Ho ikoetlisa ho phahamisa dopamine D2 receptor ka mohlala oa mouse oa Parkinsons lefu In vivo imaging le (18F) fallypride (2010)

Sepheo sa phuputso ea morao-rao e ne e le ho hlahloba liphetoho tsa polelo ea dopamine D2 receptor (DA-D2R) polelo e ka hare ho basal ganglia ea litoeba tsa MPTP tse behiloeng ka mokhoa o matla oa ho ikoetlisa. Ho sebelisa tlhahlobo ea immunoblotting Bophirimela ea synaptoneurosomes le Ka vivo positron emission tomography (PET) monahano o sebelisang ligand e khethehileng ea DA-D2R [¹⁸F] e fallypride, re fumane hore boikoetliso bo matla ba ho hatakela litlhapi bo entse hore ho eketsehe polelo e hlaselang ea DA-D2R e neng e bitsoa ka ho fetisisa ho MPTP ha e bapisoa le litoeba tse tšoaroang ka letsoai. Liphetoho tse bakoang ke ho etsa lintho tse itseng ho DA-D2R li-basal ganglia tse senyehileng tsa dopamine li lumellana le karolo e ka 'nang ea e-ba teng ea mokelikeli ona oa ho tsamaisa mokhoa oa sekhahla oa spiny neurons (MSNs) le mokhoa oa ho hlaphoheloa ha boitšoaro. Habohlokoa, liphuputso tse tsoang thutong ena li tšehetsa mohopolo oa ho sebelisa litšoantšo tsa PET ka [¹⁸F] ka ntle ho naha ho hlahloba liphetoho tsa DA-D2R ho batho ba nang le Mafu a Parkinson (PD) a nang le koetliso e phahameng ea ho hatakela litlhapi.

Liphoofolo, Lihlopha tsa Kalafo, le MPTP Administration

Monna C57BL / 6 litoeba tsa libeke tsa 8 (Charles River Laboratories, Wilmington, MA) e ne e le sehlopha-se ne se le ka kamoreng e laoloang ke mocheso tlas'a 12 h light / 12 h mosi o lefifi. Mekhoa eohle e ile ea etsoa ho latela tataiso ea NIH bakeng sa tlhokomelo le tšebeliso ea liphoofolo tsa liphoofolo tse amoheloang ke USC IACUC. Kakaretso ea litoeba tsa 164 li ne li sebelisoa ka lihlopha tse 'nè tsa phekolo: (1) saline (n = 42), (2) saline hammoho le boikoetliso (n = 55), (3) MPTP (n = 57), le (4) MPTP hammoho boikoetliso (n = 42). Bakeng sa ho silafala, litoeba li fumane liente tse 'nè tsa intraperitoneal tsa 20 mg / kg MPTP (setsi sa mahala, Sigma-Aldrich, St. Louis, MO) se qhibilihantsoeng ka 0.9% saline, nakong ea 2-h kapa liente tse' nè tsa intrperitoneal tsa 0.1 ml 0.9% NaCl e le taolo. Ho hlajoa ke litsi ho ile ha tiisoa ke HPLC ho hlahloba litekanyetso tse hlaselang tsa dopamine. Matsatsing a 10 ka mor'a tsamaiso ea MPTP, ho ne ho e-na le 82.2% dopamine e felisoang ke litoeba tsa MPTP (48.0 ± 8.4 ng / mg ea protheine) ha e bapisoa le litoeba tsa saline (269.5 ± 24.9 ng / mg ea protheine). Qetellong ea thuto, ho ne ho se na phapang e khōlō lihlopheng tsa maqheku tsa lipopali pakeng tsa MPTP le lipilisi tsa ho ikoetlisa (69.8 ± 11.7 ng / mg ea protheine) ha e bapisoa le MPTP (77.9 ± 12.0 ng / mg ea protheine). Leha ho le joalo, ho ne ho e-na le keketseho e khōlō ea ho tsoala lefu la sopamine le saleng ea ho ikoetlisa (315.2 ± 9.0 ng / mg ea protheine) ho bapisoa le saline (246.9 ± 19.8 ng / mg ea protheine) (F_(3,16) = 7.78; P <0.05).

Boikoetliso ba Treadmill

Ho ikoetlisa ho qalile matsatsi a 5 ka mor'a ho hlajoa ke lefu. Litoeba tse tsoang lihlopheng tse peli tsa boikoetliso (saline hammoho le boikoetliso ba 'mele le MPTP hammoho le boikoetliso ba' mele) li koetliselitsoe ho tsamaisa maoto a tsamaisoang ka 100-cm (Exer 6M, Columbus Instruments, OH) ka lebelo le eketsehileng la libeke tsa 6 (matsatsi a 5 / beke) ho fihlela nako ea 60 min / letsatsi le lebelo la 18-20 m / min.^5,6

Litšoantšo tsa Magnetic Resonance

Tlhaloso ea likarolo tse tharo tse nang le boima ba motlakase (MR) ea T1 (MR) e nang le boima ba motlakase (MR) e fumanoe ka 7-T micro-MRI system (Bruker Biospin, Billerica, MA). Mekhahlelo ea ho fumana setšoantšo e ne e le: TE = 46.1 ms, TR = 6292.5 ms, sekhahla sa slice sa 0.4-mm, bophara ba 0.45-mm interslice, 128 × 128 × 128 matrix boholo.

Radiochemistry

Synthesis of [¹⁸F] sephetho se entsoeng ka pele se hlalositsoe ka mokhoa o ts'oanang oa phetoho oa motlakase oa motsoako oa tosyl o nang le [¹⁸F] ho sebelisa lisebelisoa tsa radiochemistry tse entsoeng ka tloaelo.¹² Ho hloekisoa ho ne ho finyelloa ke HPLC ea phaseletso ea phapanyetsong ho C8 (2) Phenomenex Luna mohala ka ho sebelisa acetonitrile le sodium phosphate buffer e le mochine oa mobile (55: 45). Tšenyo ea UV e ne e lekanngoa ho 254 nm le AUFS 0.05. Tlhōrō ea motlakase (nako ea ho boloka 17 min) e lumellanang le [¹⁸F] ka ntle ho naha, e ne e bokelloa 'me motsoako o tlosoa ka mouoane oa rotary. Sehlahisoa sa ho qetela se ile sa lekoa bakeng sa pyrogenicity, stterility, pH, le ho tlosoa ha metsoako ea lihlahisoa ka khase ea chromatography. Ketsahalo e khethehileng le bohloeki ba seea-le-moeeng li ile tsa hlahlojoa ka mokhoa oa metsi oa metsi a HPLC e sebelisa tlhahlobo ea C8 (2) Phenomenex Luna. Ketsahalo e khethehileng e ne e le ka har'a 3,000-12,000 Ci / mmol.

Litekanyo tsa PET le Boithuto ba Litšoantšo

Ho sebelisitsoe litšoantšo tse mashome a mabeli bakeng sa litšoantšo tsa PET (n = saline ea 6; n = saline ea 3 hammoho le boikoetliso; n = 5 MPTP; le n = 6 MPTP hammoho le boikoetliso). Litšoantšo li ile tsa fumanoa ka scanner ea Concorde microPET R4 (CTI Concorde Microsystems, Knoxville, TN) e nang le mokhoa oa XMUMX oa mechine ea ho fumana mekhoa ea ho fumana melaetsa ka morao ho 60 metsana ea ho fetisetsa mochine bakeng sa khalemelo ea tlhokomelo ⁶⁸Mohloli. [¹⁸F] fallypride (10.92-11.28 MBq) e ne e kenngoa ka mothapo oa mohatla (bolus e le 'ngoe) qalong ea ho hlahisa mochine. Litoeba li ne li haelloa ke 2% isofluorane le 98% oksijene. Lenane le matla la boemo ba boemo bo hlophisitsoeng le ile la hlophisoa ho latela likarolo tsa 26 (6 × 20 sec, 4 × 40 sec, 6 × 1 min, le 10 × 5 min) le ho tsosolosoa ka litlhahiso tse peli tsa OSEM (e laetsoeng hore ho be le tebello e kholo ea tebello) e lateloa ke 18 litšebeletso tsa MAP (boholo ba posteriori) algorithm ea ho tsosolosa.¹⁴ Litšoantšo tse tsosolositsoeng li ne li koahetsoe hore li be le hlooho le li-linearly tse ngotsoeng ka har'a eona Z-direction ho hlahisa 128 × 128 × 63 setšoantšo ka isotropic 0.4 × 0.4 × 0.4 mm³ li-voxels. Litšoantšo tse nang le bokhoni bo phahameng ba ho etsa lipapali (BP) tsa statum li ne li ngotsoe ho tloha litšoantšong tse matla tse nchafatsoang ho sebelisa mefuta e mengata¹⁵ le Logan plots¹⁶ ka mosebetsi o phahameng oa striatum le mosebetsi o tlase haholo ho cerebellum (sebaka sa boitsebiso). Libaka tse thahasellisang tsa thahasello (striatum le cerebellum) li ne li hlalositsoe ka letsoho liphatlalatsong tse peli tsa litšoantšo tsa PET tse kenyelletsoeng ka MRI ho sebelisa Rview (version 8.21Beta).¹⁷ Quantification ea ho tlamisoa ka ho toba ha [¹⁸F] ka ntle ho statum ea mouse e ne e etsoa ka boleng ba BP e fanang ka tekanyo ea karo-karolelano ea tikoloho e khethehileng / e sa tsotelleng ka ho lekanngoa.^18,19 E le hore ho bontšoe sebopeho se ikemetseng ho statum, litoeba tse 'nè li ne li kotuloa 60 min ka mor'a hore e sebelisoe ke ligand, kapele limela tse nang le nitrojene ea metsi, tse arohaneng le 30-μm, le likarolo tse nang le phospho-imager (Typhoon 9200, GE Healthcare Inc., Piscataway , NJ) (Feie. 1). Liphuputso li bontšitse hore [¹⁸F] e tlama ka ho khetheha ho DA-D2R, mme e le DA-D3R e nyenyane haholo e le statum, ho tlama ho bonts'a basebetsi ba DA-D2R.^9,10,12,13

 

FEIE. 1

[¹⁸F] Fallypride e bonts'a maemo a phahameng a ho reka ho mouse statum. Lehlakoreng le letšehali le bontša phetolelo ea anatomical ea karolo ea likarolo ho hoo e batlang e le boemong ba bregma 0.20. Karolo e nepahetseng e bontša moemeli oa li-autoradiograph o nang le litlhaloso tse kholo tse tsamaisanang le tsona ...

Lihlahisoa tsa Tissue bakeng sa HPLC le Protein Analysis

Qetellong ea thuto, boko bo ile ba tlosoa ka potlako 'me litsebe tsa mahlaseli li ile tsa arohana hantle le libaka tsa lipalo ho tloha bregma 1.2 ho ea 0.6 le corpus callosum e le moeli oa mahlakoreng, karolo ea lateral ea corpus callosum e le moeling o ka morao, le ka holim'a ts'ebetso ea pele moeli oa boipheliso.²⁰

HPLC Tlhahlobo ea Dopamine le Metabolite ea eona

Matšoao a dopamine ka homogenates e hlaselang (n = 4 ka sehlopha) a ne a khetholloa ke HPLC ka ho hlahloba electrochemical.⁶ Mokhoa ona o ne o e-na le sSA-auto-sampler (ESA, Chelmsford, MA) e nang le 150 × 3.2 mm khoeli ea mohato oa C-18 (3μm bophara) le CoulArray 5600A (ESA, Chelmsford, MA), e nang le tse 'nè -le cell analytical channel le bokhoni ba beha -75, 50, 220, le 350 mV.

Bophirimela ba Immunoblot Analysis

Ho ikoetlisa ka polelo ea synaptic ea DA-D1R le DA-D2R e ile ea hlahlojoa ka litokisetso tsa synaptoneurosome tse ncha ho tloha ho tse robeli tse nang le li-dorsolateral striatum.²¹ Tsamaiso ena e ne e etsoa ka litoeba tse tharo bakeng sa litlhapi tsa 24 ka sehlopha sa liteko (n = 3 preps ka sehlopha). Tlhaloso e amanang le liprotheine bakeng sa DA-D1R (~ 50 kDa), DA-D2R (~ 50 kDa), tyrosine hydroxylase (58 kDa), motlakase oa dopamine (68 kDa), le α-tubulin (50 kDa) (e le taolo ea ho laola) li ile tsa hlahlojoa ke Bophirimela ba immunoblot²² ho sebelisa li-antibodies tse ka sehloohong tse fumanehang khoebong (li-rabbit polyclonal le mouse monoclonal antibodies, Millipore, Temecula, CA). Lihlopha tsa liprotheine li ne li bonts'oa ke lihlopha tse nang le likokoana-hloko tse loantšitsoeng tsa poli e loantšang li-poli kapa li-anti-mouse tse kopantsoeng le IRDye₆₈₀ kapa IRDye₈₀₀ (Rockland, Gilbertsville, PA). Letšoao la fluorescent le fumanoe ka ho hlahloba senola ho LI-COR Odyssey haufi le sethaleng sa litšoantšo tsa infrared le se nkiloeng ka lisebelisoa tsa Odyssey 2.1 (LI-COR Biotechnology, Lincoln, NE). Liphetho li bontšoa e le lipapiso tse amanang le kamano e bapisoang le sehlopha sa saline (se behiloeng ho 100%).

Tlhahlobo ea Litekanyetso

Phapang pakeng tsa lihlopha tsa BP tsa [¹⁸F] fallypride, DA-D1R, le litekanyetso tsa protheine tsa DA-D2R li ile tsa hlahlojoa ka ho hlahloba litsela tse peli tsa ho feto-fetoha (ANOVA) ka phekolo e pakeng tsa sehlooho (saline le MPTP), le ho ikoetlisa ho ea ka taba e itseng (ha ho na boithaopo. boikoetliso). Bakeng sa tlhahlobo e potlakileng ea ho potlakela ho potoloha, nako e ne e sebelisoa e le pakeng tsa sehlooho se itseng (beke ea 1, 2, joalo-joalo) mme phekolo e ne e sebelisoa e le ka hare ho taba (saline vs. MPTP). Setlhahlobo sa post ho bonferroni se ne se sebelisetsoa ho lokisa likamano tse ngata ha ho hlahloba bohlokoa ba thahasello. Nete ​​ea bohlokoa e behiloe ho P <0.05. Ho lekola bohlokoa ba liphapang tsa sehlopha, tekanyetso ea boholo ba phapang lipakeng tsa lihlopha e ile ea baloa ho sebelisoa boholo ba litlamorao (ES) (ES = Mean_{Sehlopha 1} - Bolelang_{Sehlopha 2}/ SD_{ho kopantsoe}). ES e bonts'a litlamorao tsa kalafo ka har'a batho ba nang le tjantjello mme e tlalehiloe ho latela litekanyetso tse behiloeng e le nyane (<0.41), e mahareng (0.41-0.70), kapa e kholo (> 0.70).²³ Ho hlahlojoa ho ne ho etsoa ka Prism5 bakeng sa Windows (GraphPad, San Diego, CA).

Boikoetliso bo Phahameng ba Tšebeliso ea Litlhapi bo ntlafalitse Boitšoaro ba Mesebelisoa ho Mantsoe a MPTP-Leihlo

Pele ho MPTP-lisosa le ho qala ho ikoetlisa, mekhahlelo e tloaelehileng ea litoeba lihlopheng tse peli tsa boikoetliso e ne e tšoana le (saline hammoho le boikoetliso: 11.7 ± 1.1 m / min, le MPTP le ho ikoetlisa: 11.2 ± 1.1 m / min). Boikoetliso ba letsatsi le leng le le leng bakeng sa libeke tsa 6 bo ntlafalitse maqhubu a mangata a ho ikoetlisa a nang le saline hammoho le litoeba tsa boikoetliso tse bontšang matla a maholo haholo ho bapisoa le MPTP hammoho le litoeba tsa ho ikoetlisa ka libeke tse 1 ka 4 (Feie. 2). Leha ho le joalo, MPTP hammoho le litlhapi tsa ho ikoetlisa li ne li e-na le lebelo le lekanang le ho feta la saline hammoho le boikoetliso ba meno ka beke 5 (MPTP le ho ikoetlisa: 17.2 ± 3.6 m / min le saline le ho ikoetlisa: 22.0 ± 1.5 m / min) le beke 6 (19.2 ± 1.2 m / metsotso le 22.2 ± 0.9 m / min, ka ho latellana). Joalokaha ho boletsoe pejana, MPTP-e fumanoeng litoeba tse sa kang tsa koetlisoa ka treadmill ha lia ka tsa hlaphoheloa ka mokhoa o itekanetseng oa boitšoaro bo tsamaeang le likoloi ka lebelo la tsona le leholo la 7.0 ± 0.3 m / min qetellong ea nako ea boikoetliso ba beke ea 6.⁵

 

FEIE. 2

Ho ikoetlisa ho ntlafatsa boitšoaro ba motlakase meleng ea MPTP. Maqhubu a ho feta a saline (n = 12) le MPTP (n = 12) litoeba tse tsamaeang ka maoto li ile tsa lekoa qetellong ea beke le beke. Litebelisoa tsa motheo tsa treadmill velocities li ne li lekantsoe pele ho sekolo sa MPTP. ...

Boikoetliso bo Phahameng ba Tšebeliso ea Boithaopo Ho eketseha ha Striatal DA-D2R empa ha e na Protheine ea DA-D1R

Boikoetliso bo phahameng ba sekhahla sa litlhapi bo ama likarolo tse fapaneng tse amanang le DA-D2R le maemong a DA-D1R ka litokisetso tsa synaptoneurosomal ho tloha ho li-rorsa striatum joalokaha ho bontšoa tlhahlobo ea lefutso la Bophirimela (Feie. 3). MPTP hammoho le ho ikoetlisa ka litoeba ho ne ho eketseha ka 48.8% ho DA-D2R e hlaselang ha e bapisoa le litoeba tsa MPTP (Setšoantšo sa 3B), le likamano tse kholo pakeng tsa boikoetliso le litheko tsa MPTP ka boemo ba protein ea DA-D2R (F_(1,8) = 6.0; P <0.05). Ka lehlakoreng le leng, ho ne ho se na tšusumetso ea boikoetliso maemong a protheine ea DA-D1R lipakeng tsa lihlopha (Setšoantšo sa 3A; F_(1,8) = 0.1, P = 0.78). Ho hlaphoheloa ha MPTP feela ha hoa ka ha fetola DA-D2R (F_(1,8) = 0.0; P = 0.88) kapa polelo ea DA-D1R (F_(1,8) = 0.0; P = 0.92). Ho phaella moo, liprotheine tse peli tse fapaneng tsa li-midbrain dopaminergic fibers botšepehi, tyrosine hydroxylase (TH; Setšoantšo sa 3C) le dopamine transporter (DAT; Setšoantšo sa 3D), e bontšitse hore MPTP e theohile haholo ho theola protheine ea TH (F_(1,8) = 757.3; P <0.05) le polelo ea DAT (F_(1,8) = 218.0; P <0.05).

 

FEIE. 3

Etsa boipiletso ba ho laola DA-D2R empa eseng protheine e hlaselang DA-D1R. Panel (A) e bontša mokhoa oa Bophirimela oa immunoblot oa tlhahlobo ea synaptoneurosome ho tloha ho dorsal striatum bakeng sa protheine ea DA-D1R. Ho ne ho se na phapang ea statistically pakeng tsa ...

Boikoetliso bo Phahameng ba Tšebeliso ea Boithaopo Boithaopo bo eketsehileng ba Striatal [¹⁸F] Ho khoneha ho qhekella ka sekhukhu (BP)

Le hoja tlhahlobo ea immunoblotting ea Bophirimela ea lipuo tsa protheine ea polelo e lekanyelitse li-epitopes tse feletseng tsa likokoana-hloko (bobeli ba mabenkele a ka hare le a ka hare) Ka vivo PET-e nahanang ka li-radioligand tse khethehileng tsa DA-D2R [¹⁸F] ka ntle ho naha e ka hlahisa liphello tsa boikoetliso ho fumaneha ha DA-D2R ho tlama ligand (Feie. 4). Tlhahlobo ea statistical e bontšitse hore ho bile le phello e kholo ea boikoetliso (F_(1,16) = 12.3; P <0.05) hammoho le leqeba la MPTP (F_(1,16) = 160.3; P <0.05) ntle le tšebelisano e kholo lipakeng tsa MPTP le boikoetliso (F_(1,16) = 3.5; P = 0.07) ho [¹⁸F] fallypride BP. Tlhaloso ea poso ea Bonferroni e bontšitse phapang e khōlō lipakeng tsa BP pakeng tsa MPTP le MPTP hammoho le litoeba tsa ho ikoetlisa (t = 1.1, Df = 1, 16; P <0.01), 'me ha ho na phapang e kholo lipakeng tsa letsoai le letsoai hammoho le litoeba tsa boikoetliso (t = 4.1, Df = 1, 16; P > 0.05). Haholo-holo, litoeba tsa MPTP hammoho le boikoetliso li bile le keketseho ea 73.1% ho [¹⁸F] flippride BP ha e bapisoa le litoeba tsa MPTP (lipalo tsa bobeli ba BP bakeng sa MPTP le ho ikoetlisa: 7.1 ± 0.7; palo ea boipheliso ea BP bakeng sa litoeba tsa MPTP: 4.1 ± 0.3) (Setšoantšo sa 4B). Ho phaella moo, saline hammoho le litlhapi tsa ho ikoetlisa li na le keketseho ea 8.2% [¹⁸F] fallypride BP (13.2 ± 1.0) ha e bapisoa le litoeba tsa saline (XUMUM ± 12.2). Tumellanong le liphuputso tsena "litekanyetso tsa boholo" li senola matla a maholo a ho ikoetlisa pakeng tsa lihlopha tsa MPTP (ES = 0.3) ho feta tse pakeng tsa lihlopha tsa saline (ES = 2.61).

 

FEIE. 4

Ho ikoetlisa ho eketsa ka mokhoa o khethollang [¹⁸F] bokhoni ba ho bokella ka ho feletseng (BP) ho striatum ea litoeba tsa MPTP. Panel (A) e bontša [¹⁸F] litšoantšo tsa boemeli ba BP fallypride ka mahlakoreng a mahlakoreng (lehlakoreng le letšehali) le mahlakoreng a holimo (ka lehlakoreng le letona). Sebaka sa tekanyo ...

Mosebetsi ona o ne o tšehetsoa ke lenane la USC CTSI Full Pilot Grant Program, le chelete e fanoang ke Parkinson's Disease Foundation, Team Parkinson (Los Angeles), Parkinson Alliance, Whittier Parkinson's Disease Education Group, NINDS RO1 NS44327-1, NIA ( AG 21937) le US Army NETRP W81XWH-04-1-0444. MGV ke mofani oa lenaneo la USC Neuroscience Graduate Program ea Merit Fellowship. Re lakatsa ho leboha Ryan Park le Dr. Peter Conti ho tloha USC Small Animal Imaging Core bakeng sa ho thusoa ke litšoantšo tsa micro-PET le Dr. Rex Moats tse tsoang Setsing sa Lipatlisiso sa Liphoofolo Tse Nyenyane Setsing sa Saban sa Thuso bakeng sa thuso ea MRI ea mouse. Re rata ho leboha Yi-Hsuan (Lilian) Lai bakeng sa thuso ka ho ikoetlisa, le Avery Abernathy bakeng sa tsebo ea hae ea tlhahlobo ea HPLC. Re leboha Metsoalle ea Sehlopha sa Lipatlisiso sa mafu a USC Parkinson, ho akarelletsa George le MaryLou Boone, Walter le Susan Doniger, le Roberto Gonzales ka thuso ea bona e fanoeng ka seatla se bulehileng.

Likhohlano tse ka 'nang tsa etsahala: Ha ho letho le lokelang ho tlaleha.

Tlhokomeliso e kentsoe ka bopaki: Sehlooho sena se hatisitsoe Inthaneteng ka 19 October 2010. Phoso e ile ea tsejoa hamorao. Tlhokomeliso ena e kenyelelitsoe li-inthaneteng le ho hatisa liphetolelo ho bontša hore ka bobeli li lokisoe.

Litlhaloso tsa lichelete: Lenaneo la Thupelo ea USC Neuroscience Graduate Program (MV), NINDS RO1 NS44327-1 (MV, CW, JW, MJ le GP), Lenaneo la US Pilotte la Pilotte la USC CTSI (QL, AN, MJ, GP).