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

Författarna tackar Olga Sharma, Theresa Hundt, och personalen i DZNE mikroskopi och djuranläggningar för teknisk support.

Alla förfaranden, inklusive djur utfördes med godkännande av djurskyddskommittén Nordrhein-Westfalen State miljöbyrån (LANUV). Djuren inhystes och vårdas enligt standardförhållanden med en 12 timmar ljus / mörker-cykel och fri tillgång till mat och vatten. 1. Djurmodell <ol><li> Intern korsning hemizygot Tg (GFAP -luc +/-) möss med hemizygot Tg (M83 +/-) möss för att generera hemizygota bigenic Tg (M83 +/-: GFAP -luc +/-...

<ol>
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(61), (2012).</li><li>Jackson-Lewis, V., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+MPTP+mouse+model+of+Parkinson's+disease.">Protocol for the MPTP mouse model of Parkinson's disease.</a> Nat Protoc. 2 (1), 141-151 (2007).</li><li>Mathews, S. T., Plaisance, E. 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Perifer injektion av alfa-synuklein fibriller i peritoneum hos Tg (M83 +/-: GFAP -luc +/-) möss representerar en enkel metod för att inducera neurologisk sjukdom åtföljs av neuroinflammation att rekapitulera viktiga egenskaper hos synukleinopatier. På liknande sätt, representerar tungan injektion annan väg för neuroinvasion genom alfa-synuklein prionoids i transgena möss, men är mindre effektiv. Vi valde att avsluta våra experiment vid 420 dagar efter injektion och vi kan inte u...

Finns det växande bevis för att a-synuklein har egenskaper som liknar de hos prionproteinet, i synnerhet i dess förmåga att själv-utsäde och utbreder felveckning mellan celler och längs nervbanorna. Denna egenskap av alfa-synuklein är även kallad &#39;prion-liknande&#39; eller &#39;prionoid&#39;, och stöds av observationer i transplantationsexperiment, som antyder överförbar av felvikt alfasynuklein från sjuka neuroner till nyligen transplanterade friska nervceller 1, 2 , 3, 4. Även direkt injektion av felvikt alfa-synuklein i hjärnan eller periferin, t.ex. bakbensmuskeln eller tarmväggen, resulterar i en spridning av alfa-synuklein patologi till distala delar av CNS 5, 6, 7, &lt;sup class = &quot;xref&quot;&gt; 8, 9, 10. Vi analyserade överföring av alfa-synuklein prionoids via perifera rutter mer i detalj och adresserade frågan huruvida felvikt alfa-synuklein kan neuroinvade CNS efter en enda intraglossal eller intraperitoneal injektion, en funktion som tidigare hade visats för prioner men inte för felvikt alfa- synuklein. Efter injektion av prioner i tungan, är neuroinvasion av CNS uppnås via utbredning längs hypoglossalnerven av tungan som leder till kärnan hos hypoglossalnerven, som ligger i hjärnstammen 11. Som en musmodell vi valde Tg (M83 +/-: GFAP -luc +/-) möss som överuttrycker den A53T mutanten av humant alfa-synuklein från prion promotorn, och luciferas eldfluga under kontroll av en GFAP-promotor för att övervaka astrocytisk aktivering genom bioluminescens, såsom tidigare visats i hjärnan hosprion-infekterade möss 12. I våra händer bigenic Tg (M83 +/-: GFAP -luc +/-) möss inte utvecklar sjukdomen förrän 23 månaders ålder som har visats av andra 13. En enda injektion av humana alfa-synuklein fibriller via intraglossal eller intraperitoneal väg inducerad neurologisk sjukdom med patologi i hjärnan och ryggmärgen av Tg (M83 +/-: GFAP -luc +/-) möss som stöder hypotesen att alfa-synuklein prionoids dela viktiga egenskaper 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

För att studera prionliknande beteende felvikt alfa-synuklein, är musmodeller behövs som tillåter snabb och enkel transmission av alfa-synuklein prionoids, som orsakar neuropatologi i det centrala nervsystemet (CNS). Här beskriver vi att intraglossal eller intraperitoneal injektion av alfa-synuklein fibriller in bigenic Tg (M83 +/-: GFAP -luc +/-) möss, vilka överuttrycker humant alfa-synuklein med A53T mutation från prionproteinpromotorn och luciferas eldfluga från promotorn för gliafibrillärt surt protein (GFAP), är tillräcklig för att inducera neuropatologisk sjukdom. I jämförelse med homozygot Tg (M83 + / +) möss som utvecklar svåra neurologiska symtom börjar vid en ålder av 8 månader, heterozygot Tg (M83 +/-: GFAP -luc +/-) djur förblir fri från spontan sjukdom tills de når en ålder av 22 månader. Interestingly, injektion av alfa-synuklein fibriller via intraperitoneal rutt inducerad neurologisk sjukdom med förlamning i fyra av fem Tg (M83 +/-: GFAP -luc +/-) möss med en median inkubationstid av 229 ± 17 dagar. Sjuka djur visade allvarliga fyndigheter av fosforylerat alfa-synuklein i deras hjärnor och ryggmärg. Ansamlingar av alfa-synuklein var sarkosyl-olöslig och samlokaliserades med ubikvitin och p62, och åtföljdes av en inflammatorisk respons som resulterar i astrocytisk glios och microgliosis. Överraskande, inokulering av alfa-synuklein fibriller in i tungan var mindre effektiv i att orsaka sjukdom med endast en av fem injicerade djur som visar alfa-synuklein patologi efter 285 dagar. Våra resultat visar att ympning via intraglossal vägen och mer så via den intraperitoneala vägen är lämplig för att framkalla neurologisk sjukdom med relevanta kännetecken synukleinopatier i Tg (M83 +/-: GFAP -luc +/-) möss. Detta ger en ny modell för att studera prion-liknande patogenes framkallad av alfa-synuklein prionoids i större detalj.

Perifer injektion av alfa-synuklein prionoids via tungan eller peritoneum inducerad neuropatologi i CNS av bigenic Tg (M83 +/-: GFAP -luc +/-) möss (tabell 1 och figur 1). Efter en enda intraperitoneal injektion med alfa-synuklein fibriller, fyra av fem möss utvecklade neurologisk sjukdom med en median inkubationstid av 229 ± 17 dagar. Överraskande, endast en av fem möss utvecklade CNS-sjukdom efter intraglossal injekt...

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 av alfa-synuklein fibriller in i bukhinnan eller tunga av Tg (M83 +/-: GFAP -luc +/-) möss, vilka uttrycker humant alfa-synuklein med familjär A53T mutation och eldflugeluciferas, kan inducera neuropatologi, inklusive neuroinflammation , i deras centrala nervsystemet.

Bioluminescence avbildning av Neuroinflammation i transgena möss efter perifer ympning av alfa-synuklein 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

En tunn-skalle Fönster Teknik för kronisk två-photon In vivo Imaging av murina mikroglia i modeller av 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

Oral administrering av Rotenon med en sondmatning och bildanalys av alfa-synuklein införanden i det enteriska nervsystemet

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

Live avbildning av dorsala Axoner efter 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 av Lokalt Transgena råttor genom<em&gt; I livmodern</em&gt; Elektroporering och<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 ...

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

Transplantation av Lukt Ensheathing celler att utvärdera funktionell återhämtning efter perifer nervskada

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 och Bioluminescence avbildning av Subcellulär Ca<sup&gt; 2+</sup&gt; I Aged hippocampus nervceller

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

Sekventiell Utvinning av lösliga och olösliga alfa-synuklein från Parkinson Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

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

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

Imaging Neural aktivitet i den primära somatosensoriska Cortex med Thy1-GCaMP6s transgena möss

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

Exogena administrering av mikrosomer-associerade alfa-synuklein aggregat till primära nervceller som en kraftfull Cell modell av fibriller bildas

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