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 de ansatte på DZNE mikroskopi og dyre fasiliteter for teknisk støtte.

Alle prosedyrer inkludert dyrene ble utført med godkjenning av dyrevern komiteen i Nordrhein-Westfalen State Environment Agency (LANUV). Dyrene ble huset og stelles i henhold til standardbetingelser med en 12 timers lys / mørke syklus og fri adgang til mat og vann. 1. Animal Model <ol><li> Intercross hemizygous Tg (GFAP -luc +/-) mus med hemizygous Tg (M83 +/-) mus for å generere hemizygous bigenic Tg (M83 +/-: GFAP -luc +/-)...

<ol>
	<li>Aguzzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology:+Beyond+the+prion+principle.">Cell biology: Beyond the prion principle.</a> Nature. 459 (7249), 924-925 (2009).</li><li>Kordower, J. H., Chu, Y., Hauser, R. A., Freeman, T. B., Olanow, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body-like+pathology+in+long-term+embryonic+nigral+transplants+in+Parkinson's+disease.">Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease.</a> Nat Med. 14 (5), 504-506 (2008).</li><li>Li, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+bodies+in+grafted+neurons+in+subjects+with+Parkinson's+disease+suggest+host-to-graft+disease+propagation.">Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation.</a> Nat Med. 14 (5), 501-503 (2008).</li><li>Hansen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Synuclein+propagates+from+mouse+brain+to+grafted+dopaminergic+neurons+and+seeds+aggregation+in+cultured+human+cells.">alpha-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells.</a> J Clin Invest. 121 (2), 715-725 (2011).</li><li>Masuda-Suzukake, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+spreading+of+pathological+alpha-synuclein+in+brain.">Prion-like spreading of pathological alpha-synuclein in brain.</a> Brain. 136 (4), 1128-1138 (2013).</li><li>Holmqvist, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+Parkinson+pathology+spread+from+the+gastrointestinal+tract+to+the+brain+in+rats.">Direct evidence of Parkinson pathology spread from the gastrointestinal tract to the brain in rats.</a> Acta Neuropathol. 128 (6), 805-820 (2014).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+alpha-synuclein+transmission+initiates+Parkinson-like+neurodegeneration+in+nontransgenic+mice.">Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice.</a> Science. 338 (6109), 949-953 (2012).</li><li>Sacino, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+CNS+alpha-synuclein+pathology+by+fibrillar+and+non-amyloidogenic+recombinant+alpha-synuclein.">Induction of CNS alpha-synuclein pathology by fibrillar and non-amyloidogenic recombinant alpha-synuclein.</a> Acta Neuropathol Commun. 1, 38 (2013).</li><li>Sacino, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramuscular+injection+of+alpha-synuclein+induces+CNS+alpha-synuclein+pathology+and+a+rapid-onset+motor+phenotype+in+transgenic+mice.">Intramuscular injection of alpha-synuclein induces CNS alpha-synuclein pathology and a rapid-onset motor phenotype in transgenic mice.</a> Proc Natl Acad Sci U S A. 111 (29), 10732-10737 (2014).</li><li>Mougenot, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+acceleration+of+a+synucleinopathy+in+a+transgenic+mouse+model.">Prion-like acceleration of a synucleinopathy in a transgenic mouse model.</a> Neurobiol Aging. 33 (9), 2225-2228 (2012).</li><li>Bartz, J. C., Kincaid, A. E., Bessen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prion+neuroinvasion+following+tongue+infection.">Rapid prion neuroinvasion following tongue infection.</a> J Virol. 77 (1), 583-591 (2003).</li><li>Tamg&#252;ney, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+prions+by+bioluminescence+imaging.">Measuring prions by bioluminescence imaging.</a> Proc Natl Acad Sci U S A. 106 (35), 15002-15006 (2009).</li><li>Watts, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+multiple+system+atrophy+prions+to+transgenic+mice.">Transmission of multiple system atrophy prions to transgenic mice.</a> Proc Natl Acad Sci U S A. 110 (48), 19555-19560 (2013).</li><li>Breid, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinvasion+of+alpha-Synuclein+Prionoids+after+Intraperitoneal+and+Intraglossal+Inoculation.">Neuroinvasion of alpha-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation.</a> J Virol. 90 (20), 9182-9193 (2016).</li><li>Giasson, B. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+alpha-synucleinopathy+with+severe+movement+disorder+in+mice+expressing+A53T+human+alpha-synuclein.">Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein.</a> Neuron. 34 (4), 521-533 (2002).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+GFAP+expression+after+neuronal+damage+in+mice.">Non-invasive imaging of GFAP expression after neuronal damage in mice.</a> Neurosci Lett. 367 (2), 210-212 (2004).</li><li>Bernis, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+propagation+of+human+brain-derived+alpha-synuclein+in+transgenic+mice+expressing+human+wild-type+alpha-synuclein.">Prion-like propagation of human brain-derived alpha-synuclein in transgenic mice expressing human wild-type alpha-synuclein.</a> Acta Neuropathol Commun. 3 (1), 75 (2015).</li><li>Gunther, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+testing+and+spinal+cord+removal+in+a+mouse+model+for+amyotrophic+lateral+sclerosis+(ALS).">Clinical testing and spinal cord removal in a mouse model for amyotrophic lateral sclerosis (ALS).</a> J Vis Exp. (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. P., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+systems+for+westerns:+chemiluminescence+vs.+infrared+detection.">Imaging systems for westerns: chemiluminescence vs. infrared detection.</a> Methods Mol Biol. 536, 499-513 (2009).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+bacterial+endotoxin+generates+a+distinct+strain+of+alpha-synuclein+fibril.">Exposure to bacterial endotoxin generates a distinct strain of alpha-synuclein fibril.</a> Sci Rep. 6, 30891 (2016).</li><li>Keller, A. F., Gravel, M., Kriz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+amyotrophic+lateral+sclerosis+pathogenesis:+disease+onset+is+characterized+by+marked+induction+of+GFAP+in+Schwann+cells.">Live imaging of amyotrophic lateral sclerosis pathogenesis: disease onset is characterized by marked induction of GFAP in Schwann cells.</a> Glia. 57 (10), 1130-1142 (2009).</li><li>St&#246;hr, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+synthetic+Abeta+prion+strains+producing+different+amyloid+deposits+in+bigenic+mice.">Distinct synthetic Abeta prion strains producing different amyloid deposits in bigenic mice.</a> Proc Natl Acad Sci U S A. , (2014).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+inoculation+of+pathological+alpha-synuclein+initiates+a+rapidly+progressive+neurodegenerative+alpha-synucleinopathy+in+mice.">Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice.</a> J Exp Med. 209 (5), 975-986 (2012).</li><li>Recasens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+extracts+from+Parkinson+disease+brains+trigger+alpha-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys.">Lewy body extracts from Parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys.</a> Ann Neurol. 75 (3), 351-362 (2014).</li><li>Sacino, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+CNS+alpha-synuclein+pathology+by+fibrillar+and+non-amyloidogenic+recombinant+alpha-synuclein.">Induction of CNS alpha-synuclein pathology by fibrillar and non-amyloidogenic recombinant alpha-synuclein.</a> Acta Neuropathol Commun. 1 (1), 38 (2013).</li><li>Peelaerts, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Synuclein+strains+cause+distinct+synucleinopathies+after+local+and+systemic+administration.">alpha-Synuclein strains cause distinct synucleinopathies after local and systemic administration.</a> Nature. 522 (7556), 340-344 (2015).</li></ol>

Perifer injeksjon av alfa-synukleinfibriller i peritoneum til Tg (M83 +/-: GFAP -luc +/-) mus representerer en lettvint fremgangsmåte for å indusere nevrologisk sykdom ledsaget av nevroinflammasjon å rekapitulere viktige kjennetegn ved synukleinopatier. Tilsvarende, representerer tunge injeksjon annen rute for neuroinvasion av alfa-synuclein prionoids i transgene mus, men er mindre effektiv. Vi valgte å si opp våre forsøk på 420 dager etter injeksjon, og vi kan ikke utelukke at fler...

Det er økende bevis for at alpha-synuclein har egenskaper som er lik de av prion-protein, spesielt i dens evne til selv-frø og forplanter misfolding mellom celler og langs nervebaner. Denne egenskapen av alfa-synuclein blir også referert til som &#39;prion-lignende&#39; eller &#39;prionoid&#39;, og er støttet av observasjoner i transplantasjonsforsøk, noe som tyder transmissibilitet misfoldet alpha-synuclein fra syke neuroner til nylig transplantert friske nerveceller 1, 2 , 3, 4. Også direkte injeksjon av misfoldet alpha-synuclein inn i hjernen eller i periferien, f.eks bakbenområdet muskel eller tarmveggen, resulterer i en spredning av alfa-synuclein patologi for å distale deler av CNS 5, 6, 7, &lt;sup class = &quot;ekstern referanse&quot;&gt; 8, 9, 10. Vi analyserte overføring av alpha-synuclein prionoids via perifere ruter i mer detalj, og om spørsmålet hvorvidt misfoldet alpha-synuclein kan neuroinvade CNS etter en enkelt intraglossal eller intraperitoneal injeksjon, en funksjon som tidligere hadde blitt vist for prioner, men ikke for misfoldet alfa- synuclein. Etter injeksjon av prioner inn i tungen, blir neuroinvasion av CNS oppnås via forplantning langs hypoglossus nerve av tungen som fører til kjernen av hypoglossus nerve, som er lokalisert i hjernestammen 11. Som en musemodell valgte Tg (M83 +/-: GFAP -luc +/-) mus som overuttrykker den A53T mutant av human alfa-synuclein fra prion-promoteren, og ildflueluciferase under kontroll av en promoter for å overvåke GFAP astrocytic aktivering av bioluminescens, som tidligere vist i hjernen hosprion-infiserte mus 12. I våre hender bigenic Tg (M83 +/-: GFAP -luc +/-) mus ikke utvikler sykdommen før 23 måneders alder som har blitt vist av andre 13. En enkelt injeksjon av humane alfa-synukleinfibriller via intraglossal eller intraperitoneal vei indusert nevrologisk sykdom med patologi i hjernen og ryggmargen av Tg (M83 +/-: GFAP -luc +/-) mus som støtter den hypotese at alpha-synuclein prionoids dele viktige 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

For å studere prion-lignende oppførsel av misfoldet alpha-synuclein, blir musemodeller nødvendig for at muliggjøre rask og enkel overføring av alpha-synuclein prionoids, som forårsaker nevro innenfor sentralnervesystemet (CNS). Her beskriver vi at intraglossal eller intraperitoneal injeksjon av alfa-synukleinfibriller til bigenic Tg (M83 +/-: GFAP -luc +/-) mus som overuttrykker humant alfa-synuclein med A53T mutasjon fra den prionprotein promoteren og ildflueluciferase fra promoteren for glialt fibrillært surt protein (GFAP), er tilstrekkelig til å indusere neuropathologic sykdom. I forhold til homozygot Tg (M83 + / +) mus som utvikler alvorlig nevrologiske symptomer som begynner ved en alder på 8 måneder, heterozygot Tg (M83 +/-: GFAP -luc +/-) dyr forblir fri for spontan sykdom inntil de når en alder av 22 måneder. Interessant, injeksjon av alfa-synukleinfibriller via intraperitoneal rute indusert nevrologisk sykdom med lammelse i fire av fem Tg (M83 +/-: GFAP -luc +/-) mus med en median inkubasjonstid på 229 ± 17 dager. Syke dyr viste alvorlige forekomster av fosforylert alfa-synuclein i hjernen og ryggmargen. Ansamlinger av alpha-synuclein ble sarkosyl-uløselig og colocalized med ubiquitin og p62, og ble fulgt av en inflammatorisk respons som resulterer i astrocytic gliosis og microgliosis. Overraskende, inokulering av alfa-synukleinfibriller inn i tungen var mindre effektivt til å forårsake sykdom med bare en av fem injiserte dyr som viser alpha-synuclein patologi etter 285 dager. Våre funn viser at vaksinasjonen via intraglossal rute og mer via intraperitonealt er egnet til å fremkalle nevrologisk sykdom med relevante kjennetegnene ved synukleinopatier i Tg (M83 +/-: GFAP -luc +/-) mus. Dette gir en ny modell for å studere prion-lignende patogenesen indusered av alfa-synuclein prionoids i større detalj.

Perifer injeksjon av alpha-synuclein prionoids via tungen eller peritoneum indusert nevro i CNS av bigenic Tg (M83 +/-: GFAP -luc +/-) mus (tabell 1 og figur 1). Etter en enkel intraperitoneal injeksjon med alfa-synukleinfibriller, fire av fem mus utviklet nevrologisk sykdom med en median inkubasjonstid på 229 ± 17 dager. Overraskende er bare en av fem mus utviklet CNS-sykdom etter intraglossal injeksjon med alfa-synuklei...

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 injeksjon av alfa-synukleinfibriller i peritoneum eller tunge av Tg (M83 +/-: GFAP -luc +/-) mus som uttrykker human alpha-synuclein med familiær A53T mutasjonen og ildflueluciferase, kan indusere neuropatologi, inkludert nevroinflammasjon , i deres sentralnervesystemet.

Bioluminesens Imaging av nevroinflammasjon i transgene mus etter Peripheral Inokulering av Alfa synukleinfibriller

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 av Topically Transgene Rats etter<em&gt; I utero</em&gt; Electroporation og<em&gt; In vivo</em&gt; Bioluminesens 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

Transplantasjon av Olfactory Ensheathing Cells å evaluere Funksjonell Recovery etter 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 Bioluminesens Imaging av subcellulære Ca<sup&gt; 2+</sup&gt; I Aged hippocampus nevroner

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

Sekvensiell Utvinning av løselig og uløselig Alpha-synuclein fra 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 nevrale aktivitet i primære Somatosensory Cortex ved hjelp av 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

Eksogene administrasjon av Microsomes-assosiert Alpha-synuclein-aggregater til primære Neurons som kraftig celle modell Fibrils formasjonen

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