Quantification Ntawm Neurite Degeneration Nrog Txhim Kho Qhov Tseeb Thiab Ua Tau Zoo hauv Tus Qauv Hauv Vitro Ntawm Parkinson's Disease Part 2

Cov txiaj ntsig

Tsim kom muaj tus qauv kab lis kev cai ntawm tes ntawm neurite degeneration txuam nrog PD

Txhawm rau tsim ib txoj hauv kev tshawb xyuas neurite degeneration hauv cell kab lis kev cai qauv ntawm PD, peb siv cov kab ke LUHMES, cov pejxeem ntawm cov neeg tsis txawj tuag, tib neeg cov hlwb mesencephalic. Raws li kev sib txawv, LUHMES hlwb dhau los ua postmitotic, txais yuav cov noob qhia cov yam ntxwv ntawm cov dopaminergic neurons paub tab, thiab tsim cov neuronal morphology nrog neurites uas ntev txog li 500 hli (Fig. 1A; Lotharius li al., 2005; Scholz li al. , 2011; Kraemer et al., 2021).

Neurodegeneration, ib yam mob uas cov paj hlwb hloov los yog tuag, muaj feem xyuam nrog kev nco. Hauv cov qauv kab lis kev cai ntawm tes, peb tuaj yeem tswj cov kab lis kev cai kom txo qis kev qhia ntawm neurodegeneration thiab yog li txhim kho kev ua haujlwm ntawm kev nco.

Cov kev tshawb fawb tau pom tias homeostasis ntawm paj hlwb ua lub luag haujlwm tseem ceeb hauv kev ciaj sia ntawm tes thiab lub cev ua haujlwm. Peb tuaj yeem txhim kho kev ruaj ntseg ntawm cov hlwb los ntawm kev tswj cov khoom xws li cov as-ham thiab kev loj hlob ntawm cov kab lis kev cai nruab nrab. Nyob rau tib lub sijhawm, ua tib zoo saib xyuas kev huv thiab ntsuas kub ntawm cov kab lis kev cai ib puag ncig kom tiv thaiv cov hlwb los ntawm kev cuam tshuam thiab puas los ntawm ib puag ncig.

Tsis tas li ntawd, nyob rau hauv cov qauv kab lis kev cai ntawm tes, peb kuj tuaj yeem txhaj tshuaj qee qhov sib txuas lossis tshuaj rau hauv lub hlwb kom txo qis kev qhia ntawm neuromodulation, yog li txhim kho cov ciaj sia taus thiab kev nco ua haujlwm ntawm cov hlwb. Cov txheej txheem no kuj tau siv dav hauv kev kawm txog cov kab mob neurodegenerative.

Nyob rau hauv luv luv, neurodegeneration muaj ib qho tseem ceeb cuam tshuam rau cell stability thiab system functionality, thiab cell kab lis kev cai technology yog ib tug zoo qauv kev tshawb fawb txoj kev, uas yuav ua tau nyob rau hauv-tob tshawb nrhiav thiab kev tshawb fawb ntawm kev sib raug zoo ntawm neurodegeneration thiab nco. Peb tuaj yeem txo qhov kev ua tau zoo neuromodulation los ntawm kev cog qoob loo ib puag ncig, txhaj tshuaj rau hauv cov hlwb, thiab lwm yam, txhawm rau txhim kho cell ciaj sia taus thiab nco kev ua tau zoo thiab muab lub hauv paus zoo rau kev kawm txog cov kab mob neurodegenerative. Nws pom tau tias peb yuav tsum txhim kho peb lub cim xeeb. Cistanche tuaj yeem txhim kho peb lub cim xeeb zoo, vim Cistanche 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, cov nqaij kuj 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 qhov tseem ceeb thiab kev ua haujlwm ntawm lub hlwb.

Nyem paub ntxiv los txhim kho kev nco

Vim tias qhov zoo tshaj plaws kho qhov muag ntawm iav piv nrog cov yas, cov kab lis kev cai tau tsim nyob rau hauv cov iav slides uas muaj yim chav xov xwm. Raws li cov ntaub ntawv dhau los (Lotharius li al., 2005; Scholz li al., 2011; Kurowska et al., 2014), peb pib sim tsim kab lis kev cai ntawm chamber slides coated nrog PLO thiab fibronectin; Txawm li cas los xij, cov kab lis kev cai no tau nthuav tawm qhov adhesion tsis txaus thiab tsis muaj kev vam meej. Raws li qhov tshwm sim, peb tau sim seb puas yog txheej txheej nrog cov substrates ntxiv yuav txhim kho qhov muaj txiaj ntsig ntawm LUHMES cell kab lis kev cai tsim los ntawm cov iav nto.

LUHMES cell kab lis kev cai tau tsim nyob rau hauv cov slides uas yog uncoated los yog coated nrog PLO thiab fibronectin, PDL thiab laminin, los yog ib tug ua ke ntawm tag nrho plaub substrates. Kev soj ntsuam ntawm cov nuclei siv tau los ntawm fluorescence microscopy tau qhia tias cov txheej txheem swb tau cuam tshuam rau kev muaj peev xwm ntawm cov kab lis kev cai [Friedman x2 (3)=18, p, 0.0001], nrog rau ntau qhov kev sib piv kev soj ntsuam qhia pom tias cov slides coated nrog kev sib xyaw ua ke ntawm tag nrho plaub lub substrates uas tau hais los saum no ua rau muaj txiaj ntsig ntau dua ntawm cov hlwb siv tau thaum piv nrog cov swb tsuas yog coated nrog PLO thiab fibronectin (p= 0.0437; Fig. 1B, C).

Tom qab txheeb xyuas qhov zoo tshaj plaws txheej substrates rau culturing LUHMES hlwb ntawm iav slides, peb soj ntsuam cov teebmeem ntawm cell ceev ntawm qhov haum ntawm cov kab lis kev cai rau neurite degeneration ntsuas. LUHMES cov hlwb raug coj los rau hauv qhov sib txawv nruab nrab nrog cov plating ntom ntawm 57,500 hlwb ntawm ib qhov dej, 115, 000 cov hlwb hauv ib qhov dej, lossis 230, 000 hlwb ib qhov zoo, thiab nyob rau hnub thib tsib ntawm kev sib txawv, cov hlwb tau kho nrog 4% PFA thiab duab los ntawm theem-qhov sib piv microscopy.

Qhov ceev ntawm 57,500 hlwb ib qho zoo ua rau cov kev coj noj coj ua thiab cov kab lis kev cai tsis zoo ntawm 230, {} Cov neeg neurities, muaj cov neurite, muaj kev ntsuas tsis zoo rau kev ntsuas. Qhov nruab nrab ntawm tes ntom ntom ntawm 115,000 cov cell ib qhov zoo muab kev muaj peev xwm zoo thiab sib cais axon tracts uas tsim nyog rau kev ntsuas qhov tseeb ntawm neurite degeneration (Daim duab 1D).

Txhawm rau ua qauv PD-koom nrog neurite degeneration hauv vitro, peb nthuav tawm cov hlwb sib txawv ntawm LUHMES rau 6-OHDA, siv dav neurotoxin uas txhawb nqa oxidative kev nyuaj siab thiab degeneration ntawm catecholaminergic neurons (Bové thiab Perier, 2012). Kev nthuav tawm ntawm cov hlwb sib txawv ntawm LUHMES rau 6-OHDA txhawb kev nce koob tshuaj ntawm cov neurite degeneration, nrog 5.0 mM 6-OHDA thiab 7.5 mM 6-OHDA inducing nruab nrab thiab loj neurite degeneration, feem (Daim duab . 2F). Txhawm rau kom muaj nuj nqis neurite degeneration nyob rau hauv kab lis kev cai ntawm LUHMES hlwb raug rau 6-OHDA, peb siv ib txoj kev hais dav dav nyob rau hauv uas theem-zoo sib xws dluab ntawm kab lis kev cai yog binarized, cell lub cev nyob rau hauv cov duab yog digitally tshem tawm, thiab particle analyzer algorithm ntawm ImageJ yog siv los ntsuas thaj tsam ntawm neurite fragments. Cov cheeb tsam neurite uas tau tawg yog tom qab ntawd suav nrog thiab muab faib los ntawm tag nrho thaj chaw neurite los txiav txim siab DI (Fig. 2A–E; Sasaki li al., 2009; Kraemer et al., 2014; Di Stefano et al., 2015; Hill et al. ., 2018; Geisler et al., 2019).

Raws li qhov xav tau, qhov sib txawv ntawm LUHMES hlwb rau 6- OHDA ua rau muaj kev nce koob tshuaj nyob hauv DI (F(3,24)= 119.8, p=0.0001, R{ {8}}.9374; Fig. 2G). Txawm li cas los xij, thaum kev txheeb xyuas cov hlwb raug rau 6-OHDA, peb tau pom ntau qhov teeb meem uas tshwm sim los cuam tshuam qhov raug thiab ua tau zoo ntawm DI ntsuas. Yog li, peb tau nrhiav kev ua kom zoo dua qhov kev ntsuas no rau kev ntsuas DI, uas peb tam sim no xa mus rau txoj kev DI ib txwm muaj, los tsim cov txheej txheem ua kom muaj txiaj ntsig zoo dua ntawm kev ntsuas ntawm neurite fragmentation.

Txo cov khoom siv dag zog cuam tshuam nrog micrograph binarization

Thaum siv cov txheej txheem DI ib txwm muaj, peb nquag pom ib qho teeb meem uas cov neurites tsis zoo nyob rau hauv cov duab theem sib piv tau tshwm sim hauv lawv cov duab binarized sib raug (Fig. 3A). Qhov kev sib cais no feem ntau cuam tshuam rau neurites nrog qhov tsis tshua muaj txiaj ntsig. Yog li, peb tau tshawb xyuas seb qhov teeb meem puas tuaj yeem raug txo los ntawm cov txheej txheem uas txhim kho cov duab sib piv. Txhawm rau txhim kho cov duab sib piv, peb thawj zaug ntes cov duab ntawm neurites siv ntau lub teeb siv los txiav txim siab qib ntawm cov duab ci uas yuav ua rau pom qhov sib txawv.

Cov duab uas tau ntes nrog lub teeb pom kev zoo yog tom qab ntawd raug rau qhov sib txawv ntxiv los ntawm kev siv ImageJ los siv LUT nrog qhov tsawg kawg nkaus thiab qhov siab tshaj plaws ntawm 90 thiab 205, feem. Kev sib koom ua ke ntawm cov kev sib txawv no tau muab qhov piv txwv muaj tseeb ntawm cov neurites hauv cov duab binarized tsim los ntawm cov duab theem sib piv (Fig. 3B). Txawm li cas los xij, kev txheeb xyuas ntau ntawm cov duab binarized ntawm cov neurites noj qab haus huv tau qhia tias qhov kev sib txawv ntawm kev txhim kho tsis tau txo qis kev puas tsuaj (p=0.9999; Fig. 3D). Yog li, peb txuas ntxiv mus tshawb xyuas seb puas siv cov duab immunofluorescence yuav tsim cov duab nrog qhov sib txawv zoo dua thiab yog li pab txhawb kev tsim cov duab binarized neurite ntau dua nrog txo cov khoom tawg.

Cov slides piav nyob rau hauv daim duab 2, featuring tsau LUHMES hlwb raug mus rau ntau yam concentrations ntawm 6- OHDA, twb immunostained rau lub cytoskeletal protein b III-tubulin, duab siv fluorescence microscopy, thiab soj ntsuam los ntawm ib tug dig muag soj ntsuam rau dag fragmentation (Fig . 3C). Kev txheeb xyuas ntau ntawm cov duab binarized tau los ntawm fluorescence micrographs thiab cov theem sib piv micrographs tau qhia tias hom microscopy cuam tshuam rau qib ntawm cov khoom sib cais [Friedman x2 (3)=15.44, p, 0.0001] . Tshwj xeeb tshaj yog, fluorescence micrographs tsim cov duab binarized nrog txo qis cov khoom sib xyaw ua ke piv nrog cov ntawm cov theem sib piv micrographs (p=0.0016; Fig. 3D).

Yog li ntawd, cov duab ntawm cov kab lis kev cai noj qab haus huv tau ua rau cov qhab nia DI qis dua thaum siv cov fluorescence micrographs es tsis yog theem sib piv micrographs (F(1,8)= 131.4, p=0.{{12} }001, R2=0.1442; Fig. 3E). Tsis tas li ntawd, ib qho tseem ceeb tag nrho yog qhov hloov pauv ntawm qhov txhais tau tias DI qhov tseem ceeb cuam tshuam nrog kev noj qab haus huv (tsheb-kho) kab lis kev cai thiab kab lis kev cai nthuav tawm neurite degeneration (5.0 lossis 7.5 mM 6-OHDA-kho) tau ntau dua thaum ntsuas siv fluorescence micrographs dua li thaum tau los ntawm theem sib piv micrographs (Fig. 3E). Qhov no tshwm sim txawm hais tias fluorescence micrographs ntawm kab lis kev cai nrog nruab nrab neurite degeneration (vim los ntawm raug rau 5 mM 6-OHDA) muaj cov qhab nia DI qis dua li cov duab theem sib xws vim tias txo qis ntawm cov khoom sib cais (p=0.0001 , d=0.6312).

Optimizing parameters rau fragmented neurite thiab tag nrho neurite nrhiav kom tau
Cov txheej txheem ib txwm ua rau kev ntsuas DI suav nrog kev siv Analyze Particles plugin rau ImageJ nrog qhov loj me ntawm 20 10, 000 pixels txhawm rau txheeb xyuas cov neurite fragments. Txawm li cas los xij, peb tau pom tias feem ntau ntawm cov khoom neurite tsis raug kuaj pom los ntawm cov particle analyzer thaum siv cov kev txwv dav dav no (Shin et al., 2012; Di Stefano et al., 2015; Sasaki et al., 2016; Loreto et al. , 2020; Shin thiab Cho, 2020) los ntsuas neurite degeneration nyob rau hauv theem-zoo sib xws micrographs ntawm LUHMES hlwb raug rau 6-OHDA, generated raws li tau piav nyob rau hauv daim duab 2.

Yog li, peb rov txheeb xyuas cov duab los ntawm cov ntaub ntawv ntawd siv qhov sib txawv qhov tsawg kawg nkaus txwv rau kev kuaj pom cov kab mob los txiav txim siab qhov tsis zoo rau kev txheeb xyuas cov kab mob neurite. Los ntawm kev soj ntsuam zoo ntawm cov duab binarized tsim los ntawm theem-qhov sib piv micrographs, qhov loj me ntawm 5–10, 000 pixels txhim kho kev tshawb pom ntawm neurite fragments, tab sis kuj tseem cuam tshuam nrog kev tsis ncaj ncees ntawm lub suab nrov tom qab, cov duab kos duab, lossis lwm yam me me. cov khoom uas tsis yog neurite li neurite fragments. Piv nrog rau cov kev siv dav dav ntawm 20–10, 000 pixels, txawm li cas los xij, qhov tsis muaj 10–10, 000 pixels txhim kho qhov kev tshawb pom ntawm neurite fragments yam tsis muaj qhov tshwm sim loj zuj zus hauv qhov tsis zoo (Fig. 4A , sab laug).

Cov kev soj ntsuam zoo sib xws kuj tau ua nrog cov duab binarized tau los ntawm fluorescence micrographs ntawm LUHMES hlwb immunolabeled rau b III-tubulin. Particle analyzer loj qhov ntsuas ntawm 5–10,000 pixels tau yooj yim rau kev tshawb nrhiav pom cov khoom neurite tshaj plaws thiab, txij li cov fluorescence micrographs muaj cov khib nyiab me me, cov kev ntsuas no tsis pom tseeb ua rau qhov tsis zoo ntawm cov khoom tsis yog neurite li neurite fragments. (Daim duab 4A, sab xis).

Cov txheej txheem ib txwm ua rau kev ntsuas DI, los ntawm kev siv cov khoom ntsuas particle loj ntawm 20– 10, 000 pixels, tsis suav cov khoom me dua 20 pixels los ntawm kev ntsuas cov khoom neurite.

Txawm li cas los xij, cov khoom me me no feem ntau ua yuam kev suav nrog tag nrho cov neurite cheeb tsam kev ntsuas, txij li txoj kev ntsuas DI ib txwm suav nrog suav tag nrho thaj chaw neurite los ntawm kev suav tag nrho cov pixels dub hauv cov duab binarized. Txhawm rau kov yeej qhov teeb meem no, peb siv Particle Remover plugin rau ImageJ, sau los ntawm ImageJ tus tsim tawm Wayne Rasband, tshem tawm cov khoom me me, tsis yog neurite los ntawm cov duab binarized. Daim ntawv thov ntawm cov plugin no thaum ua DI ntsuas siv cov theem sib piv micrographs ntawm LUHMES cov hlwb raug rau 6-OHDA, tsim tawm raws li tau piav qhia hauv daim duab 2, ua rau muaj qhov nce ntxiv hauv DI ntsuas thoob plaws txhua qhov kev tawm tsam neurotoxin (F(1 ,8)=83.43, p=0.0001, R2=0.0416).
Tsis tas li ntawd, ib qho kev cuam tshuam tseem ceeb tau qhia tias qhov nce hauv DI tshwm sim los ntawm kev siv cov khoom tshem tawm tau ua kom muaj zog ntawm cov ntsiab lus siab dua (F(1.881,15.05)=59.97, p=0.0001, R{ {8}}.0094; Fig. 4B, C). Peb kuj tau teeb tsa tib lub swb ntawm cov hlwb los ntawm fluorescence microscopy thiab tshuaj xyuas qhov cuam tshuam ntawm kev tshem tawm me me ntawm DI ntsuas tau los ntawm fluorescence micrographs. Txawm hais tias qhov kev tshem tawm me me muab DI qhov tseem ceeb uas yog qhov tseeb dua, qhov txiaj ntsig ntawm qhov nruab nrab tsis txawv ntawm qhov tau txais yam tsis tau siv cov khoom tshem tawm cov khoom siv thaum ntsuas siv fluorescence micrographs (F(1,8)=2. 613, p=0.1446, R2=0.0013; Fig. 4D).

Automation ntawm DI ntsuas los ntawm fluorescence micrographs

Kev ua tau zoo ntawm DI kev ntsuas uas siv cov txheej txheem ib txwm siv yuav tsum tau ua cov duab tedious thiab siv sij hawm, siv cov cuab yeej freehand kom tshem tawm ib tus neeg lub cev ntawm tes thiab ntsuas cov fragmented neurite thiab tag nrho cov neurite thaj chaw ntawm txhua daim duab. Yog li, peb tau nrhiav kev txhim kho qhov ua tau zoo ntawm txoj kev los ntawm kev sau ib qho macro uas ua tiav DI analyses tag nrho. Txhawm rau automate soma tshem tawm, macro, lub npe ANDI, ua tiav binarization ntawm cov duab ntawm tes nuclei, sau los ntawm nuclear stain DAPI, ua raws li kev hloov kho ntawm cov duab binarized xws li cov cheeb tsam nuclear tau nthuav dav kom suav nrog tag nrho cheeb tsam ntawm soma. Cov duab zoo li no nrog binarized thiab loj nuclei raug rho tawm los ntawm cov fluorescence micrographs sib thooj ntawm cov neurons immunolabeled rau b III-tubulin, tsim cov duab tshwj xeeb uas muaj cov neurites (Fig. 5A, B).

Txhawm rau kho qhov sib txawv ntawm qhov ntau thiab tsawg los ntawm nuclear shrinkage lossis fragmentation cuam tshuam nrog neurodegeneration, binarized dluab ntawm nuclei yog loj los ntawm kev sib xyaw ntawm dilations thiab erosions uas ua ke nuclear fragments thiab nthuav binarized cheeb tsam nyob rau thaj tsam loj dua lub somas ntawm txawv LUHMES hlwb. . Cov duab tshwj xeeb uas muaj cov neurite yog tom qab ntawd binarized thiab raug rau DI tsom xam los ntawm kev ua haujlwm tsis siv neeg (Daim duab 5C, D). Txhawm rau ntsuas qhov tseeb ntawm cov haujlwm macro uas cuam tshuam nrog kev tshem tawm ntawm cov duab, peb muab piv rau DI cov qhab nia tau txais tom qab phau ntawv tshem tawm ntawm lub cev ntawm cov fluorescence micrographs siv cov cuab yeej pub dawb ntawm ImageJ (raws li tau piav qhia hauv daim duab 4D), rau DI cov qhab nia. tsim los ntawm kev txheeb xyuas ntawm cov duab zoo ib yam tom qab kev tshem tawm ntawm lub cev ntawm lub cev siv cov kev tshem tawm soma uas muaj nyob hauv ANDI Macro. Peb cov kev txheeb xyuas tau qhia tias DI cov qhab nia suav nrog kev tshem tawm lub cev ntawm lub cev muaj feem cuam tshuam nrog qhov sib xws nrog DI kev ntsuas tau los ntawm cov duab raug rau phau ntawv soma tshem tawm (r(34)=0.980, p, 0.0001; Fig. 5 E).

Txhawm rau ua kom qhov tseeb ntawm cov kev tshuaj ntsuam xyuas, peb suav nrog ntau qhov kev hloov kho yav dhau los uas tau txhim kho DI ntsuas rau hauv ANDI. Lub macro nta kev sib piv kev ua haujlwm, kev siv Particle Remover plugin los tshem tawm cov teeb meem tsis yog neurite los ntawm cov duab neurite thiab cov teeb meem tsis yog nuclear los ntawm cov duab ntawm nuclei, thiab tshawb pom cov neurite fragments siv qhov loj me ntawm 5-10,000 rau Analyze Particles plugin.

Peb xav tias, kev sib sau ua ke, kev siv cov fluorescence microscopy, kev ua haujlwm zoo sib xws, tshem tawm cov khoom me me nrog cov khoom tshem tawm, thiab qhov ua kom zoo tshaj plaws rau kev tshawb nrhiav particle yuav ua rau muaj kev ntsuas ntau dua ntawm neurite degeneration. Txhawm rau ntsuas qhov kev xav no, cov duab theem-qhov sib piv ntawm LUHMES hlwb raug rau ntau qhov sib txawv ntawm 6-OHDA, tsim tawm raws li tau piav qhia hauv daim duab 2, tau txheeb xyuas los ntawm qhov muag tsis pom thiab cov qhab nia uas siv tus nqi ntau ntawm 0. 0 thiab 1. Peb mam li ntsuam xyuas qhov sib txheeb ntawm cov qhab nia ntawm cov qhab nia thiab cov kev ntsuas DI tau txais los ntawm cov txheej txheem ib txwm muaj, nrog rau kev sib raug zoo ntawm cov qhab nia ntawm cov qhab nia thiab DI ntsuas tau siv ANDI Macro.

Thaum cov kev ntsuas DI tau txais los ntawm kev siv cov qauv ib txwm muaj cuam tshuam nrog cov qhab nia (r {{0}}}.7850, p,0.0001; Fig. 5F), ANDI Macro yielded Kev ntsuas kev sib raug zoo ntau dua nrog cov kev ntsuas ntsuas (r 2=0.978, p, 0.0001; Fig. 5G). Kev sib piv ncaj qha qhia tau hais tias cov qhab nia tau los ntawm ANDI Macro muaj kev sib raug zoo nrog cov kev ntsuam xyuas kev kawm ntau dua li cov qhab nia tau txais los ntawm txoj kev ib txwm muaj (z=6.626, p, 0.0001; Fig. 5F, G). Kev txheeb xyuas ntxiv tau qhia tias macro pab txhawb kev ua haujlwm ntawm DI ntsuas nrog kev txhim kho tseem ceeb hauv lub sijhawm ua haujlwm. DI kev soj ntsuam ua tiav siv ANDI Macro yuav tsum muaj lub sijhawm nruab nrab ntawm 12.75 s ib daim duab txheeb xyuas, piv nrog lub sijhawm nruab nrab ntawm 5.27 min rau ib daim duab txheeb xyuas los ntawm cov txheej txheem ib txwm muaj [Friedman x2 (1)=9, p {{22} }.0027] ib. Ua ke, cov txiaj ntsig no qhia tau hais tias ANDI tau txais txiaj ntsig sai ntawm DI kev ntsuas uas, piv nrog cov qhab nia tau los ntawm cov txheej txheem ib txwm muaj, ntau ze ze rau qhov degeneration uas cov neeg tshawb xyuas pom tau los ntawm kev soj ntsuam zoo ntawm cov duab neurite.

Tom qab txiav txim siab qhov raug thiab ua tau zoo ntawm ANDI Macro rau kev ntsuas neurite degeneration nyob rau hauv sib txawv LUHMES hlwb raug rau 6-OHDA, peb ntsuas qhov tsim nyog ntawm macro rau kev sim nrog rau lwm yam cell kab lis kev cai qauv. Kev tshawb nrhiav thawj zaug tau ua los ntsuas qhov tseeb ntawm ANDI hauv kev ua DI ntsuas siv micrographs ntawm thawj kab lis kev cai sympathetic immunolabeled rau b III-tubulin thiab DAPI. Interestingly, txawm li cas los xij, lub cev ntawm lub cev ntawm cov neeg mob siab rau cov neurons loj dua li cov hlwb sib txawv ntawm LUHMES, thiab yog li cov haujlwm hauv ANDI ntsig txog kev tshem tawm soma tsis tshem tawm tag nrho cov soma (cov ntaub ntawv tsis qhia).

Txhawm rau kov yeej qhov teeb meem no, peb tau hloov kho ANDI kom muaj lub thawv sib tham ua rau cov neeg siv xaiv qhov loj ntawm soma tshem tawm los ntawm kev tswj cov sij hawm uas binarized micrographs ntawm nuclei dilated. Qhov no version ntawm ANDI, version 1.1, tom qab ntawd siv los ua DI ntsuas los ntawm micrographs qhia txog kev noj qab haus huv, tsheb kho mob hlwb lossis degenerating sympathetic neurons raug hydrogen peroxide. Cov duab ntawm cov neurons raug hydrogen peroxide tau txais txiaj ntsig ntau dua DI cov qhab nia piv nrog cov duab ntawm lub tsheb kho neurons (F(1,3)=71.2, p=0.0035, R2=0 .9301; Fig. 6A, C). Ntxiv mus, peb piv cov qhab nia DI uas tau siv ANDI rau DI kev ntsuas tau los ntawm tib daim duab teeb tsa tom qab phau ntawv tshem tawm ntawm lub cev ntawm cov fluorescence micrographs siv lub cuab yeej dawb ntawm ImageJ. Tsis muaj qhov sib txawv tseem ceeb hauv DI cov qhab nia suav tom qab phau ntawv soma tshem tawm piv nrog cov qhab nia tau siv ANDI (F(1,3)=0.1082, p=0.7639, R2=0. 0000; Fig. 6A–C). Cov kev ntsuam xyuas tom qab hoc kuj tau lees paub tias DI cov qhab nia tau siv ANDI tsis txawv qhov txawv ntawm ob qho tib si ntawm cov neurons tsis kho (p=0.0972) thiab nyob rau hauv rooj plaub ntawm cov neurons raug hydrogen peroxide (p=0). 9986 ib.).

Tsis tas li ntawd, DI cov qhab nia tsim los ntawm ANDI sib raug zoo ib yam nrog DI cov qhab nia tau txais tom qab phau ntawv tshem tawm ntawm lub cev ntawm tes los ntawm cov micrographs sib xws (r(34)=0.991, p, 0.0001). Cov ntaub ntawv no qhia tau tias ANDI yog qhov tsim nyog rau kev ntsuas neurite degeneration hauv kab lis kev cai ntawm thawj cov neurons, nrog rau kev tshem tawm soma uas tus neeg siv tuaj yeem hloov kho kom haum rau ntau hom neuron. Tsis tas li ntawd, kev tshawb pom ntawm neurite fragments nyob rau hauv kab lis kev cai raug hydrogen peroxide qhia tau hais tias qhov kev siv ntawm macro rau kev siv nyob rau hauv kev sim nrog neurite degeneration induced los ntawm lwm yam tshaj li 6-OHDA. Txhawm rau txheeb xyuas ntxiv txog kev siv ANDI hauv kev kuaj pom neurite degeneration cuam tshuam nrog ntau yam ua rau, peb kuj tau siv cov macro los ntsuas neurite degeneration hauv micrographs ntawm cov kab lis kev cai noj qab haus huv lossis kab lis kev cai raug tshem tawm NGF. Raws li qhov xav tau, cov duab ntawm kab lis kev cai raug tshem tawm NGF tau txais txiaj ntsig DI ntau dua piv nrog cov duab ntawm kab lis kev cai noj qab haus huv (F(1,2)= 287, p= 0.0035, R2=0. 958; Fig. 6D, E). Ua ke, cov kev tshawb pom no qhia tau tias ANDI tuaj yeem siv los txheeb xyuas cov neurite degeneration cuam tshuam nrog ntau yam kev lom neeg.

Txij li staining rau TH feem ntau yog siv los pom cov kab mob catecholaminergic neurons, peb kuj tau soj ntsuam qhov tsim nyog ntawm micrographs piav qhia TH staining rau ntsuas neurite degeneration siv ANDI. Kab lis kev cai ntawm kev noj qab haus huv sympathetic neurons kho nrog lub tsheb daws los yog degenerating sympathetic neurons raug hydrogen peroxide raug kho thiab raug rau immunofluorescence labeling rau TH thiab b III-tubulin, nrog rau counterstaining rau DAPI. Neurite dluab depicting staining rau TH los yog b III-tubulin, thiab coj cov dluab ntawm nuclei featuring DAPI labeling, ces raug ntes ntawm qhov zoo sib xws ntawm saib. ANDI raug tua, thiab cov duab uas muaj TH staining lossis b III-tubulin staining raug xaiv ua cov duab neurite. Peb cov kev txheeb xyuas tau qhia tias cov qhab nia DI tau siv cov duab ntawm TH staining sib cuam tshuam nrog cov qhab nia tau los ntawm cov duab ntawm b III-tubulin staining (r(28)= 0.962, p, 0.0001; Fig. 6 F, G). Cov ntaub ntawv no qhia tau hais tias qhov kev ntsuas DI muaj tseeb tuaj yeem tau txais los ntawm ANDI siv lwm txoj kev staining uas sau tag nrho cov neurites, xws li immunolabeling rau TH.

Txhawm rau ua kom pom cov txiaj ntsig ntawm txoj kev txhim kho rau kev ua DI ntsuas hauv kev sim tshawb fawb, peb tau siv txoj kev tshiab los ntsuas lub luag haujlwm ntawm JNK hauv oxidative stress-induced neurite degeneration hauv tib neeg cov hlwb mesencephalic. Sib txawv LUHMES hlwb raug pretreated rau 1 h nrog JNK inhibitor SP600125 los yog tsheb tov. Tom qab ntawd cov hlwb raug kho rau 24 teev nrog 6- OHDA kom ua rau muaj kev ntxhov siab oxidative lossis nrog kev daws tsheb. Cov hlwb ruaj khov tau raug txhaj tshuaj tiv thaiv kab mob rau b III-tubulin thiab DAPI staining, thiab ANDI tau siv los muab DI ntsuas los ntawm fluorescence micrographs. Thaum cov kab lis kev cai tsis muaj pretreatment nrog JNK inhibitor tau nthuav tawm neurite degeneration tom qab raug rau 6-OHDA, JNK inhibition ua rau muaj qhov cim thiab txo qis hauv 6- OHDA-induced neurite degeneration (p= 0.0001 , d= 2.0209; Fig. 7). Cov kev tshawb pom no txhawb nqa lub luag haujlwm tseem ceeb rau JNK teeb liab hauv neurite degeneration vim yog oxidative kev nyuaj siab hauv tib neeg cov hlwb mesencephalic, thiab qhov tseem ceeb, ua kom pom kev siv hluav taws xob ntawm peb txoj kev tshiab los tsim cov kev tshawb fawb tshawb pom los ntawm kev ua kom yooj yim thiab raug ntsuas ntawm neurite degeneration.

Kev sib tham

Neurite degeneration yog txuam nrog ntau yam ntawm cov kab mob neuropathological, tab sis cov txheej txheem molecular hauv qab qhov degradation ntawm neurites tseem tsis to taub. Cov txheej txheem ua kom muaj tseeb thiab muaj txiaj ntsig kev tshuaj xyuas ntawm neurite degeneration yog qhov tseem ceeb rau kev txheeb xyuas qhov zoo ntawm cov xwm txheej tshiab tswj hwm qhov xwm txheej tseem ceeb ntawm tes. Hauv txoj kev tshawb fawb tam sim no, peb nthuav tawm ntau qhov chaw ntawm kev ua yuam kev cuam tshuam nrog ib txoj hauv kev siv los txheeb xyuas cov neurite degeneration. Peb tshaj tawm cov ntaub ntawv tshawb fawb los txhawb cov txheej txheem hloov kho uas tuaj yeem siv los txo DI kev txheeb xyuas qhov yuam kev, thiab cov kev hloov kho no tau muab tso rau hauv ImageJ macro tshiab los muab cov cuab yeej rau kev ceev, raug, thiab lub hom phiaj DI kev tshuaj xyuas siv qhib qhov chaw thiab dawb software. Ntxiv mus, peb qhia tau hais tias yuav ua li cas txoj kev txhim kho tuaj yeem siv los ntsuas neurite degeneration hauv cell kab lis kev cai qauv ntawm PD, ib qho teeb meem nyob rau hauv uas axonal fragmentation nyob rau hauv dopaminergic neurons yog ib qho kev tshwm sim thaum ntxov uas ua ntej qhov tshwm sim neuronal poob (Tagliaferro thiab Burke, 2016) .

Hauv txoj kev tshawb fawb tam sim no, peb qhia txog kev siv hluav taws xob ntawm ANDI Macro hauv kev ntsuas neurite degeneration hauv cov qauv kab lis kev cai ntawm PD uas muaj cov hlwb sib txawv ntawm LUHMES raug rau 6-OHDA.

Peb tshaj tawm cov kev tshawb nrhiav pom uas txhawb nqa qhov pom kev zoo tshaj plaws uas cov kab lis kev cai yuav tsum tau tsim, nrog rau ua kom pom cov plating ntom ntom uas tsim nyog tshaj plaws rau kev tshuaj ntsuam neurite degeneration. Kev siv cov qauv no los kawm txog neurite degeneration muaj ntau yam zoo. Cov hlwb tsim cov neurites uas muaj kev sib txuas zoo thiab feem ntau .500 hli ntev, thiab cov kab lis kev cai raug cuam tshuam rau neurodegeneration induced classic, neurotoxin qauv ntawm PD xws li 6-OHDA, 1-methyl{{4 }}phenylpyridinium ion (MPP1), thiab rotenone (Lotharius et al., 2005; Zhang et al., 2014; Smirnova et al., 2016; Kraemer et al., 2021). Tsis tas li ntawd, tib neeg lub hauv paus chiv keeb ntawm LUHMES hlwb nce qhov kev txhais ntawm kev tshawb pom cuam tshuam nrog tus qauv, thiab cov xwm txheej tsis txawj tuag ua rau muaj kev hloov pauv sai thiab nthuav tawm ntawm kab lis kev cai (Scholz li al., 2011). Yog li, ua ke nrog kev tshuaj ntsuam xyuas tsis siv neeg ua tiav siv ANDI, LUHMES cov hlwb tuaj yeem siv los ua cov kev tshawb pom tshiab ntsig txog PD-koom nrog neurite degeneration thaum pab txhawb kev muaj txiaj ntsig ntau dua piv nrog cov kev tshuaj ntsuam tsis siv neeg ua nrog cov kab lis kev cai hauv lub paj hlwb.

Ntau pab pawg tshawb fawb tau ua DI tshuaj ntsuam xyuas siv cov duab theem sib piv kom tau txais kev nkag siab txog yam uas tswj cov neurite degeneration (Xovxwm thiab Milbrandt, 20{{10}}9; Kraemer li al., 2014 ; Di Stefano et al., 2015; Hill et al., 2018; Geisler et al., 2019). Ntawm no, peb qhia txog qhov teeb meem tseem ceeb uas cuam tshuam nrog txoj kev siv dav dav rau kev ntsuas neurite degeneration: binarization ntawm theem sib piv cov duab ua rau muaj feem cuam tshuam ntawm cov neurite tsis zoo tshwm sim. Txawm hais tias theem ntawm qhov sib txawv uas yuav ua tiav thaum kev ntes micrographs txawv nyob ntawm lub tshuab tsom iav muaj rau cov neeg soj ntsuam, qhov teeb meem no muaj nyob rau hauv ntau pawg kev tshawb fawb, raws li DI ntawm 0.2 feem ntau lees txais raws li qhov pib saum toj no cov kab lis kev cai raug suav tias muaj degenerating. axons, thiab feem ntau cov ntaub ntawv tshawb fawb featuring DI ntsuas los ntawm theem sib piv micrographs tau tshaj tawm cov qhab nia DI rau cov neurons noj qab haus huv ntawm 0.1 thiab 0.3 (Sasaki li al., 2009, 2016; Di Stefano et al., 2015; Hill et al., 2018. Loreto et al., 2020). Peb qhov kev soj ntsuam DI tau txais los ntawm fluorescence micrographs, nrog rau cov qhab nia ntawm cov neeg tshawb xyuas qhov muag tsis pom, qhia tias 10-20% neurite fragmentation tsis muaj nyob hauv cov kab lis kev cai noj qab haus huv. Hloov chaw, cov kab lis kev cai no tsuas muaj 2.5% neurite fragmentation ntawm qhov nruab nrab. Yog li, peb qhov kev tshawb pom pom tau hais tias yuav tsum tau siv txoj hauv kev los txhim kho qhov tseeb ntawm neurite degeneration tsom xam los ntawm kev txo qis qhov kev ntsuas qhov ua yuam kev no.

Thaum feem ntau ntawm cov kev tshawb fawb txog DI kev ntsuas tau siv cov duab sib piv los ua cov kev ntsuam xyuas neurite degeneration, ntau pab pawg tshawb fawb tsis ntev los no tau ua DI ntsuas siv cov duab ntawm neurons immunolabeled rau cytoskeletal filaments xws li neurofilament nruab nrab polypeptide lossis b-b-tubulin (Wakatsuki et al., 2011; Li et al., 2017; Pease-Raissi et al., 2017; Hernandez et al., 2018; Sundaramoorthy et al., 2020). Txawm li cas los xij, daim ntawv microscopy twg yog qhov zoo tshaj plaws rau kev ntsuas neurite degeneration tseem tsis tau paub meej, vim tias kev tshawb nrhiav tau xav tau kom nkag siab seb puas muaj kev hloov pauv hauv cheeb tsam ntawm cov cytoskeletal proteins thaum lub sij hawm neurite degeneration tsim nyog tus qauv tag nrho cov kev hloov hauv neurite morphology, nrog rau kev soj ntsuam cov degree uas DI ntsuas tau nrog fluorescence micrographs sib cuam tshuam nrog cov kev ntsuas zoo sib xws tau los ntawm theem-contrast micrographs. Ntawm no, peb qhia tau hais tias fluorescence micrographs depicting b III-tubulin staining tsis yog tsuas yog raug sawv cev rau neurite fragmentation, tab sis kuj pab txhawb DI kev ntsuas ntau dua vim lawv qhov zoo sib xws thiab tsis tshua muaj kev cuam tshuam rau kev sib cais ntawm binarization. Yog li, peb cov txiaj ntsig tau hais txog qhov txiaj ntsig ntawm kev siv b III-tubulin staining los ntsuas neurite degeneration.

Ntawm cov kev tshawb fawb luam tawm uas suav nrog DI kev tshuaj ntsuam xyuas los ntsuas neurite degeneration, qhov qhia txog qhov loj thiab qhov sib txawv uas siv los txheeb xyuas cov kab mob neurite muaj ntau yam sib txawv. Piv txwv li, ib txoj kev tshawb fawb tsis ntev los no nrog rau kev ntsuam xyuas ntawm neurite degeneration hauv kab lis kev cai hippocampal neurons siv qhov loj me ntawm 4–900 pixels (Li li al., 2017), thaum txoj kev tshawb fawb uas muaj kab lis kev cai DRG neurons siv cov ntsuas ntsuas ntawm 0–10, 000 pixels (Wakatsuki et al., 2011). Ib qho laj thawj rau qhov kev hloov pauv no yog tias qhov ua tau zoo ntawm qhov ntsuas particle loj tsis pom hauv kev kuaj pom cov neurite fragments nws txawv nyob ntawm qhov kev daws teeb meem ntawm daim duab vim tias qhov loj me rau qhov ntsuas particle yog txuam nrog pixel units. Hmoov tsis zoo, feem ntau cov kev tshawb fawb luam tawm uas cuam tshuam nrog kev tshuaj ntsuam DI tsis suav nrog cov lus piav qhia ntawm cov duab daws teeb meem siv rau kev tshuaj ntsuam. Yog li, tsim kom muaj cov qauv ntsuas ntsuas ntawm cov txheej txheem uas nthuav tawm cov ntsiab lus tseem ceeb xws li kev pom zoo cov duab daws teeb meem tau xav tau. Ntawm no, peb tshaj tawm hais tias qhov feem ntau hais tsis tau rau neurite fragment detection, 20– 10, 000 pixels (Shin et al., 2012; Di Stefano et al., 2015; Sasaki et al., 2016; Loreto et al. al., 2020; Shin thiab Cho, 2020), ua rau cov feem ntau ntawm cov paj hlwb tsis raug kuaj pom. Txo qhov tsawg kawg nkaus qhov ntsuas rau kev tshawb nrhiav neurite fragment txhim kho qhov rhiab heev ntawm fragment nrhiav kom tau, thaum ib txhij nce tus naj npawb ntawm cov duab artifacts thiab non-neurite cov khib nyiab uas tsis raug kuaj pom tias neurite fragments. Hauv kev tshawb nrhiav qhov tsis sib xws uas yuav zoo tshaj qhov sib npaug ntawm kev tshawb pom rhiab heev thiab qhov tsis tseeb-positivity tus nqi, peb tau txheeb xyuas qhov ntsuas ntawm 10–10,000 raws li muab qhov tseeb tshaj plaws tshawb pom ntawm neurite fragments hauv binarized dluab tau los ntawm theem sib piv micrographs, thaum tsis pub dhau 5– 10,000 pab txhawb qhov tseeb tshaj plaws ntawm kev tshawb pom ntawm neurite fragments hauv binarized dluab tau los ntawm fluorescence micrographs. Thaum cov kev txiav txim siab no tau tsim los siv cov duab daws teeb meem ntawm 1280 1024, cov kev txwv no yuav tsum ua kom muaj qhov tseeb neurite fragment nrhiav pom hauv cov duab ntes ntawm lwm qhov kev daws teeb meem uas muaj qhov loj ntawm 1024 pixels, xws li cov kev daws teeb meem puv npo ntawm {{ 27}}, txij li cov neurons tau piav qhia hauv cov duab zoo li no yuav zoo ib yam pixel loj.

Ntau pab pawg tshawb fawb tau siv qhov tsawg kawg nkaus qhov ntsuas qhov ntsuas qhov ntsuas qhov ntsuas kom tsis txhob muaj cov khib nyiab me me, uas tsis yog neurite los ntawm kev suav nrog hauv kev ntsuas neurite fragment (Shin et al., 2012; Cosker et al., 2013; Di Stefano et al., 2015; Sasaki et al., 2016; Li et al., 2017; Loreto et al., 2020; Shin and Cho, 2020; Sundaramoorthy et al., 2020). Txawm li cas los xij, cov kev tshawb fawb no tau ua yuam kev suav nrog cov teeb meem me me, tsis yog tawg nyob rau hauv qhov ntsuas ntawm tag nrho cov neurite cheeb tsam, vim tias qhov kev ntsuas no tau txais los ntawm kev suav cov cheeb tsam ntawm tag nrho cov pixels dub hauv daim duab. Ntawm no, peb ua qauv qhia txog kev siv hluav taws xob ntawm Particle Remover plugin kom tshem tawm cov khib nyiab me me, tsis yog tawg ntawm daim duab ua ntej DI tsom xam. Kev siv lub plugin ua rau muaj kev nkag siab zoo ntawm neurite degeneration ntsuas tau los ntawm cov duab theem sib piv. Yog li, peb cov txiaj ntsig tau qhia txog tus nqi ntawm Particle Remover plugin rau cov neeg tshawb xyuas ua DI kev tshuaj ntsuam xyuas siv cov theem sib piv micrographs. Interestingly, fluorescence micrographs feature me me, uas tsis yog-neurite cov khib nyiab, thiab yog li, qhov qis me me ntawm tsib pixels tuaj yeem siv rau kev tshawb nrhiav tawg. Thaum tshem tawm cov khoom uas tsis yog-neurite cov khib nyiab tsawg dua tsib pixels qhov loj me tsis cuam tshuam rau DI kev ntsuas tau los ntawm fluorescence micrographs, cov haujlwm no muab cov txiaj ntsig uas muaj qhov tseeb dua thiab yog li tau suav nrog hauv ANDI.

Txij li thaum kev soj ntsuam DI yuav tsum tau ua nrog cov duab tshwj xeeb uas muaj cov neurites, daim ntawv thov ntawm txoj kev tsom xam feem ntau tau txwv rau kev siv nrog cov kab lis kev cai uas muaj peev xwm tshem tawm ntawm lub cev, xws li cov kab lis kev cai uas lub cev ntawm lub cev tuaj yeem raug tshem tawm (Catenaccio li al. , 2017; Shin thiab Cho, 2020) lossis kab lis kev cai hauv cov khoom siv microfluidic pab txhawb kev sib cais ntawm soma thiab axon compartments (Cosker et al., 2013; Pease-Raissi et al., 2017; Tan et al., 2018). Xwb ua DI kev soj ntsuam nrog cov kab lis kev cai sib txawv tuaj yeem ua tiav los ntawm kev ua haujlwm hnyav thiab siv sij hawm ua haujlwm nrog kev tshem tawm cov xov tooj ntawm tes los ntawm micrographs (Kraemer li al., 2014). Hauv txoj kev tshawb fawb tam sim no, peb nthuav tawm tias ANDI ua rau kev ua tau zoo ntawm DI kev ntsuas los ntawm micrographs uas muaj LUHMES cov hlwb uas sib cais thiab mosaically faib thoob plaws 2D kab lis kev cai. Peb qhov kev tshawb pom kuj qhia tau hais tias ANDI pab txhawb rau 24-fold txo lub sij hawm yuav tsum tau ua cov kev ntsuam xyuas, thiab cov hauj lwm hauv ANDI muaj feem xyuam rau soma tshem tawm tawm los DI tsom xam cov txiaj ntsig uas cuam tshuam 98% nrog cov kev soj ntsuam zoo sib xws tau ua raws li phau ntawv soma tshem tawm siv cov freehand tool ntawm ImageJ. Yog li, cov macro tau nce qhov ua tau zoo ntawm kev txheeb xyuas los ntawm kev ua haujlwm ntawm soma tshem tawm ntawm cov txheej txheem uas tsis txi qhov tseeb, thiab cov haujlwm zoo li no nthuav dav qhov tsim nyog ntawm txoj hauv kev rau kev txheeb xyuas cov kab lis kev cai sib txawv nrog cov mosaically faib lub cev.

Ntxiv nrog rau kev ua kom yooj yim tshem tawm lub cev ntawm lub cev los ntawm cov duab, ANDI nta ntau qhov kev hloov kho rau cov txheej txheem ib txwm ua rau kev soj ntsuam DI, suav nrog kev siv cov fluorescence micrographs, hloov kho qhov ntsuas qhov ntsuas, thiab siv cov khoom tshem tawm kom tshem tawm cov khib nyiab me me, tsis yog neurite. los ntawm cov duab. Peb qhov kev tshawb pom qhia tau hais tias qhov kev ua kom zoo tshaj plaws no ua ke muab cov txiaj ntsig DI uas ze ze dua qhov txiaj ntsig tau los ntawm qhov muag tsis pom thiab raug qhab nia. Yog li, ntxiv rau txo cov sij hawm xav tau los ua cov kev tshuaj ntsuam xyuas, ANDI pab txhawb DI kev ntsuas uas, piv nrog cov txheej txheem ib txwm muaj, muaj tseeb dua qhov cuam tshuam ntawm neurite degeneration uas cov neeg tshawb xyuas tau tshaj tawm los ntawm kev txheeb xyuas cov duab zoo.

Ib qho kev tsis zoo ntawm cov txheej txheem ib txwm ua rau kev soj ntsuam DI yog tias cov kev tshuaj ntsuam no xav tau kev cob qhia hauv kev siv ImageJ kom tus neeg siv paub txog cov txheej txheem tedious ntawm ImageJ kev ua haujlwm. Tsis tas li ntawd, txhawm rau ua qhov kev soj ntsuam kom zoo, kev cob qhia ntxiv yog xav tau rau tus neeg siv los ua batch tsom xam lossis sau ntawv rau ib nrab-automate qhov kev ua tiav ntawm cov kauj ruam tshwj xeeb. Los ntawm tag nrho automating qhov kev tshuaj ntsuam, ANDI pab txhawb DI ntsuas nyob rau hauv tsuas yog ob peb kauj ruam hais txog kev rub tawm thiab ua tiav cov macro. Txhawm rau kom cov neeg siv tau yooj yim dua, cov macro suav nrog cov lej los muab qhov kev sib txuas rau xaiv cov npe uas muaj cov duab los txheeb xyuas, nrog rau pab txhawb cov txiaj ntsig ntawm cov duab thiab cov ntaub ntawv cov lus rau cov npe ntawm tus neeg siv xaiv. Ntxiv mus, tsab ntawv muaj ntau yam qauv tsim los tiv thaiv kev ntsuas qhov tsis raug lossis cov neeg siv yuam kev, suav nrog kev tshem tawm ntawm cov txiaj ntsig yav dhau los ImageJ; tshem tawm cov ntaub ntawv teev cov duab kom ntseeg tau tias kev ntsuas tau ua tiav siv pixel units; Kev teeb tsa tsim nyog ntawm ImageJ xim teeb tsa, ntsuas qhov ntsuas, thiab ntsuas particle ntsuas tsis; Cov lus qhia yuam kev yog tias cov neeg siv yuam kev xaiv cov npe uas muaj cov lej tsis sib xws ntawm cov duab piav qhia b III-tubulin staining thiab DAPI staining; cov lus piav qhia txog kev txheeb xyuas kev nce qib; thiab coding los tiv thaiv kab mob cuam tshuam nrog automated .ini cov ntaub ntawv tsim los ntawm Windows 10. Ua ke, cov yam ntxwv no ua rau DI tsom xam cov neeg siv ntau dua, uas yuav txhawb kev txaus siab rau cov kev tshawb fawb ntsig txog neurite degeneration.

Txhawm rau ua kom pom nws cov txiaj ntsig hauv qhov kev sim, peb siv ANDI los ntsuas qhov kev koom tes ntawm JNK rau neurite degeneration hauv LUHMES cell kab lis kev cai raug rau 6-OHDA. JNK yog ib qho kev ntxhov siab-activated kinase uas tau tshaj tawm tias yog tus yuam sij rau neurite degeneration hauv peripheral neurons ntawm nonprimate keeb kwm (Shin li al., 2012), tab sis xav tau kev tshawb fawb ntxiv kom nkag siab txog lub luag haujlwm ntawm kinase hauv neurite degeneration hauv mesencephalic tib neeg hlwb. . Ntawm no, peb qhia tau tias inhibition ntawm JNK ho tiv thaiv tib neeg mesencephalic hlwb los ntawm neurite degeneration txuam nrog oxidative kev nyuaj siab. Cov kev tshawb pom zoo li no tsis tsuas yog nthuav tawm lub luag haujlwm tseem ceeb rau JNK hauv neurite degeneration cuam tshuam nrog tus qauv ntawm tib neeg tus kab mob tab sis kuj ua piv txwv rau kev siv hluav taws xob ntawm ANDI rau kev ua haujlwm automated thiab lub hom phiaj duab tsom mus rau kev tshawb pom tshiab.

Thaum ANDI tau tsim los ua kom yooj yim thiab raug ntsuas ntawm neurite degeneration nyob rau hauv kab lis kev cai ntawm txawv LUHMES hlwb raug rau 6-OHDA, peb qhov kev tshawb pom kuj qhia tau hais tias cov macro tuaj yeem siv los tshawb xyuas neurite degeneration induced los ntawm lwm yam ua rau, xws li raug rau hydrogen peroxide los yog tshem tawm neurotrophin. Tsis tas li ntawd, version 1.1 ntawm macro nta lub thawv sib tham ua rau cov neeg tshawb nrhiav los kho cov haujlwm tshem tawm soma rau cov neurons ntawm qhov ntau thiab tsawg. Peb cov ntaub ntawv tseem txhawb nqa txoj kev staining uas tsis yog immunolabeling rau b III-tubulin tuaj yeem siv los ntsuas qhov tseeb ntawm neurite degeneration. Txawm hais tias peb pom zoo kom siv cov kev tshawb fawb sim los txheeb xyuas qhov tseeb ntawm DI kev ntsuas thaum siv ANDI nrog cov xov tooj ntawm tes lossis cov txheej txheem staining uas tsis tau sim yav dhau los, cov kev tshawb pom no txhawb nqa tias macro yog cov cuab yeej siv dav dav rau kev ntsuas neurite degeneration hauv kab lis kev cai neurons. Txij li thaum ANDI tsis tau ua kom zoo rau kev ntsuas neurite degeneration hauv cov ntaub so ntswg, peb tam sim no tsuas yog pom zoo kom siv nws rau kev tshuaj xyuas cov neurite degeneration cuam tshuam nrog cov kab lis kev cai. Txawm li cas los xij, cov cai rau ANDI tau tshaj tawm rau pej xeem thiab yog li kuj tseem yuav pab tau rau cov neeg tshawb nrhiav xav hloov cov ntawv kom haum rau hauv vivo kev tshawb fawb lossis lwm yam kev siv.

Hauv kev xaus, tsab xov xwm no nthuav tawm cov ntaub ntawv nthuav tawm ntau qhov chaw ntawm kev ua yuam kev cuam tshuam nrog kev siv dav dav rau kev txheeb xyuas cov neurite degeneration. Cov kev sim tau siv los txhim kho txoj hauv kev, thiab cov kev txhim kho zoo li no tau muab tso rau hauv qhov chaw pub dawb thiab qhib ImageJ macro uas tuaj yeem siv los ua kom nrawm, raug, thiab lub hom phiaj tsom xam ntawm fluorescence micrographs rau neurite degeneration. Peb qhov kev tshawb pom tau nthuav tawm qhov ua tau zoo ntawm ANDI rau kev txheeb xyuas cov neurite degeneration hauv micrographs piav qhia txog kab lis kev cai ntawm tes ntawm PD, nrog rau muab pov thawj ntawm lub hauv paus ntsiab lus tias macro tuaj yeem kuaj pom neurite degeneration cuam tshuam nrog lwm cov ntsiab lus lom neeg. Tus neeg siv-phooj ywg macro yuav txo lub sij hawm xav tau rau cov neeg tshawb xyuas los kawm kom muaj nuj nqis ntawm neurite fragmentation thaum nce kev nkag mus thiab qhov tseeb ntawm cov kev tshawb fawb soj ntsuam cov xwm txheej hauv qab neurite degeneration. ANDI Macro muaj nyob rau ntawm qhov URL hauv qab no: https://github.com/kraemerb/kraemerlab.

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