Name: adult mouse drg explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection
Uploaded: 2018-03-09T13:30:00
Description: Watch this Scientific Journal Video about Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection at JoVE.co

Vi takker Imaging core-facilitet på Midwestern Universitet (Mae) og gruppen af studerende [Chanmoly Seng, Christopher Dipollina, Darryl Giambalvo og Casey Sigerson] for deres bidrag i cellekultur og billedbehandling arbejde. Denne forskningsarbejde blev støttet af Mae Intramural tilskud til MF og forskning start-up midler til V.T.

Alle de procedurer, herunder brugen af dyrene er blevet godkendt af institutionelle review board-godkendte protokoller (IACUC-Midwestern Universitet).
1. høst DRG fra mus embryoner
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Aflive de voksne mus ved kvælning metode (CO2) efterfulgt af halshugning. Straks videre til kirurgisk fjerne rygsøjlen.
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<li>Udsætte rygsøjlen ved at skære ned den hud lag dorsalt ved hjælp af fine sakse. Isolere rygsøjlen ved at skære gennem ribben på hver side ...

<ol>
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B., Boyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+effects+of+nerve+injury+on+estimates+of+sensory+neuron+number+in+juvenile+bullfrog.">Transient effects of nerve injury on estimates of sensory neuron number in juvenile bullfrog.</a> J Comp Neurol. 410 (2), 171-177 (1999).</li><li>Geuna, S., Borrione, P., Poncino, A., Giacobini-Robecchi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+morphometrical+changes+in+dorsal+root+ganglion+neurons+innervating+the+regenerated+lizard+tail.">Morphological and morphometrical changes in dorsal root ganglion neurons innervating the regenerated lizard tail.</a> Int J Dev Neurosci. 16 (2), 85-95 (1998).</li><li>La Forte, R. A., Melville, S., Chung, K., Coggeshall, R. 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A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trip+of+the+tip:+understanding+the+growth+cone+machinery.">The trip of the tip: understanding the growth cone machinery.</a> Nat Rev Mol Cell Biol. 10 (5), 332-343 (2009).</li><li>Dickson, B. 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Ex vivo DRG model er yderst nyttigt at undersøge et bredt spektrum af begivenheder såsom neuron-glia interaktion og påvirkning af mikromiljø begge neuronal og glial stofskifte37. Yderligere, DRG-modellen kunne anvendes som et omkostningseffektivt redskab til at behandle relevante spørgsmål vedrørende sygdomsfremkaldende mekanisme og associeret markører ved at udvikle ex vivo systemer for akut kronisk og latente fase af infektion eller i en given sygdom. Derudover kunne en...

I dette manuskript rapporterer vi en metode til at opnå en organotypic ex vivo model af en mus afledt DRG modelsystem som en bevaret væv-lignende mikromiljø at undersøge en bred vifte af biologiske svar til PNS fornærmelser spænder fra neuron-glial interaktion, neuroplasticity, inflammatoriske markører, virusinfektion. Desuden har vi yderligere udviklet en protokol for at oprette en primær Co kultur af DRG-afledte enkelt sensoriske neuroner og satellit celler.
DRG er satellit grå-sagen-enheder er beliggende uden for det centrale nervesystem (CNS) langs de dorsale spinale rødder af spinal nerver. DRG, beliggende i nærhed af intervertebral foramina, house pseudounipolar sensoriske neuroner og satellit glial celler. De pseudounipolar neuroner har en enkelt neurite, der opdeler i en perifer transporterer somatiske og visceralt input fra perifere mål til cellen kroppen, og en central proces, der sender sensorisk information fra cellen organ i CNS. En bindevævs kapsel definerer og isolerer denne perifere klynge af neuroner og gliaceller fra CNS. Ingen postnatal celle migration til eller fra DRG er nogensinde blevet beskrevet og en lokal stamcelle niche er ansvarlig for neurogen begivenheder gennem hele livet1. Derfor, denne model er særligt velegnet til at studere voksen neurogenese, axonogenesis, reaktion på traumatiske læsioner, og celle død2,3,4,5,6,7 ,8,9 .
Inden for neuroregeneration DRG høstet fra in vivo og eksplanterede in vitro- gengiver axonotmesis, en skade tilstand i hvilken axoner er fuldt afskåret og neuronal cellen kroppen er afbrudt fra innerverede målet10 ,11. Det er velkendt at perifer nerveskade kan forårsage nedsat og øget genekspression i DRG og mange af disse ændringer skyldes af regenerative processer, men mange kan også være et resultat af immunrespons eller et andet svar fra ikke-neuronale celler. Ved hjælp af en ex vivo system af isolerede DRG, nogle af denne kompleksitet er fjernet og mekanistiske veje kan undersøges mere let.
Udover sin centrale rolle i at formidle sensoriske input til CNS, overfloden af receptorer for mange neurotransmittere, herunder GABA12,13,14,15 på samme niveau som den neuronale soma samt dokumentation af interneuronal Kors-excitation kan tyde på at DRG er sofistikeret foreløbige integratorer sensoriske input16,17. Disse nye fund giver til DRG eksplantat Karakteristik af en mini-neuronal netværkssystem svarer til andre "mini hjerne" modeller, som er nervøs-væv-specifikke organoids anvendes til bredere forsøgsmarker undersøgelse og terapeutiske tilgang til neurologiske sygdomme18,19. Disse beviser sammen med, at DRG er en diskret og veldefinerede klynge af neuronal væv omgivet af et bindevæv kapsel, laver det en egnet organ for ex vivo transplantation.
Dyrkningsbaserede mus DRG præsenterer flercellede tiltrækkende model menneskelige pathophysiologies som følge af strukturelle og genetiske ligheder mellem arterne. Derudover er et stort lager af Transgene mus stammer yderst befordrende for fremtidige Mekanistiske undersøgelser. Neurite udvidelse både under udvikling og efter skade kræver mekanisk interaktioner mellem vækst kegle og substrat20,21. Nano - og mikro-mønstrede underlag har været brugt som værktøjer til direkte neurite udvækst og vise deres evne til at reagere på topografiske egenskaber i deres microenvironments. Neuroner har vist sig at overleve, overholde, flytte og orientere deres axoner til at navigere overflade funktioner såsom riller i substrater22,23. Men disse undersøgelser har typisk udnyttet kulturperler cellelinjer og det er vanskeligt at forudsige, hvordan primære neuronale celler vil reagere på veldefinerede, fysiske stikord i vivo eller ex vivo.
Ex vivo eksplantat model af musen DRG anvendes til dette forslag efterligner den virkelige celle-celle interaktion og biokemiske stikord omkring voksende axoner. Blandt mange forskellige eksperimentelle paradigmer spænder fra cytoskeletale regenerering, neurosphere produktion, til neuroinflammation, DRG explant fortsætter model med at fungere som et værdifuldt redskab til at undersøge den viral infektion og latency aspekt inden for sensorisk ganglier24,25,26,27.
Nervesystem (NS) er generelt mål for virusinfektioner28,29,30. De fleste virus inficere epitel og endotel celle overflader og gøre deres vej fra den overflade væv til NS via perifer nerve sensoriske og motoriske fibre. Især herpes simplex virus type 1 (HSV-1) efter en indledende infektion i epitelceller etablerer en livslang ventetid i de sensoriske ganglier helst, DRG PNS31,32. HSV-1 neuroptropic kapacitet for at inficere PNS i sidste ende fører til neurologiske sygdomme33.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adult Mice NIH/Swiss</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm petri dish</td>
			<td>Cell Treat</td>
			<td>229635</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel ECM</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
			<td>gelatinous protein mixture</td>
		</tr>
		<tr>
			<td>F12 Media</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F6178</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22um filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Supplement for neuronal culture</td>
		</tr>
		<tr>
			<td>PSN antibiotics</td>
			<td>Gibco</td>
			<td>15640-055</td>
			<td>*Part of SFM media 
			Antibiotic mixture</td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Alomone Labs</td>
			<td>N-100</td>
			<td>Nerve growth factor</td>
		</tr>
		<tr>
			<td>Laminin coated coverslide</td>
			<td>Neuvitro</td>
			<td>GG-14-Laminin</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG subtrate</td>
			<td>Pierce</td>
			<td>34055</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Invitrogen</td>
			<td>15520034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-B-tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T8328</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Antibody anti-peripherin</td>
			<td>Millipore</td>
			<td>AB1530</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Hoechst dye</td>
			<td>Thermo Fisher</td>
			<td>62249</td>
			<td>1.5 &#181;M final concentration</td>
		</tr>
		<tr>
			<td>Anti-heparan sulfate</td>
			<td>US Biological</td>
			<td>H1890-10</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>Anti gD antibody</td>
			<td>Virostat</td>
			<td>196</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-100G</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>BME</td>
			<td>Gibco</td>
			<td>21010-046</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>KIT (Insulin-transferrin-Selenium-A)</td>
			<td>Gibco</td>
			<td>51300-044</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Vitamin-C</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>488 (goat anti-mouse)</td>
			<td>Life Technologies</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 (goat anti-rabbit)</td>
			<td>Jackson Immunoresearch laboratories</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat serum&#160;</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin Solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT5014-120ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield</td>
			<td>Vector</td>
			<td>H-1500</td>
			<td>Flurescence mount</td>
		</tr>
		<tr>
			<td>Diamond White Glass Coverslides</td>
			<td>Globe Scientific</td>
			<td>1380-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

adult mouse drg explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection

Denne protokol beskriver en ex vivo model af mus-afledte dorsalrodsganglier (DRG) eksplantat og in vitro- DRG-afledte Co kultur af dissocierede sensoriske neuroner og gliaceller satellit. Disse er nyttige og alsidig modeller til at undersøge en række biologiske reaktioner forbundet med fysiologiske og patologiske forhold i det perifere nervesystem (PNS) spænder fra neuron-glial interaktion, neuroplasticitet, neuroinflammation, og viral infektion. Anvendelse af DRG eksplantat er videnskabeligt fordelagtigt i forhold til forsimplede enkelt celler modeller af flere grunde. For eksempel, som en organotypic kultur tillader DRG eksplantat ex vivo overførsel af et helt neuronal netværk herunder den ekstracellulære mikromiljø, der spiller en væsentlig rolle i alle de neuronal og glial funktioner. Yderligere, DRG explants kan også opretholdes ex vivo for flere dage og dyrkningsbetingelserne kan blive rystet som ønsket. Derudover kan høstede DRG yderligere adskilles i en in vitro- co kultur af primære sensoriske neuroner og satellit glial celler at undersøge neuronal-glial interaktion, neuritogenesis, cytoskeletale kegle samspil med det ekstracellulære mikromiljø, og mere generelt, alle aspekter forbundet med neuronal stofskiftet. Derfor, DRG-eksplantat system tilbyder stor fleksibilitet til at studere en bred vifte af begivenheder relateret til biologiske, fysiologiske og patologiske forhold på en omkostningseffektiv måde.

Flere aspekter af neuroplasticitet og neuron-miljø interaktion kan undersøges ved hjælp af DRG og en enkelt dissocierede celle kultur model. Vi begyndte at undersøgelser ved at isolere en DRG eksplantat og DRG-afledte dissocierede celler som skematisk repræsenteret i figur 1. Både væv og enkelt celler modeller kan analyseres ved hjælp af en række molekylærbiologiske teknikker såsom immunofluorescens, vestlige skamplet, genomisk assays og andre analytiske teknikker afhængigt af ar...

Watch this Scientific Journal Video about Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection at JoVE.co

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

I denne betænkning, er fordelene ved organotypic kulturer og dissocierede primære kulturer af mus-afledte dorsalrodsganglier fremhævet for at undersøge en række mekanismer forbundet med neuron-glial interaktion, neuroplasticitet, neuroinflammation, og svar på viral infektion.

Voksen mus DRG Explant og adskilles Cell modeller for at undersøge neuroplasticitet og svar til miljømæssige fornærmelser herunder virusinfektion

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

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JoVE Journal

Neuroscience

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