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

Forfatterne takker Olga Sharma, Theresa Hundt, og personalet i de DZNE mikroskopi og dyrefaciliteter for teknisk support.

Alle procedurer, herunder dyr blev udført med godkendelse af dyrebeskyttelse udvalg af Nordrhein-Westfalen Stat Miljøagentur (LANUV). Dyrene blev holdt og plejes i overensstemmelse med standardbetingelser med en 12 timers lys / mørkecyklus og fri adgang til foder og vand. 1. Animalske Model <ol><li> Intercross hemizygote Tg (GFAP -Luc +/-) mus med hemizygote Tg (M83 +/-) mus til at generere hemizygote bigenic Tg (M83 +/-: GFAP -Luc <sup...

<ol>
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Perifer injektion af alfa-synuclein fibriller i peritoneum af Tg (M83 +/-: GFAP -Luc +/-) -mus betegner en let metode til at inducere neurologisk sygdom ledsaget af neuroinflammation at rekapitulere vigtige kendetegn ved synucleinopatier. Tilsvarende tungen injektion repræsenterer en anden rute for neuroinvasion med alfa-synuclein prionoids i transgene mus, men er mindre effektiv. Vi valgte at opsige vores eksperimenter på 420 dage efter injektion, og vi kan ikke udelukke, at flere eller...

Der er stigende tegn på, at alfa-synuclein har egenskaber, der ligner dem af prionproteinet, især i dets evne til at selv-frø og udbreder misfoldning mellem celler og langs nervebaner. Denne egenskab af alfa-synuclein er også benævnt &#39;prion-lignende&#39; eller &#39;prionoid&#39;, og er støttet af observationer i transplantationsforsøg, der antyder muligheden for overførsel af fejlfoldet alfa-synuclein fra syge neuroner til nyligt transplanterede sunde neuroner 1, 2 , 3, 4. Også direkte injektion af misfoldet alfa-synuclein i hjernen eller i periferien, f.eks bagbenet muskel eller tarmvæggen, resulterer i en spredning af alfa-synuclein patologi til distale dele af CNS 5, 6, 7, &lt;sup class = &quot;xref&quot;&gt; 8, 9, 10. Vi analyserede transmission af alfa-synuclein prionoids via perifere ruter mere detaljeret og behandlet spørgsmålet om, hvorvidt misfoldet alfa-synuclein kan neuroinvade CNS efter en enkelt intraglossal eller intraperitoneal injektion, en funktion, der tidligere var blevet vist for prioner, men ikke for misfoldet alfa- synuclein. Efter injektion af prioner i tungen, er neuroinvasion af CNS opnås via udbredelse langs nervus hypoglossus af tungen, der fører til kernen af nerven under tungen, som er placeret i hjernestammen 11. Som en musemodel vi valgte Tg (M83 +/-: GFAP -Luc +/-) mus, som overudtrykker A53T mutant af humant alfa-synuclein fra prion-promotoren, og ildflueluciferase under kontrol af en GFAP promotor for at overvåge astrocytaktivering ved bioluminescens, som tidligere vist i hjernen hosprion-inficerede mus 12. I vores hænder bigenic Tg (M83 +/-: GFAP -Luc +/-) gjorde musene ikke udvikle sygdommen indtil 23 måneder, som er blevet vist af andre 13. En enkelt injektion af humane alfa-synukleinfibriller via intraglossal eller intraperitoneale vej induceret neurologisk sygdom med patologi i hjernen og rygmarven hos Tg (M83 +/-: GFAP -Luc +/-) mus understøtter den hypotese, at alfa-synuclein prionoids dele vigtige karakteristika med prioner 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

For at undersøge prion-lignende opførsel af misfoldet alfa-synuclein, er brug musemodeller, der tillader hurtig og enkel transmission af alfa-synuclein prionoids, som forårsager neuropatologi i centralnervesystemet (CNS). Her beskriver vi, at intraglossal eller intraperitoneal injektion af alfa-synuclein fibriller i bigenic Tg (M83 +/-: GFAP -Luc +/-) mus, der overudtrykker humant alfa-synuclein med A53T mutation fra prionprotein-promotoren og ildflueluciferase fra promotoren for glial fibrillært surt protein (GFAP), er tilstrækkelig til at inducere neuropatologisk sygdom. I sammenligning med homozygot Tg (M83 + / +) mus, der udvikler alvorlige neurologiske symptomer begynder i en alder af 8 måneder, heterozygote Tg (M83 +/-: GFAP -Luc +/-) dyr forblive fri for spontan sygdom, indtil de når en alder på 22 måneder. Interessant injektion af alfa-synukleinfibriller via intraperitoneal rute induceret neurologisk sygdom med lammelse i fire af fem Tg (M83 +/-: GFAP -Luc +/-) mus med en median inkubationstid på 229 ± 17 dage. Syge dyr viste alvorlige forekomster af phosphoryleret alfa-synuclein i deres hjerner og rygmarv. Ophobninger af alfa-synuclein var sarkosyl-uopløselige og co-lokaliseret med ubiquitin og p62, og var ledsaget af en inflammatorisk respons, der resulterer i astrocytisk gliose og mikrogliose. Overraskende podning af alfa-synuclein fibriller i tungen var mindre effektiv til at forårsage sygdom med kun én af fem injicerede dyr viser alfa-synuclein patologi efter 285 dage. Vores resultater viser, at podning via intraglossal rute og mere så via den intraperitoneale vej er egnet til at fremkalde neurologisk sygdom med relevante kendetegnende for synucleinopatier i Tg (M83 +/-: GFAP -Luc +/-) mus. Dette giver en ny model til at studere prion-lignende patogenese fremkalded fra alfa-synuclein prionoids mere detaljeret.

Perifer injektion af alfa-synuclein prionoids via tungen eller bughinden induceret neuropatologi i CNS af bigenic Tg (M83 +/-: GFAP -Luc +/-) mus (tabel 1 og figur 1). Efter en enkelt intraperitoneal injektion med alfa-synukleinfibriller, fire af fem mus udviklede neurologiske sygdom med en median inkubationstid på 229 ± 17 dage. Overraskende kun én af fem mus udviklede CNS sygdom efter intraglossal injektion med alfa-sy...

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

Perifer injektion af alfa-synuclein fibriller i peritoneum eller tunge af Tg (M83 +/-: GFAP -Luc +/-) mus, som udtrykker humant alfa-synuclein med familiær A53T mutation og ildflueluciferase, kan inducere neuropatologi, herunder neuroinflammation , i det centrale nervesystem.

Bioluminescens Billeddannelse af Neuroinflammation i transgene mus efter perifer Inokulering af alfa-synuclein Fibriller

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

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

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

Generering af Topisk Transgene rotter ved<em&gt; In Utero</em&gt; Elektroporation og<em&gt; In vivo</em&gt; Bioluminescens Screening

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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Transplantation af olfaktoriske Ensheathing Cells at evaluere Funktionel Recovery efter perifer nerveskade

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

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

Fluorescens og Bioluminescens Imaging af Subcellulær Ca<sup&gt; 2+</sup&gt; I Aged hippocampusneuroner

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

Sekventiel Udvinding af opløselige og uopløselige Alpha-Synuclein fra Parkinson Brains

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Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

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Imaging neurale aktivitet i den primære somatosensoriske Cortex ved hjælp af Thy1-GCaMP6s Transgene mus

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

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

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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 ...