Name: gene-environment interaction models to unmask susceptibility mechanisms in parkinson's disease
Uploaded: 2014-01-07T13:45:00
Description: Watch this Scientific Journal Video about Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease at JoVE.com

Dette arbeidet ble finansiert av National Institutes of Health NIGMS 056062.

Merk: Alle dyreprosedyrer og dyrepleiemetoder bør godkjennes av institusjonens institusjonelle dyrepleie- og brukskomité (IACUC). Studien som er beskrevet her, ble utført i henhold til retningslinjene fastsatt av SRI Internationals IACUC. 
1. Oppkjøp og vedlikehold av LOX-mangelfulle mus
<ol><li>Kjøp 5-LOX-mangelfulle eller 12/15-LOX-mangelfulle mus og respektive belastnings- og sextilpassede kontroller i alderen 7-8 uker og la det ...

Utformingen av denne gen-miljø interaksjonsstudien tillot oss å få ny informasjon om den doble naturen til 5-LOX isozymet i nigrostriatalveien. Ved å utføre HPLC for å måle striatale monoaminer etter saltvanns- eller MPTP-behandling hos transgener som mangler 5-LOX-isozymet og deres wildtype-kull, kunne vi merke oss at mangelen ser ut til å være beskyttende under giftige forhold (figur 1), men under normale forhold reduserer mangel på enzymet striatal dopaminnivåer og kan være skadelig. Der...

Bruk av gen-miljø interaksjonsmodeller gir en tilnærming til å etterligne risikofaktorer som sannsynligvis påvirker idiopatisk Parkinsons sykdom (PD) og gir en mulighet til å skjelne mekanistisk innsikt som sannsynligvis ikke vil bli belyst ved bruk av et genetisk eller giftig system alene1,2. Her illustrerer vi dette punktet og beskriver anvendelsen av 1-metyl-4-fenyl-1,2,3,6-tetrahydropyridin (MPTP) musemodell av nigrostriatal degenerasjon3 for bedre å forstå selektiviteten til lipoksygenase (LOX) isozymaktivitet på nevroinflammasjon og toksisitet4. Mens en rolle for LOX isozymer har blitt mye evaluert i perifere lidelser5,6 samt CNS sykdom inkludert hjerneslag7 og Alzheimers sykdom8,9, rollen som familien av isozymer i nigrostriatal funksjon og degenerasjon relatert til PD er ikke godt forstått og garanterer studie. MPTP-nevrotoksinet demonstrerer preferansedegenerering av nigrostriatalbanen og rekapitulerer striatal dopaminnedbrytning og nigral dopaminerg celletap som ligger til grunn for motoriske svekkelser hos PD-pasienter10. Selv om denne modellen ikke reproduserer hele kaderet av ikke-motorisk og motorisk PD-oppførsel og frank α-synuclein-positiv Lewy kroppspatologi, har det vært nyttig å belyse nye mekanistiske mål som bidrar til nigrostriatal skade og for tidlig-stadium translasjonell testing som det er den best karakteriserte ikke-invasive modellen tilgjengelig for pålitelig å produsere nigral celledød ledsaget av striatal dopamin tap11-15. Bred bruk av MPTP-musen, med paradigmer som spenner fra akutt, subakutt til kronisk16-18, har tillatt standardisering av dosering for å resultere i mild til alvorlig nigrostriatal skade19,20 med aktivering av forskjellige mekanismer for toksisitet avhengig av behandlingsregimet18,21,22. Følgelig tillater dette et "vindu med lesjonering" som kan føre til forbedret eller redusert nigrostriatal skade avhengig av terapeutisk middel eller transgen modell som brukes23-25.
Også avgjørende for translasjons- og oppdagelsesbiologistudier er teknikkene som brukes til å vurdere skade og bevisene slike metoder gir. For MPTP-musemodellen er etablerte beregninger for å evaluere lesjoner måling av markører av striatal dopaminerg tone, inkludert dopamin og dets metabolitter av HPLC, og vestlig blotteanalyse av tyrosinhydroksylase (TH), det hastighetsbegrensende enzymet i dopaminsyntese og indikatorer på degenerative hendelser som glial aktivering ved hjelp av vestlig blotanalyse og immunohistokjemi4. Selv om disse er klassiske nevrokjemiske, biokjemiske og histologiske prosedyrer, gir teknikkene kritiske og reproduserbare avlesninger om skadeomfanget innenfor den nigrostriatale dopaminerge banen, indikerer mekanismer for toksisitet, og har vist seg å være verdifulle verktøy for å forstå degenerative hendelser i PD.

<table border="0" cellpadding="0" cellspacing="0" width="1227">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:481px;">1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP-HCL)</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:121px;">M0896</td>
			<td style="width:351px;">for PD modeling</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% Formaldehyde (paraformaldehyde) solution, phosphate-buffered (PFA)</td>
			<td>American MasterTech Scientific</td>
			<td>BUP0157</td>
			<td>for immersion fixation</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid ACS reagent, 70% (PCA)</td>
			<td>Sigma-Aldrich</td>
			<td>244252</td>
			<td>for HPLC acid extraction</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminotetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E1644</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphatase inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P5726</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>for Lowry protein assay</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose, molecular biology, &#8805;99.5% (GC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>for cryoprotection</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline, powder, pH 7.4 (for 0.01 M PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BCA Protein Assay Kit</td>
			<td>Pierce/Thermo</td>
			<td>23225</td>
			<td>for protein determination</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex 12% Tris-Glycine Mini Gels 1.0 mm, 12-well</td>
			<td>Invitrogen/Life Technologies</td>
			<td>EC60052BOX</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NuPAGE LDS Sample Buffer (4x)</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0007</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex Sharp Prestained Protein Standard&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>LC5800</td>
			<td>protein ladder</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate, electrophoresis, 98.5% (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methyl Alcohol, Anhydrous, Reagent&#160;</td>
			<td>American MasterTech Scientific</td>
			<td>SPM1057C</td>
			<td>methanol for transfer</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride (NaCl), ACS reagent</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>saline and buffers</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nonfat dry milk powder</td>
			<td>Carnation</td>
			<td>n/a</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ponceau S solution in 5% acetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P7170</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Tyrosine Hydroxylase (TH), sheep polyclonal</td>
			<td>Chemicon/Millipore</td>
			<td>AB1542</td>
			<td>for immunofluorescence&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Tyrosine Hydroxylase (TH), rabbit polyclonal</td>
			<td>Pel-Freez Biologicals</td>
			<td>P40101-0</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-&#946; Actin, rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>A2066</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Glial Fibrillary Acidic Protein (GFAP), rabbit polyclonal</td>
			<td>Chemicon/Millipore</td>
			<td>AB5804</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Glial Fibrillary Acidic Protein (GFAP), mouse monoclonal</td>
			<td>Covance Inc.</td>
			<td>SMI-22R</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated&#160;</td>
			<td>Fisher Scientific</td>
			<td>31462</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">goat anti-sheep, peroxidase conjugated</td>
			<td>Pierce/Thermo</td>
			<td>31480</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">goat anti-mouse, peroxidase conjugated</td>
			<td>Pierce/Thermo</td>
			<td>31430</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Pierce/Thermo</td>
			<td>34078</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CL-XPosure Film 7 in x 9.5 in&#160;</td>
			<td>Pierce/Thermo</td>
			<td>34089</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Restore Western Blot Stripping Buffer&#160;</td>
			<td>Pierce/Thermo</td>
			<td>21059</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric acid monohydrate, ACS reagent, &#8805;99.0%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Normal Donkey Serum</td>
			<td>Millipore</td>
			<td>S30-100ML</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Polyvinylpyrrolidone (PVP)</td>
			<td>Sigma-Aldrich</td>
			<td>P5288</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Bovine Serum Albumin (BSA), lyophilized</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-01</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Donkey anti-Rabbit IgG, Alexa Fluor 568-labeled&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A10042</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Sheep IgG (H+L), FITC&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>713-095-147</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASHIELD Hard-Set Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Goat Serum</td>
			<td>Millipore</td>
			<td>S26-100ML</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASTAIN ABC Kit (Rabbit IgG )&#160;</td>
			<td>Vector Laboratories</td>
			<td>PK-4001</td>
			<td>for IHC; 10 &#181;l each of solutions A and B per 1 ml PBS (per instructions )</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">DAB Peroxidase Substrate Kit, 3,3&#8217;-diaminobenzidine</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>for IHC; per 5 ml cold ddH2O, add 2 drops buffer stock solution, 2 drops DAB, and 1 drop H2O2 (H2O2 is added immediately before use)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrogen peroxide, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>216763</td>
			<td>for quench step in IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-Iba1</td>
			<td>Biocare Medicals</td>
			<td>CP290A</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:481px;">Cresyl Violet Solution, Regular Strength&#160;</td>
			<td>FD Neurotechnologies</td>
			<td>PS102-01&#160;</td>
			<td>counterstain for Iba1 IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">95% Ethanol, reagent alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R8382</td>
			<td>dehydration for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Absolute ethanol</td>
			<td>Mallinckrodt&#160;</td>
			<td>7019-10</td>
			<td>dehydration for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>destaining for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>clearing agent for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPX Mountant</td>
			<td>Sigma-Aldrich</td>
			<td>06522</td>
			<td>mounting medium for DAB IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O.C.T. Compound - Frozen Section Embedding Medium&#160;</td>
			<td>American MasterTech Scientific</td>
			<td>EMOCTCS</td>
			<td>embeddium medium for cryostat cutting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium permanganate</td>
			<td>Sigma-Aldrich</td>
			<td>223468</td>
			<td>to decontaminate DAB solution</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dopamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,4-Dihydroxyphenylacetic acid (DOPAC)</td>
			<td>Sigma-Aldrich</td>
			<td>850217</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homovanillic acid (HVA)</td>
			<td>Sigma-Aldrich</td>
			<td>H1252</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid (PCA) - 70%</td>
			<td>Sigma-Aldrich</td>
			<td>244252</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dihydrogen phosphate monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>71504</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Octanesulfonic acid sodium salt (OSA)</td>
			<td>Sigma-Aldrich</td>
			<td>O8380</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E1644</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>EMD</td>
			<td>AX0145-1</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC-grade distilled deionized water (ddH2O)</td>
			<td>Millipore</td>
			<td></td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.22 &#181;m GSTF membrane</td>
			<td>Millipore</td>
			<td></td>
			<td>for filtration</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Netwells</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3477</td>
			<td>polystyrene insert with polyester mesh bottom, for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cell disrupter (Soniprep 150)</td>
			<td>MSE</td>
			<td>MSE.41371.274</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5414R</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ESA MD-150 reverse-phase column&#160;</td>
			<td>ESA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Pump (Ultimate 3000)</td>
			<td>Dionex</td>
			<td>ISO-3100BM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Autosampler (Ultimate 3000)</td>
			<td>Dionex</td>
			<td>WPS-3000TSL</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electrochemical detector</td>
			<td>ESA</td>
			<td>Coulochem III</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Guard Cell</td>
			<td>ESA</td>
			<td>5020</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analytical Cell</td>
			<td>ESA</td>
			<td>5011A</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromeleon software</td>
			<td>Dionex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse E400</td>
			<td>Nikon</td>
			<td>E400</td>
			<td>light/fluorescent microscope</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable mouse cage</td>
			<td>Ancare</td>
			<td>N10HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfilter top</td>
			<td>Ancare</td>
			<td>N10MBT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-LOX- deficient mice</td>
			<td>The Jackson Laboratory</td>
			<td>004155</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12/15-LOX-deficient mice</td>
			<td>The Jackson Laboratory</td>
			<td>002778</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

gene-environment interaction models to unmask susceptibility mechanisms in parkinson's disease

Lipoksygenase (LOX) aktivitet har vært involvert i nevrodegenerative lidelser som Alzheimers sykdom, men dens effekter i Parkinsons sykdom (PD) patogenese er mindre forstått. Gen-miljø interaksjonsmodeller har nytte i å avdekke virkningen av spesifikke cellulære veier i toksisitet som kanskje ikke observeres ved hjelp av en utelukkende genetisk eller toksisk sykdomsmodell alene. For å vurdere om distinkte LOX-isozymer selektivt bidrar til PD-relatert nevrodegenerasjon, kan transgene (dvs. 5-LOX og 12/15-LOX mangelfulle) mus utfordres med et giftstoff som etterligner celleskade og død i lidelsen. Her beskriver vi bruken av et nevrotoksin, 1-metyl-4-fenyl-1,2,3,6-tetrahydropyridin (MPTP), som produserer en nigrostriatal lesjon for å belyse de distinkte bidragene fra LOX-isozymer til nevrodegenerasjon relatert til PD. Bruken av MPTP i mus, og ikke-umenneskelig primat, er veletablert for å rekapitulere nigrostriatal skade i PD. Omfanget av MPTP-indusert lesjon måles ved HPLC-analyse av dopamin og dets metabolitter og semi-kvantitative vestlige blotanalyse av striatum for tyrosinhydroksylase (TH), det hastighetsbegrensende enzymet for syntesen av dopamin. For å vurdere inflammatoriske markører, som kan demonstrere LOX isozyme-selektiv følsomhet, utføres glial fibrillært surt protein (GFAP) og Iba-1 immunhiistokjemi på hjerneseksjoner som inneholder substantia nigra, og GFAP Western blotanalyse utføres på striatale homogenater. Denne eksperimentelle tilnærmingen kan gi ny innsikt i gen-miljø interaksjoner underliggende nigrostriatal degenerasjon og PD.

Dette toksineksponeringsparadigmet kan produsere en betydelig og påviselig 20% striatal dopaminuttømming hos MPTP- vs. saltvannsinjiserte dyr. Det er viktig å merke seg at forskjellige mange MPTP kan gi litt mer eller mindre lesjon; For bedre presisjon anbefales derfor et foreløpig eksperiment i wildtype mus før bruk i transgener når en ny masse nevrotoksin brukes. Bruken av mild til moderat lesjon gjør det mulig å observere virkningen av transgene; en alvorlig lesjon kan gi en "gulveffekt" med skade for robust ...

Watch this Scientific Journal Video about Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease at JoVE.com

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoksygenase (LOX) isozymer kan generere produkter som kan øke eller redusere nevroinflammasjon og nevrodegenerasjon. En gen-miljø interaksjonsstudie kan identifisere LOX isozym-spesifikke effekter. Ved hjelp av 1-metyl-4-fenyl-1,2,3,6-tetrahydropyridin (MPTP) modell av nigrostriatal skade i to LOX isozyme-mangelfulle transgene linjer tillater sammenligning av bidrag av LOX isozymer på dopaminerg integritet og betennelse.

Gen-miljø interaksjonsmodeller for å avdekke følsomhetsmekanismer ved Parkinsons sykdom

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Research

JoVE Journal

Medicine

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

Utvikling av en ensidig-lesioned 6-OHDA mus modell av Parkinsons sykdom

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI Imaging massespektrometri av nevropeptider i Parkinsons sykdom

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Skin Punch Biopsi Eksplantering Kultur for Utledning av primære humane fibroblaster

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Bruk av primære humane fibroblaster for overvåking Mitokondrielle Fenotyper i feltet av Parkinsons sykdom

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Kontrollere Parkinsons sykdom med Adaptive dyp hjernestimulering

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

Tilpasse menneske Videofluoroscopic Swallow Study Metoder for å påvise og karakterisere Dysfagi i murine sykdomsmodeller

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ex Vivo Tarm Sacs å vurdere slimhinner Permeabilitet i modeller av gastrointestinal sykdom

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

Utvikling av en Alpha-synuclein Based Rat Modell for Parkinsons sykdom via Stereo Injeksjon av en rekombinant adenoassosiert Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

kvantitativ 3D<em&gt; I Silico</em&gt; Modeling (q3DISM) av cerebral amyloid-beta Fagocytose i gnagermodeller av Alzheimers sykdom

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Gjentatt måling av respiratorisk muskelaktivitet og ventilasjon i musemodeller av neuromuskulær Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...