qis Glial Scar Formation Tom Qab Hypoxic-ischemic Brain Injury hauv Neonatal nas Part 1

Abstract

Cov kev tshawb fawb yav dhau los tau pom tias sevoflurane postconditioning tuaj yeem muab kev tiv thaiv neuroprotection tom qab hypoxic-ischemic raug mob thiab txhim kho kev kawm thiab nco ua haujlwm hauv kev tsim cov hlwb nas.

Hypoxic-ischemic raug mob yog hais txog lub xeev pathological tshwm sim los ntawm cerebral ischemia, hypoxia, metabolic teeb meem, thiab paj hlwb puas vim muaj kev cuam tshuam ntawm cov hlab plawv thiab cerebrovascular systems. Nrog rau kev vam meej thiab kev ua neej hloov pauv, kev raug mob hypoxic-ischemic tau dhau los ua ntau dua. Cov mob no tuaj yeem ua rau muaj kev phom sij ntau rau tib neeg kev noj qab haus huv, suav nrog kev cuam tshuam rau ib tus neeg txawj ntse thiab kev nco.

Txawm hais tias hypoxia thiab ischemia tuaj yeem ua rau lub hlwb tsis ua haujlwm, nws tsis tau txhais hais tias kev nco yuav cuam tshuam tsis zoo. Ntawm qhov tsis sib xws, peb tuaj yeem txhim kho kev nco thiab ua kom lub hlwb ua haujlwm tau zoo los ntawm txoj kev raug. Nov yog qee txoj hauv kev los pab txhim kho koj lub cim xeeb:

1. Noj qab nyob zoo: Qhov cuam tshuam ntawm kev noj haus ntawm kev nco yog qhov tseem ceeb heev. Cov khoom noj uas muaj protein ntau, vitamins, thiab minerals tuaj yeem pab tswj kev noj qab haus huv ntawm lub paj hlwb thiab kev xav.

2. Kev tawm dag zog ntau dua: Kev tawm dag zog lub cev tuaj yeem txhawb cov ntshav ncig, ua kom cov pa oxygen txaus, txhim kho lub cev tiv thaiv kab mob, thiab txo cov kab mob. Los ntawm kev tawm dag zog, koj tuaj yeem txhim kho koj lub peev xwm nco.

3. Ua kom muaj kev ntseeg siab rau tus kheej: Kev ntseeg siab yog qhov tseem ceeb hauv tib neeg lub cim xeeb. Yog tias koj ntseeg tias koj tuaj yeem ua tiav ib txoj haujlwm, koj yuav ua tiav cov txiaj ntsig koj xav tau yooj yim, thiab koj tuaj yeem txhim kho koj lub cim xeeb zoo.

Nws tuaj yeem pom tau tias kev raug mob hypoxic-ischemic tsis yog ib yam mob uas yuav ua rau tib neeg nco tsis tau. Tsuav peb paub cov hau kev kom raug thiab saib xyuas peb lub hlwb kom zoo, peb tuaj yeem txais kev noj qab haus huv yav tom ntej nrog tus cwj pwm zoo. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche deserticola tuaj yeem txhim kho kev nco zoo vim Cistanche deserticola yog cov khoom siv tshuaj hauv Suav teb uas muaj ntau yam teebmeem, ib qho ntawm kev txhim kho kev nco. Kev ua tau zoo ntawm Cistanche deserticola los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog tannic acid, polysaccharides, flavonoid glycosides, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv los ntawm ntau txoj hauv kev.

Nyem paub 10 txoj hauv kev los txhim kho kev nco

Cov qauv Classical Rice-Vannucci tau siv los ua kom muaj cov kab mob hypoxic-ischemicinjury thiab cov menyuam yug tshiab (hnub yug hnub 7) nas tau kho nrog 2.4% sevoflurane rau 30 feeb tom qab raug mob hypoxic-ischemic.

Peb cov txiaj ntsig tau pom tias sevoflurane postconditioning tau txhim kho kev kawm thiab kev nco ua haujlwm ntawm nas, txo qis astrogliosis thiab glialscar tsim, nce tus naj npawb ntawm dendritic spines, thiab tiv thaiv histomorphology ntawm hippocampus. Mechanistically, sevofluranepostconditioning txo qhov kev qhia ntawm von Hippel-Lindau ntawm hypoxia-inducible yam tseem ceeb -1 thiab nce qhov kev qhia ntawm DJ-1.

Kev txhaj tshuaj ntawm 1.52 ug ntawm hypoxia-inducible yam tseem ceeb -1 inhibitor YC-1 (Lificiguat) rau sab laug sab laug ventricle 30 feeb ua ntej hypoxic-ischemic raug mob thim rov qab cov neuroprotection induced los ntawm sevoflurane. Qhov kev tshawb pom no qhia tias sevoflurane tuaj yeem txo qis astrogliosis hauv hippocampus thiab txo cov kev kawm thiab kev nco tsis zoo uas tshwm sim los ntawm glial caws pliav tom qab raug mob hypoxic-ischemic.

Cov txheej txheem hauv qab no tuaj yeem cuam tshuam nrog kev tswj hwm DJ-1 kev qhia, txo qis ubiquitination ntawm hypoxia-inducible factor-1 , thiab stabilized hypoxia-inducible factor-1 qhia. Txoj kev tshawb no tau pom zoo los ntawm Laboratory Animal Care Committee ntawm Tuam Tshoj Medical University, Tuam Tshoj (pom zoo No. 2016PS337K) thaum lub Kaum Ib Hlis 9, 2016.

Lo lus tseem ceeb: mob hlwb; hlwb; central paj hlwb; hauv vivo; raug mob; qauv; plasticity; nas; rov qab los; rov tsim dua tshiab; kho Suav Tsev Qiv Qeb No. R453; r741; ib. R614 2+1.

Taw qhia

Neonatal hypoxic-ischemic encephalopathy (HIE) yog ib qho teeb meem tshwm sim nyob rau hauv lub sij hawm neonatal tshwm sim los ntawm ntau yam, xws li neonatal asphyxia, intrauterine distress, thiab hyalinemembrane kab mob (Douglas-Escobar thiab Weiss, 2015; Barkhuizen li al., 2017).

HIE yog qhov ua rau cov me nyuam mos tuag thiab yog qhov tseem ceeb ntawm cov kab mob neurological sequelae (Edwards li al., 2010; Descloux li al., 2015). Cuaj caum feem pua ​​​​ntawm HIE cov neeg muaj txoj sia nyob muaj kev ua haujlwm ntev lub paj hlwb, xws li kev kawm, kev paub, thiab lub cev muaj zog, thiab mob vwm (Doi li al., 2012; Davies et al., 2019). Kev kho tam sim no ntawm HIE feem ntau yog tsom rau kev kho cov tsos mob, suav nrog kev siv tshuab ua pa, kho hypotension, ntxiv cov piam thaj, thiab txwv cov dej kom tsawg, tsim nyog.

Txawm li cas los xij, yuav tsum muaj kev kho mob zoo uas muaj peev xwm thim rov qab lossis txo qis kev puas hlwb mus sij hawm ntev (Stankowski thiab Gupta, 2011; Davidson li al., 2015).Raws li ib feem tseem ceeb ntawm kev kawm thiab kev lees paub, hippocampus, suav nrog cov hniav gyrus (DG) , CA1, thiab CA3 cheeb tsam, koom nrog hauv kev hloov pauv thiab sib koom ua ke cov ntaub ntawv peripheralin rau hauv cov hlab ntsha (Morris li al., 2012; Jung li al., 2020).

Lub hippocampus yog hypersensitive rau hypoxic-ischemic (HI) kev puas tsuaj, uas tej zaum yuav provoke apoptosis ntawm hippocampalneurons, txhawb ntau reactive gliosis, thiab ua rau glialscar tsim (Hopkins thiab Haaland, 2004; Wang li al., 2012). Kev loj hlob ntawm astrocytes yog qhov tseem ceeb rau kev sib khi ntawm qhov chaw ntawm infarction, kho cov qauv hippocampus, thiab tswj lub cev tiv thaiv kab mob hauv zos thaum lub sijhawm mob hnyav (Rolls li al., 2009).

Txawm li cas los xij, hypertrophyand glial caws pliav ua rau muaj kev tsis txaus ntseeg neurogenesis, cuam tshuam kev loj hlob ntawm tus txha caj qaum, thiab hamper synapse zoo, ntxiv kev cuam tshuam kev kawm thiab kev paub txog kev ua haujlwm (Yiuand He, 2006; Wanner li al., 2008; Burda thiab Sofroniew, 2014al. , 2014; Shi et al., 2017).

Glial fibrillary acidicprotein (GFAP) thiab chondroitin sulfate proteoglycans, xws li neurocan, yog cov cim kab mob ntawm astrogliosis thiab glialscarring uas nws cov lus qhia tuaj yeem hloov kho los ntawm cov astrocytes siab heev (Pekny thiab Nilsson, 2005; Choudhuryand Ding, 201). Yog li, inhibition ntawm astrogliosis thiab glialscarring tej zaum yuav yog lub hom phiaj kho mob rau HIE thiab nws cov sequelae mus sij hawm ntev.

Daim ntawv thov ntawm sevoflurane tau pom zoo kom txo tau HI raug mob hauv nas. Cov kev tshawb fawb soj ntsuam qhov tseem ceeb ntawm sevoflurane postconditioning (SPC) kom txo qis kev puas tsuaj los ntawm kev txhim kho hypoxia-inducible yam tseem ceeb -1 (HIF-1 ) hauv cov nas laus thiab cov nas me me tau txheeb xyuas ntau lub peev xwm mechanisms (Wang et al., 2019). al., 2019; Du et al., 2020; Liu and Gong, 2020).

Raws li ib qho tseem ceeb ntawm lub cev muaj zog ntawm hypoxia, HIF-1 yog ib qho transcriptionfactor nrog ntau pua lub hauv qab molecules koom nrog inischemic kam rau ua mechanisms, xws li vascular endothelialgrowth factor thiab erythropoietin (Na et al., 2015). Hypoxicpostconditioning tau raug txheeb xyuas kom txo qis astrocyte thiab microglial ua kom tom qab HI nyob rau hauv neonatal nas hlwb (Teo li al., 2015).

Yog li ntawd, peb xav tias HIF-1 tuaj yeem ua rau txo qis kev tsim ntawm glial nti thiab astrogliosis.HIF-1 tuaj yeem muaj nyob hauv cov neurons, yog li nws nrawm nrawm hauv kev qhia thiab khaws cia hauv qab hypoxicconditions. Txawm li cas los xij, thaum cov pa oxygen rov qab mus rau qhov qub, HIF1 kev qhia tsis tuaj yeem txhawb nqa ntawm qhov siab txaus kom tsis tu ncua ua rau cov tshuaj tiv thaiv neuroprotective vim tias cov pa oxygen degradation los ntawm prolyl-hydroxylase proteins thiab tom qab ubiquitylation los ntawm von Hippel-Lindau (VHL) proteins (Berra li al., 2003; Zhang et al., 2018).

DJ-1 (encodedby Park7) tau tshaj tawm tias muaj cov protein tiv thaiv kab mob, tshwj xeeb tshaj yog nyob rau hauv cov xwm txheej oxidative (Aleyasin li al., 2007, 2010).VHL protein lub cev cuam tshuam nrog DJ-1, raws li kev txiav txim siab los ntawm qhov tsis pom zoo ntawm pawg spectrometry screen thiab tau lees paub hauv tus qauv aParkinson's disease (PD). Tsis tas li ntawd, Parsanejad et al.(2014) tau qhia tias DJ-1 inhibits VHL-dependent ubiquitylationand stabilizes HIF-1 qhia.

Yog li, peb xav tias sevoflurane stabilizes HIF-1 protein nyob rau hauv lub hippocampus byupregulating DJ-1 inhibit HIF-1 ubiquitylation, yog li ntawd txhim kho kev kawm mus sij hawm ntev thiab nco muaj nuj nqi.

Txhawm rau tshawb xyuas lub luag haujlwm ntawm HIF-1 hauv kev sib raug zoo ntawm sevoflurane thiab astrogliosis, peb siv YC-1 (Lificiguat), aselective antagonist ntawm HIF-1 , kom tag nrho inhibit HIF-1 qhia ntawm qib post-transcriptional. Nyob rau hauv txoj kev tshawb no, weinvested lub luag hauj lwm ntawm HIF-1ib tug nyob rau hauv neuroprotection ntawm SPCtreated HIE neonatal nas.

Cov ntaub ntawv thiab cov txheej txheem

Tsiaj txhu
Kev tshawb fawb tsiaj tau ua nyob rau theem synaptic ntawm ratdevelopment, uas pib rau hnub postnatal 7 (P7), sib xws rau lub sijhawm loj hlob ntawm tib neeg cov menyuam mos txij li cev xeeb tub mus rau 3 xyoos tom qab yug me nyuam (Jevtovic-Todorovic li al., 2003). Xya-hnub Sprague-Dawley nas uas hnyav 12-16g raug xaiv los ntawm 20 tus nas cev xeeb tub (yuav los ntawm Lub Tsev Kho Mob ntawm Shengjing, Tuam Tshoj Medical University, Suav; txiv neej / poj niam piv, 1: 1).

Txhua tus nas tuaj yeem nkag mus rau hauv dej thiab zaub mov dawb, thiab khaws cia ntawm cov av noo thiab qhov kub thiab txias hauv chav kuaj, nrog 12- teev lub teeb / tsaus ntuj (8 teev sawv ntxov / 8 teev tsaus ntuj). Txhua qhov kev sim tsiaj tau ua raws li National Institutes of Health (Bethesda, MD, USA) Cov Lus Qhia rau Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaum Cai.

Pawg Saib Xyuas Tsiaj Tsiaj ntawm Tuam Tshoj Medical University tau pom zoo cov kev sim piav qhia (Shenyang, Tuam Tshoj; pom zoo No. 2016PS337K) thaum lub Kaum Ib Hlis 9, 2016.

Los ntawm 25 tus nas cev xeeb tub, 249 P7 nas raug xaiv thiab muab faib ua pawg raws li cov lus sib txawv (Wang etal., 2019a). Peb qhov kev ntsuam xyuas zaum kawg suav nrog tag nrho 201 tus menyuam yug tshiab; Qhov kev tuag tom qab kho HI yog 19%.

201 cov nas yug tshiab no tau muab faib ua plaub pawg: sham(n=54), HI (n=54), HI + sevoflurane (HIS) (n=54), thiab HIS+ YC{{ 6}} (n=39) pawg. Nyob rau hauv txhua pab pawg, 24 nas tau siv rau sab hnub poob blot assay, 5 tau siv rau immunohistochemistry thiab Golgi staining, thiab 10 tau siv rau kev coj tus cwj pwm.Lavxias teb sab 45 nas nyob rau hauv sham, HI, thiab HIS pawg tau siv rau reverse transcription polymerase saw cov tshuaj tiv thaiv (RT- PCR).

Kev sim tsim thiab raug tshuaj loog

Tus qauv ua haujlwm HIE tau piav qhia yav dhau los (Zhao etal., 2007; Grandvuillemin li al., 2017). Raws li qhov kev ncua deb ntawm lub qhov quav thiab cov plab hnyuv siab raum, kev sib deev ntawm cov nas tom qab yug me nyuam tau ua tiav. Txhawm rau tsim cov qauv HIE, lub taub hau ntawm txhua tus nas P7 tau muab tso rau hauv cov yeeb nkab pob tshab, uas tau ntim nrog paj rwb ua ntej sevofluranewas tso tawm (Wang li al., 2019a; Xue et al., 2019).

Cov hlab ntsha carotid sab laug ntawm txhua tus nas tau txuas mus tas li, thiab nruab nrab ntawm ob txoj kab, cov hlab ntsha raug txiav; Txhua qhov kev phais tau ua tiav hauv 5 feeb. Tom qab ntawd, cov ratsawoke tsis cuam tshuam los ntawm kev siv tshuaj loog thiab tau muab tso rau hauv lawv niam lub tawb rau 2 teev.Rau kev tswj hwm ntawm SPC, nas tau muab tso rau hauv lub pob tshab (Lingzhi Company, Hangzhou, Tuam Tshoj) txuas nrog asevoflurane vaporizer; ib lub duct tau pab nyob rau hauv qhov cua, hos lwm tus xa cov pa qauv tawm ntawm lub chamber mus rau lub monitor.

Lub chamber tau tso cua nrog 30% O2 thiab 70% N2 rau 2 teev rau pawg sham, thaum 8% O2 thiab 92% N2 tau muab rau 2 teev rau HI pawg. SPC tau teeb tsa tam sim ntawd tom qab HI: 2.4% sevoflurane (1 yam tsawg kawg nkaus alveolarconcentration) tau nqus los ntawm cov nas hauv chav nrog anatmosphere ntawm 30% O2 thiab 70% N2 thiab airflow ntawm 2 L / min rau 30 feeb. Lub pas dej ua cua sov stabilized lub ntsuas kub hauv lub chamber ntawm 37 degree.

Kev tswj hwm tshuaj

Raws nraim 30 feeb ua ntej HI, 1.52 ug ntawm HIF-1 inhibitor (YC1; Sigma-Aldrich, St. Louis, MO, USA) tau txhaj rau sab laug sab ventricle (Paxinos thiab Franklin, 2013) siv ib 5-μLHamilton syringe (Yingweida Technology, Beijing, Suav). Raws li tau piav qhia, YC-1 tau yaj hauv cov kua cerebralspinal ntawm qhov concentration ntawm 0.304 ug/μL (Shen li al., 2012; Na et al., 2015).

Western blot tsom xam

P7 nas raug tshuaj loog los ntawm 2% sevoflurane, thiab cov ntaub so ntswg hippocampal tau muab rho tawm los ntawm cov menyuam yaus ntawm 12, 24, thiab 48 teev, thiab 28 hnub tom qab kev phais. Tom qab rho tawm, cov ntaub so ntswg tau muab tso rau hauv dej khov tam sim ntawd.

Tag nrho cov supernatant proteins raug cais los ntawm kev ntxiv xov tooj cua-immunoprecipitation assayagent (Beyotime, Haimen, Tuam Tshoj). Protein concentrations tau txiav txim siab nrog Ib Cov Khoom Siv BCA (Beyotime).

Tom ntej no, cov qauv raug cais ntawm 12.5% ​​sodium dodecylsulfate-polyacrylamide gels thiab xa mus rau nitrocellulosemembranes. Tom qab thaiv nrog 5% bovine serum albumin (Sigma-Aldrich) ntawm 4 degree, lub membrane tau incubated nrog cov thawj antibodies uas tsim nyog, suav nrog anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1: 2000; Cat# 5174S; CellSignaling) , Danvers, MA, USA), anti-DJ-1 (1:1000;Cat# ab18257; Abcam, Cambridge, UK), anti-VHL (1:5000; polyclonal; Cat# ab77262; Abcam), anti -HIF-1 (1:900;polyclonal; Cat# ab2185; Abcam), anti-neurocan (1:200; monoclonal; Cat# sc-33663; Santa Cruz Biotechnology, Dallas, TX, USA) , anti-postsynaptic density protein 95 (PSD95; 1:1000; monoclonal; Cat. No 3409S; Cell Signaling Technology), antigrowth related protein 43 (GAP43; 1:2000; polyclonal; Cat# 16971-1-AP; Proteintech Biotechnology) , Chicago, IL, USA), thiab anti-GFAP (1: 5000, polyclonal, Cat# ab53554; Abcam).

Tom qab ntawd, blots tau incubated nrog anti-luav IgG (1: 5000; Cat# ZB-2301; Zhongshanjinqiao, Beijing, Tuam Tshoj) oranti-nas IgG (1: 5000; Cat# ZB-2301; Zhongshanjinqiao) rau 2 teev ntawm chav tsev kub.

Protein blots tau pom nrog txhim kho chemiluminescence nrhiav kom pom cov tshuaj tiv thaiv (SuperSignal West Pico; Pierce, Rockford, IL, USA). Protein bandswere quantified nrog ImageJ software (National Institutes of Health). Rau kev tsom xam ntawm western blots, cov ntaub ntawv tau normalized rau GAPDH.

Immunohistochemistry

Nees nkaum yim hnub tom qab kev phais, nas tau muab tshuaj loog siv cov txheej txheem tau piav qhia saum toj no, perfused nrog 4% formalin, thiab lawv lub hlwb raug tshem tawm forimmunofluorescence staining.

Lub hlwb tau muab tso rau hauv 4% paraformaldehyde ntawm 4 degree rau 48 teev, tom qab dehydrated hauv cov qib ethanol series, thiab thaum kawg embeddedin paraffin. Tom ntej no, paraffin-embedded ntaub so ntswg raug txiav rau hauv 2.5-μm-thick seem nrog vibratome, thiab cov txiaj ntsig tau muab khaws cia rau hauv chav tsev kub.

Txhua lub hlwb tau muab faib ua ntu zus, thiab peb lub hlwb tsis sib txuas tau raug xaiv rau NeuN (neuron marker), GFAP (astrocyte marker), thiab neurocan (glial scar marker) staining.Rau ob daim ntawv lo, cov ntaub so ntswg tau incubated nrog ob hom tshuaj tiv thaiv, suav nrog tshis los tiv thaiv. GFAP(1:250; polyclonal; Cat# ab53554; Abcam), luav antigens (1:100; Cat# 12943; Cell Signaling Technology), thiab/los yog nas anti-neurocan (1:200; monoclonal; Cat# sc{{ 10}};Santa Cruz Biotechnology) ntawm 4 degree thaum hmo ntuj nyob rau hauv ib tug humidifiedchamber.

Tom qab ntawd, ntu tau ntxuav nrog 0.1 Mphosphate-buffered saline thiab incubated rau 2 teev nyob rau hauv chav tsev kub nrog rau qhov tsim nyog cov tshuaj tiv thaiv theem nrab, nrog rau cov anti-luv IgG conjugated rau Alexa Fluor-594 (1:200; Lub neej Technologies, Grand Island, NY, USA), anti-nas IgGconjugated rau Alexa Fluor-594 (1:200; Life Technologies), thiab anti-tshis IgG conjugated rau fluorescein isothiocyanate (1: 200; Life Technologies).

Kev suav cov cell nuclei tau ua tiav nrog 4′, 6-diamidino-2-phenylindole (Beyotime) rau 5 feeb.

Ib qho Olympus BX51 microscope (OlympusCorporation, Tokyo, Nyiv) tau siv rau kev soj ntsuam cov seem. Peb qhov kev pom ntawm sab laug hippocampuswere randomly xaiv los ntawm txhua daim rau kev yees duab.ImageJ software tau siv los kwv yees quantitativecolocalization thiab, yog li ntawd, xam Manders 'overlapcoefficient.

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