Name: a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments
Uploaded: 2017-11-09T13:00:00
Description: Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

DGG er en NHMRC Peter Doherty og multipel sklerose forskning Australien (MSRA) tidlige karriere kolleger. JLF understøttes af et MSRA postdoc stipendium. Dette arbejde blev støttet af den australske National sundhed og Medical Research Rådet (NHMRC) projekt give JX #APP1058647.

alle procedurer, der beskrives her blev godkendt af Florey Institut for neurovidenskab og Mental sundhed (Melbourne hjernen centrum) dyr etiske komité og følger den australske adfærdskodeksen for brug af dyr til videnskabelige Formål.
 1. EAN induktion Bemærk: EAN kan induceres med succes i mandlige C57BL/6 mus i alderen mellem 6-8 uger. Induktion protokollen tager 9 dage i alt. Dag 0 refererer til dag i den første vaccination. For denne protokol, injektioner ...

<ol>
	<li>Newswanger, D. L., Warren, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 69 (10), 2405-2410 (2004).</li><li>Ruiz, E., Ramalle-Gomara, E., Quinones, C., Martinez-Ochoa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Guillain-Barre+syndrome+mortality+in+Spain+from+1999+to+2013.">Trends in Guillain-Barre syndrome mortality in Spain from 1999 to 2013.</a> Int J Neurosci. 126 (11), 985-988 (2016).</li><li>Walling, A. D., Dickson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 87 (3), 191-197 (2013).</li><li>Gonsalvez, D. 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=A+Functional+and+Neuropathological+Testing+Paradigm+Reveals+New+Disability-Based+Parameters+and+Histological+Features+for+P0180-199-Induced+Experimental+Autoimmune+Neuritis+in+C57BL/6+Mice.">A Functional and Neuropathological Testing Paradigm Reveals New Disability-Based Parameters and Histological Features for P0180-199-Induced Experimental Autoimmune Neuritis in C57BL/6 Mice.</a> J Neuropathol Exp Neurol. 76 (2), 89-100 (2017).</li><li>Berg, B., 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=Guillain-Barre+syndrome:+pathogenesis,+diagnosis,+treatment+and+prognosis.">Guillain-Barre syndrome: pathogenesis, diagnosis, treatment and prognosis.</a> Nat Rev Neurol. 10 (8), 469-482 (2014).</li><li>Koller, H., Schroeter, M., Kieseier, B. C., Hartung, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammatory+demyelinating+polyneuropathy--update+on+pathogenesis,+diagnostic+criteria+and+therapy.">Chronic inflammatory demyelinating polyneuropathy--update on pathogenesis, diagnostic criteria and therapy.</a> Curr Opin Neurol. 18 (3), 273-278 (2005).</li><li>Griffin, J. 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=Pathology+of+the+motor-sensory+axonal+Guillain-Barre+syndrome.">Pathology of the motor-sensory axonal Guillain-Barre syndrome.</a> Ann Neurol. 39 (1), 17-28 (1996).</li><li>Taylor, W. A., Hughes, 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=Experimental+allergic+neuritis+induced+in+SJL+mice+by+bovine+P2.">Experimental allergic neuritis induced in SJL mice by bovine P2.</a> J Neuroimmunol. 8 (2-3), 153-157 (1985).</li><li>Zou, L. P., 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=P0+protein+peptide+180-199+together+with+pertussis+toxin+induces+experimental+autoimmune+neuritis+in+resistant+C57BL/6+mice.">P0 protein peptide 180-199 together with pertussis toxin induces experimental autoimmune neuritis in resistant C57BL/6 mice.</a> J Neurosci Res. 62 (5), 717-721 (2000).</li><li>Miletic, H., 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=P0(106-125)+is+a+neuritogenic+epitope+of+the+peripheral+myelin+protein+P0+and+induces+autoimmune+neuritis+in+C57BL/6+mice.">P0(106-125) is a neuritogenic epitope of the peripheral myelin protein P0 and induces autoimmune neuritis in C57BL/6 mice.</a> J Neuropathol Exp Neurol. 64 (1), 66-73 (2005).</li><li>Hafer-Macko, 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=Acute+motor+axonal+neuropathy:+an+antibody-mediated+attack+on+axolemma.">Acute motor axonal neuropathy: an antibody-mediated attack on axolemma.</a> Ann Neurol. 40 (4), 635-644 (1996).</li><li>Griffin, J. 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=Early+nodal+changes+in+the+acute+motor+axonal+neuropathy+pattern+of+the+Guillain-Barre+syndrome.">Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain-Barre syndrome.</a> J Neurocytol. 25 (1), 33-51 (1996).</li></ol>

Denne betænkning skitserer en enkel metode til at fremkalde EAN bruger P0180-199 peptid i C57BL/6 mus, gør det muligt for kvantificering af centrale Neuropatologisk og funktionelle underskud i mus induceret med EAN. Særskilte EAN induktion protokollen beskrevet her er brugen af anæstesi mens de udfører immunisering injektioner. Brugen af isofluran anæstesi forbedrer i høj grad evnen til at sikre, at den samlede mængde af inokulum injiceres subkutant i den ønskede placering med minimale fejl og stress ...

Perifer neuropati kan være genetisk oprindelse eller erhvervede, med erhvervede neuropatier har enten metaboliske, kar, inflammatoriske, eller giftige precipitants. Disse sygdomme er også nyttigt klassificeret som enten cytoskeletale eller demyelinative oprindelse. De mest almindelige erhvervet demyeliniserende perifere neuropatier er akut inflammatorisk demyeliniserende polyneuropati (AIDP, også kendt som Guillain-Barré syndrom, GBS) og kronisk inflammatorisk demyeliniserende polyneuropati (CIDP)1, 2 , 3 , 4; begge er pathogenetically karakteriseret ved en autoimmun reaktion mod myelinskeden, forårsager demyelinering af de perifere nerver. I disse sygdomme, aktiverede T celler krydse blod nerve barriere og generere en immunreaktion i PNS. Aktiveringen af makrofager i nerve derefter forårsager demyelinering enten direkte via fagocyterende angreb, eller indirekte via udskilles inflammatoriske mediatorer, hvilket resulterer i klinisk handicap såsom lammelse og sensorisk dysfunktion5. Mens demyelinated axoner bevarer evnen til at være remyelinated efter demyelinering, remyelination er ofte forsinket eller ufuldstændig, resulterer i modtagelighed af de nøgne axoner til uoprettelige skader, som er den største årsag til permanent kliniske handicap. I øjeblikket de mest effektive behandlinger er immunmodulerende, men trods deres effektivitet, i mange tilfælde opsvinget er ofte langsomme og ~ 25% patienter vil opleve resterende funktionelle mangler, der væsentligt reducerer deres livskvalitet6, 7.
EAN er en udbredt dyremodel af demyeliniserende perifer neuropati, der har givet værdifulde indsigt i patogenese og et middel til at vurdere nye terapeutiske agenter4. Denne model kan være fremkaldt i forskellige arter som kaniner, rotter, mus, og marsvin, og er foranlediget af immunisering med neurogen antigener. Men i sidste ende vellykket EAN induktion afhænger af en passende immunrespons for sygdom opstår. Givet de arter (og Inter-Diesel-dyr/stamme) variationer i immunforsvaret, er flere kombinationer af antigener og adjuvanser, der udviklet til at kunne fremkalde EAN. Med hensyn til murine genetiske værktøjer er C57BL/6 den mest udbredte; men den traditionelle P2 protein peptid 57-81 (P257-81), der resulterer i sygdom i den modtagelige SJL mus stamme8 er afskåret fra ulovlige patogenese fører til funktionelle mangler i C57BL/6 stamme. Heldigvis, sensibilisering paradigmer ved hjælp af P0106-125 eller P0180-199 peptider, leveret i adjuverende kombineret med injektion af kighoste toksin kan overvinde denne barriere, at aktivere avancerede genetiske værktøjer til at blive udnyttet i den murine EAN model.
Her er en simpel metode til induktion af EAN i C57BL/6 mus præsenteret. Derudover tilbydes en omfattende detaljerede tilgang til at vurdere de funktionelle og neuropatologiske underskud forbundet med sygdommen. P0180-199 peptid9 blev valgt frem for P057-81 alternativ10. P0180-199 model er blevet beskrevet til at producere mindre alvorlige kliniske symptomer sammenlignet med P057-81 alternative10, og derfor er tilbøjelige til at modstå indførelsen af potentielt skadelige genetiske forstyrrelser, tilbagesøge kirurgiske procedurer (såsom osmotisk pumpe implantation), og er indstillet til løbebånd gangart funktion test4. Men løbebånd gangart funktion tests og histologiske protokoller er beskrevet her kan nemt anvendes når man studerer sygdom i en P057-81 induceret variant.

<table>
	<tbody>
		<tr>
			<td>C57BL/6, male, 6-8 weeks old</td>
			<td>Australian Bioresources Cenre, WA, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pertussis toxin</td>
			<td>List Biological Laboratories, Inc., CA, USA</td>
			<td>#181</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M mouse-isotonic phosphated buffered salined (MT-PBS)</td>
			<td></td>
			<td></td>
			<td>Laboratories will have their own protocol.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pharmachem, QLD, Australia</td>
			<td></td>
			<td>Laboratories will have their own protocol for administration.</td>
		</tr>
		<tr>
			<td>P0180&#8211;199 peptide</td>
			<td>Wuxi Nordisk Biotech Co. Lt. SHG, CHN</td>
			<td></td>
			<td>P0180&#8211;199, sequence S&#8211;S&#8211;K&#8211;R&#8211;G&#8211;R&#8211; Q&#8211;T&#8211;P&#8211;V&#8211;L&#8211;Y&#8211;A&#8211;M&#8211;L&#8211;D&#8211;H&#8211;S&#8211;R&#8211;S</td>
		</tr>
		<tr>
			<td>Heat killed Mycobacterium tuberculosis (strain H37RA)</td>
			<td>Difco, MI, USA</td>
			<td>#231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#39;s complete adjuvant (FCA)</td>
			<td>Difco, MI, USA</td>
			<td>#263910</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Services</td>
			<td>#15710</td>
			<td>Dilute to 4% PFA day of tissue collection.</td>
		</tr>
		<tr>
			<td>25% glutaraldheyde</td>
			<td>ProSciTech Pty Ltd, QLD, Australia</td>
			<td>#11-30-8</td>
			<td>Dilute to 2.5% glutaraldehyde day of fixation.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Chem-Supply Pty Ltd, SA, Australia</td>
			<td>SL189</td>
			<td>Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v).</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-Supply Pty Ltd, SA, Australia</td>
			<td>SA030</td>
			<td>Use at 30% (w/v).</td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (OCT) medium</td>
			<td>Sakura Finetek, CA, USA</td>
			<td>#4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Merck Millipore, MA, USA</td>
			<td>#S30-100</td>
			<td>Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory.</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma Aldrich, MI, USA</td>
			<td>#90o2-31-1</td>
			<td>Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory.</td>
		</tr>
		<tr>
			<td>rabbit anti-amyloid precursor protein (APP)</td>
			<td>Invitrogen (Life Technologies), CA, USA</td>
			<td>S12700</td>
			<td>Used at 1:400 or titrate in own lab.</td>
		</tr>
		<tr>
			<td>rabbit anti-contactin-associated protein-1 (Caspr)</td>
			<td>Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel</td>
			<td></td>
			<td>Used at 1:500 or titrate in own lab.</td>
		</tr>
		<tr>
			<td>Appropriate Alexa Fluor conjugated secondary antibodies</td>
			<td>Molecular Probes (Life Technologies), OR, USA</td>
			<td>Various</td>
			<td>Use at 1:200 or titrate in own lab. Choice of species the antibody was raised in and Alexa Fluor chosen is at the discretion of each laboratory.</td>
		</tr>
		<tr>
			<td>Aqueous mounting solution</td>
			<td>Dako (Agilent), CA, USA</td>
			<td>#S3023</td>
			<td>Each laboratory will have their own preference.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL syringe with 301/2 g needles</td>
			<td>BD</td>
			<td>#326105</td>
			<td></td>
		</tr>
		<tr>
			<td>23 g needles</td>
			<td>BD</td>
			<td>#305143</td>
			<td></td>
		</tr>
		<tr>
			<td>Red ink pad</td>
			<td></td>
			<td></td>
			<td>Any red ink pad or red food dye could be used to mark the animals&#39; feet.</td>
		</tr>
		<tr>
			<td>DigiGate apparatus (includes treadmill)</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DigiGate Imaging System</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td></td>
			<td></td>
			<td>Any timer may be used.</td>
		</tr>
		<tr>
			<td>DigiGait 8 Software</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any appropriate dissecting microscope may be used.</td>
		</tr>
		<tr>
			<td>Charged slides</td>
			<td>Superfrost Plus, Lomb Scientific Pty Ltd</td>
			<td>SF41296SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyrostat</td>
			<td>Leica</td>
			<td></td>
			<td>Any suitable cyrostat may be used.</td>
		</tr>
		<tr>
			<td>Perfusion equipment and dissecting instruments</td>
			<td></td>
			<td></td>
			<td>Labs will have their own perfusion protoctols.</td>
		</tr>
		<tr>
			<td>Opaque humified chamber</td>
			<td></td>
			<td></td>
			<td>Labs may produce their own using an opaque plastic container.</td>
		</tr>
		<tr>
			<td>PAP pene</td>
			<td>GeneTex (USA)</td>
			<td></td>
			<td>Wax pencil, or surface tension may also be used to create a well around the tissue section.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeis LSM780</td>
			<td></td>
			<td>Any confocal microscope with appropriate laser lines may be used.</td>
		</tr>
		<tr>
			<td>FIJI/Image J</td>
			<td>National Institues of Health</td>
			<td></td>
			<td>Available from www.fiji.sc</td>
		</tr>
	</tbody>
</table>

a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments

Eksperimentelle autoimmune neuritis (EAN) er et godt værdsat eksperimentel model af autoimmune perifere demyeliniserende sygdomme. EAN sygdom er fremkaldt af immuniserende mus med neurogen peptider til direkte en inflammatorisk angreb mod komponenter i det perifere nervesystem (PNS). De seneste fremskridt har aktiveret induktion af EAN i den relativt resistente C57BL/6 mus linje ved hjælp af myelin protein nul (P0)106-125 eller P0180-199 peptider leveret i adjuverende kombineret med injektion af kighoste toksin. Evnen til at fremkalde EAN i C57BL/6 stamme giver mulighed for brug af de mange genetiske værktøjer, der findes på denne mus baggrund, og dermed giver mulighed for avancerede studier af sygdommen patogenese og forhør af handlingen mekanistiske af roman therapeutics i kombination med transgene tilgange. I denne undersøgelse viser vi en enkel tilgang for at kunne fremkalde EAN bruger P0180-199 peptid i C57BL/6 mus. Vi skitsere også en protokol for vurdering af funktionelle mangler, der opstår i denne model, ledsaget af en række Neuropatologisk funktioner. Således, denne model er en kraftfuld eksperimentelle model til at studere patogenesen af menneskelige perifere demyeliniserende neuropatier, og at fastslå effekten af potentielle behandlingsformer, der har til formål at fremme myelin reparation og beskytte mod nerveskader i autoimmune neuritis.

P0180-199 peptid induceret EAN i C57BL/6 mus fører til en monofasiske sygdom med kliniske score debut fra 6 dage post første vaccination (dpi), og maksimal score sværhedsgraden er observeret fra 25 dpi efterfulgt af nogle kliniske score forbedring fra 40 dpi varighed (figur 1)4,9. Gangart funktion, mus begynder at svigte på en simpel kører opgaven så tidligt som 6 dpi, og af 35 dpi mu...

Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Denne betænkning skitserer en enkel tilgang for at med held fremkalde eksperimentelle autoimmune neuritis (EAN) ved hjælp af myelin protein nul (P0)180-199 peptid i kombination med Freunds komplet adjuvans og pertussis toksin. Vi præsenterer en sofistikeret paradigme i stand til præcist at vurdere omfanget af funktionelle mangler og neuropatologiske, der forekommer i denne EAN.

En enkel metode til at fremkalde eksperimentelle Autoimmune Neuritis i C57BL/6 mus for funktionelle og neuropatologiske vurderinger

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

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