Name: bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils
Uploaded: 2017-04-13T13:00:00
Description: Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

De auteurs danken Olga Sharma, Theresa Hundt, en het personeel van de DZNE microscopie en dierlijke faciliteiten voor technische ondersteuning.

Alle procedures met inbegrip van dieren werden uitgevoerd met toestemming van de dierenbescherming commissie van Noord-Rijnland-Westfalen State Environment Agency (LANUV). Dieren werden gehuisvest en verzorgd volgens standaardomstandigheden met een 12 uur licht / donkercyclus en vrije toegang tot voedsel en water. 1. Dierlijke Model <ol><li> Kruisen hemizygoot Tg (GFAP -Luc +/-) muizen met hemizygoot Tg (M83 +/-) muizen hemizygoot bigene Tg (M83 +/-:</su...

<ol>
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Perifere injectie van alfa-synucleine fibrillen in het peritoneum van Tg (M83 +/-: GFAP -Luc +/-) muizen is een gemakkelijke werkwijze voor neurologische ziekte gepaard gaat met inflammatoire processen induceert belangrijke kenmerken van synucleïne interageren recapituleren. Evenzo tong injectie vertegenwoordigt een andere weg voor neuroinvasion door alfa-synucleine prionoids in transgene muizen, maar minder efficiënt. We kozen ervoor om onze experimenten te beëindigen op 420 dagen na d...

Er is groeiend bewijs dat alfa-synucleine heeft kenmerken die lijken op die van het prion eiwit, met name in zijn vermogen om uitzaaien en propageren verkeerd vouwen tussen cellen en langs zenuwbanen. Deze eigenschap van alpha-synuclein wordt ook wel &#39;prion-achtige&#39; of &#39;prionoid&#39;, en wordt ondersteund door waarnemingen bij transplantatie-experimenten, waarbij de overdraagbaarheid van verkeerd gevouwen alfa-synucleine van aangetaste neuronen suggereren nieuw getransplanteerde gezonde neuronen 1, 2 , 3, 4. Ook directe injectie van verkeerd gevouwen alfa-synucleïne in de hersenen of de periferie, zoals het achterbeen spier of darmwand leidt tot een spreiding van alpha-synuclein pathologie distale delen van het CNS 5, 6, 7, &lt;sup class = &quot;xref&quot;&gt; 8, 9, 10. We analyseerden overdracht van alfa-synucleine prionoids via perifere routes nader en de vraag of verkeerd gevouwen alpha-synuclein het CNS kan neuroinvade na één intraglossal of intraperitoneale injectie, een eigenschap die eerder was aangetoond voor prionen maar niet te verkeerd gevouwen alfa- geadresseerd synucleïne. Na injectie van prionen in de tong is neuroinvasion van het CZS bereikt door voortplanting langs de hypoglossale zenuw van de tong tot de kern van de hypoglossuszenuw, die is gelegen in de hersenstam 11. Als muismodel we kozen Tg (M83 +/-: GFAP -Luc +/-) muizen die de A53T mutant van humaan alfa-synucleine van het prion promoter tot overexpressie brengen en vuurvlieg luciferase onder controle van een GFAP promotor astrocytaire activering door toezicht bioluminescentie, zoals eerder aangetoond in de hersenen vanprion-geïnfecteerde muizen 12. In onze handen bigene Tg (M83 +/-: GFAP -Luc +/-) heeft muizen niet ontwikkelen van de ziekte tot 23 maanden oud, zoals is aangetoond door anderen 13. Een enkele injectie van humaan alfa-synucleine fibrillen via intraglossal of intraperitoneale route geïnduceerde neurologische ziekte pathologie in de hersenen en ruggenmerg van Tg (M83 +/-: GFAP -Luc +/-) muizen ondersteunt de hypothese dat alfa-synucleine prionoids deel belangrijke kenmerken met prionen 14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-actin antibody</td>
			<td>Merck Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [pSyn#64]</td>
			<td>Wako</td>
			<td>015-25191</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [EP1536Y]</td>
			<td>Abcam</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFAP antibody</td>
			<td>Dako</td>
			<td>Z0334 01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA-1 antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sequestosome-1 (p62) antibody</td>
			<td>Proteintech</td>
			<td>18420-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ubiquitin antibody [Ubi-1]</td>
			<td>Merck Millipore</td>
			<td>MAB1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>100 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>10 mg/kg</td>
		</tr>
		<tr>
			<td>27-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal Healthcare</td>
			<td>PZN&#160; 4831850</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secureline lab marker&#160;</td>
			<td>Neolab</td>
			<td>25040</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin potassium salt</td>
			<td>Acris</td>
			<td>LK10000</td>
			<td>30 mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5776671</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonopuls Mini20 sonicator</td>
			<td>Bandelin</td>
			<td>3648</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image 3.0 Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>004479</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>N-laurylasarcosyl</td>
			<td>Sigma</td>
			<td>L5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>TLA-110 rotor&#160;</td>
		</tr>
		<tr>
			<td>Thickwall polycarbonate tubes</td>
			<td>Beckman Coulter</td>
			<td>362305</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAG Novex 4-12% Bis-Tris Midi Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>WG1401BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated antibody</td>
			<td>Cayman</td>
			<td>Cay10004301-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Dye 680 conjugated antibody&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34075</td>
			<td></td>
		</tr>
		<tr>
			<td>Stella 3200 imaging system</td>
			<td>Raytest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey infrared imaging system&#160;</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td>MP Biomedicals</td>
			<td>TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SA/T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF membrane</td>
			<td>Merck Millipore</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol</td>
			<td>Sigma</td>
			<td>Roth</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma</td>
			<td>SA/00216763/000500</td>
			<td>working solution 3%</td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3294-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td>working dilution 1:50,000</td>
		</tr>
		<tr>
			<td>Fluoromount media&#160;</td>
			<td>Omnilab</td>
			<td>SA/F4680/000025</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALT protease and phosphatase inhibitors</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>10516495</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24-Dual homogenizer&#160;</td>
			<td>Peqlab</td>
			<td>91-PCS24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31619</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160;BCA&#160;Protein&#160;Assay&#160;Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>10741395</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome RM2255</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils

De prion-achtige gedrag van verkeerd gevouwen alfa-synucleine bestuderen, zijn muismodellen nodig die snelle en eenvoudige overdracht van alfa-synucleïne prionoids die neuropathologie veroorzaken in het centrale zenuwstelsel (CZS) laten. Hier beschrijven we intraglossal of intraperitoneale injectie van alfa-synucleine fibrillen in bigene Tg (M83 +/-: GFAP -Luc +/-) muizen, die humaan alfa-synucleine tot overexpressie met A53T mutatie van het prioneiwit promoter en vuurvlieg luciferase van de promotor voor glia fibrillair zuur eiwit (GFAP), volstaat de neuropathologische ziekte induceren. In vergelijking met homozygote Tg (M83 + / +) muizen die ernstige neurologische symptomen beginnen op een leeftijd van 8 maanden, heterozygote Tg (M83 +/-: GFAP -Luc +/-) ontwikkelen dieren vrij blijven van spontane ziekte totdat ze een te bereiken leeftijd van 22 maanden. Interessant injectie van alfa-synucleine fibrillen via intraperitoneal route geïnduceerde neurologische ziekte met verlamming in vier van de vijf Tg (M83 +/-: GFAP -Luc +/-) muizen met een gemiddelde incubatietijd van 229 ± 17 dagen. Zieke dieren vertoonden ernstige afzettingen van gefosforyleerde alfa-synucleine in hun hersenen en ruggenmerg. Ophopingen van alfa-synucleïne waren Sarkosyl onoplosbare en colocalized met ubiquitine en p62, onder begeleiding van een ontstekingsreactie resulteert in astrocytische gliose en microgliosis. Verrassenderwijs inoculatie van alfa-synucleine fibrillen in de tong minder effectief bij het veroorzaken van ziekte met slechts een van de vijf geïnjecteerde dieren met alpha-synuclein pathologie na 285 dagen. Onze bevindingen tonen aan dat enting via de intraglossal route en nog meer via de intraperitoneale route is geschikt voor neurologische ziekte met relevante kenmerken van synucleïnopathieën induceren in Tg (M83 +/-: GFAP -Luc +/-) muizen. Dit zorgt voor een nieuw model voor het bestuderen van prion-achtige pathogenese inducerend door alfa-synucleine prionoids nader.

Perifere injectie van alfa-synucleïne prionoids via de tong of het buikvlies geïnduceerde neuropathologie in het CZS van bigene Tg (M83 +/-: GFAP -Luc +/-) muizen (Tabel 1 en Figuur 1). Na een enkele intraperitoneale injectie met alfa-synucleine fibrillen, vier van de vijf muizen ontwikkelden neurologische ziekte met een mediane incubatietijd van 229 ± 17 dagen. Verrassend is dat slechts een van de vijf muizen ontwikkeld...

Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

Perifere injectie van alfa-synucleine fibrillen in het peritoneum of de tong van Tg (M83 +/-: GFAP -Luc +/-) muizen, die humaan alfa-synucleine met familiaire A53T mutatie en vuurvlieg luciferase tot expressie brengen, kunnen neuropathologie induceren, waaronder neuroinflammatie in het centrale zenuwstelsel.

Bioluminescentie beeldvorming van ontstekingsprocessen in transgene muizen Na Perifere Enten van alfa-synucleine fibrillen

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Research

JoVE Journal

Neuroscience

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Een dunne-schedel Window Techniek voor chronische Twee-foton In vivo Imaging Murine Microglia in modellen van ontstekingsprocessen

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Live Imaging of Dorsal Root Axons after Rhizotomy

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Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generatie Topisch transgene Rats door<em&gt; In de baarmoeder</em&gt; Elektroporatie en<em&gt; In vivo</em&gt; Bioluminescence Screening

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

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Fluorescentie en Bioluminescentie beeldvorming van Subcellulaire Ca<sup&gt; 2+</sup&gt; In Aged hippocampus Neuronen

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

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The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...