Name: transplantation of olfactory ensheathing cells to evaluate functional recovery after peripheral nerve injury
Uploaded: 2014-02-23T16:00:00
Description: Watch this Scientific Journal Video about Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury at JoVE.com

Forfatterne vil gerne anerkende Adir (Aide à Hjemsted aux Insuffisants Respiratoires) og Fondation de l&#39;Avenir for deres økonomiske støtte og til Dr. Fanie Barnabé-Heider til redigering af manuskriptet.

1.. Primære kulturer af olfaktoriske slimhinde og Olfaktoriske Pærer <ol><li> Forud for dissektion <ol><li> Gør medium, 50 ml ved tilsætning af 44 ml af calcium-frit Dulbeccos Modified Eagles / Hams F12-medium (DMEM/F12) suppleret med 5 ml føtalt bovint serum (FBS) og 1 ml penicillin / streptomycin. </li><li> Coat 75 cm2 kolber med poly-L-lysin (50 ug / ml, 1,5 ug / cm 2), 1 time ved stuetemperatur. </li><li> Skyl kolber med PBS 1x. </li><li> Opbevar overtrukne kolber i køleskabet i op til ...

<ol>
	<li>Barraud, 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=Neural+crest+origin+of+olfactory+ensheathing+glia.">Neural crest origin of olfactory ensheathing glia.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 21040-21045 (2010).</li><li>Guerout, 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=Comparative+gene+expression+profiling+of+olfactory+ensheathing+cells+from+olfactory+bulb+and+olfactory+mucosa.">Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa.</a> Glia. 58, 1570-1580 (1002).</li><li>Honore, 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=Isolation,+characterization,+and+genetic+profiling+of+subpopulations+of+olfactory+ensheathing+cells+from+the+olfactory+bulb.">Isolation, characterization, and genetic profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb.</a> Glia. 60, 404-413 (2012).</li><li>Franssen, E. H., de Bree, F. M., Verhaagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+glia:+their+contribution+to+primary+olfactory+nervous+system+regeneration+and+their+regenerative+potential+following+transplantation+into+the+injured+spinal+cord.">Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord.</a> Brain Res. Rev. 56, 236-258 (2007).</li><li>Lakatos, A., Franklin, R. J., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes.">Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.</a> Glia. 32, 214-225 (2000).</li><li>Woodhall, E., West, A. K., Chuah, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+olfactory+ensheathing+cells+express+nerve+growth+factor,+brain-derived+neurotrophic+factor,+glia+cell+line-derived+neurotrophic+factor+and+their+receptors.">Cultured olfactory ensheathing cells express nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor and their receptors.</a> Brain Res. Mol. Brain Res. 88, 203-213 (2001).</li><li>Dombrowski, M. A., Sasaki, M., Lankford, K. L., Kocsis, J. D., Radtke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+nodal+formation+of+regenerated+peripheral+nerve+fibers+following+transplantation+of+acutely+prepared+olfactory+ensheathing+cells.">Myelination and nodal formation of regenerated peripheral nerve fibers following transplantation of acutely prepared olfactory ensheathing cells.</a> Brain Res. 1125, 1-8 (2006).</li><li>Guerout, 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=Transplantation+of+olfactory+ensheathing+cells+promotes+axonal+regeneration+and+functional+recovery+of+peripheral+nerve+lesion+in+rats.">Transplantation of olfactory ensheathing cells promotes axonal regeneration and functional recovery of peripheral nerve lesion in rats.</a> Muscle Nerve. 43, 543-551 (2011).</li><li>Guntinas-Lichius, O., 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=Transplantation+of+olfactory+ensheathing+cells+stimulates+the+collateral+sprouting+from+axotomized+adult+rat+facial+motoneurons.">Transplantation of olfactory ensheathing cells stimulates the collateral sprouting from axotomized adult rat facial motoneurons.</a> Exp. Neurol. 172, 70-80 (2001).</li><li>Makoukji, 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=Lithium+enhances+remyelination+of+peripheral+nerves.">Lithium enhances remyelination of peripheral nerves.</a> Proc. Natl. Acad. Sci U.S.A. 109, 3973-3978 (2012).</li><li>Guerout, 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=Co-transplantation+of+olfactory+ensheathing+cells+from+mucosa+and+bulb+origin+enhances+functional+recovery+after+peripheral+nerve+lesion.">Co-transplantation of olfactory ensheathing cells from mucosa and bulb origin enhances functional recovery after peripheral nerve lesion.</a> PloS one. 6, 22816 (2011).</li><li>Paviot, 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=Efficiency+of+laryngeal+motor+nerve+repair+is+greater+with+bulbar+than+with+mucosal+olfactory+ensheathing+cells.">Efficiency of laryngeal motor nerve repair is greater with bulbar than with mucosal olfactory ensheathing cells.</a> Neurobiol. Dis. 41, 688-694 (2011).</li><li>Girard, S. D., 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=Isolating+nasal+olfactory+stem+cells+from+rodents+or+humans.">Isolating nasal olfactory stem cells from rodents or humans.</a> J. Vis. Exp. , (2011).</li><li>Nash, H. H., Borke, R. C., Anders, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+of+purification+for+establishing+primary+cultures+of+ensheathing+cells+from+the+adult+olfactory+bulb.">New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb.</a> Glia. 34, 81-87 (2001).</li><li>Bianco, J. I., Perry, C., Harkin, D. G., Mackay-Sim, A., Feron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin+3+promotes+purification+and+proliferation+of+olfactory+ensheathing+cells+from+human+nose.">Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose.</a> Glia. 45, 111-123 (2004).</li><li>Paviot, A., Bon-Mardion, N., Duclos, C., Marie, J. P., Guerout, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Although+olfactory+ensheathing+cells+have+remarkable+potential+to+sustain+nerve+regeneration,+they+cannot+be+applied+to+a+severe+vagus+nerve+section/resection+model.">Although olfactory ensheathing cells have remarkable potential to sustain nerve regeneration, they cannot be applied to a severe vagus nerve section/resection model.</a> Muscle Nerve. 43, 919-920 (2011).</li></ol>

De teknikker, der præsenteres her gør OECS en nyttig model til at studere cellulære transplantationer i perifere modeller nerveskade. Cellekultur-protokollen er relativt ligetil og kan let udføres. , Kirurgiske procedurer, især afsnit / anastomose af RLN, kræver på den anden side erfaring og skal udføres af kvalificeret personale. De i denne protokol beskrevne procedurer fremhæver vigtige faktorer til at fokusere på for at opnå de bedst mulige resultater. Først i løbet af dissoc...

Den primære olfaktoriske system er sammensat af to særskilte dele; olfaktoriske mucosa (OM) i det perifere nervesystem og lugtekolben (OB) i det centrale nervesystem. Den primære olfaktoriske system er kendetegnet ved evnen af ​​de primære olfaktoriske neuroner (PON) til selv at forny hele livet i pattedyrarter. Denne evne er gjort mulig på grund af tilstedeværelsen af ​​neurale stamceller i OM. PON vækst og genvækst fra OM til OB lettes af specialiserede gliaceller kaldet olfaktoriske ensheathing celler (OECS). OECS er neurale kamceller, der skaber en positiv mikromiljø for neurogenese af PON fra OM til OB 1. Derved kan OECS findes i OM og i OB udgør forskellige subpopulationer af celler 2,3. De forskellige egenskaber af OECS have bly forskere til at bruge dem til cellulære transplantationer i flere nervesystem læsioner paradigmer 4. Faktisk OECS producerer vækstfaktorer, reducere glial skræmmer, PRomote axonal genvækst, og frit kan blande sig med astrocytter 5,6. Men langt de fleste af disse undersøgelser er baseret på rygmarvsskade (SCI), få af dem har brugt OECS efter perifer nerveskade (PNI) 7,8. Selvom det perifere nervesystem har en stor evne til at regenerere efter nerveskade, komplet sektioner fremkalde afvigende axonal genvækst. Faktisk efter fuldstændig transections af ansigtsbehandling eller de tilbagevendende larynx nerver (RLN) fejlsendte axoner forårsager muskulære cocontractions kaldet synkinesis. Det er derfor af største vigtighed at foreslå en model af PNI til ikke kun at kvantificere aksonal genvækst, men også for at vurdere effektiviteten af ​​de muskelsammentrækninger. I litteraturen er den mest almindelige beskrevne model er baseret på facial nerve læsion 9,10. I denne model er funktionelle evalueringer baseret på inddrivelse af knurhår bevægelser 10. Men det er vanskeligt at påvise effektiviteten af ​​movements og til at diskriminere de muskulære cocontractions fænomener. Vi foreslår her en model baseret på en RLN læsion. Denne model giver mulighed for vurdering af ikke blot axonal genvækst og bevægelser af stemmebånd, men også effektiviteten og funktionaliteten af disse bevægelser efter cellulære transplantationer 11,12. Denne protokol giver en trinvis procedure til kultur og transplantation OECS fra OM og OB i en RLN sektion / anastomosemodellen og evaluere dyrene efter operationen.

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			<td>Karl Storz Endoskope</td>
			<td>Telecam SL NLSC 20212120</td>
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transplantation of olfactory ensheathing cells to evaluate functional recovery after peripheral nerve injury

<html> Olfaktoriske ensheathing celler (OECS) er neurale kamceller som tillader vækst og genvækst af de primære olfaktoriske neuroner. Faktisk er den primære olfaktoriske system kendetegnet ved sin evne til at give anledning til nye neuroner selv i voksne dyr. Denne særlige evne er til dels på grund af tilstedeværelsen af ​​OECS som skaber et gunstigt mikromiljø for neurogenese. Denne egenskab af OECS har været anvendt til cellulær transplantation såsom rygmarvsskader modeller. Selvom det perifere nervesystem har en større evne til at regenerere efter nerveskade end centralnervesystemet, komplet sektioner fremkalde misrouting under axonal genvækst især efter facial af larynx nerve transection. Konkret fuld sektionering af den tilbagevendende larynx nerve (RLN) inducerer afvigende axonal genvækst resulterer i synkinesis af stemmebånd. I denne specifikke model, viste vi, at OECS transplantation effektivt øger axonal genvækst. ve_content &quot;&gt; OECS udgøres af flere delpopulationer stede i både det olfaktoriske slimhinder (OM-OECS), og de olfaktoriske pærer (OB-OECS). Vi præsenterer her en model af cellulær transplantation baseret på brugen af ​​disse forskellige subpopulationer af OECS i en RLN skade model. Brug dette paradigme blev primære kulturer af OB-OECS og OM-OECS transplanteret i Matrigel efter sektion og anastomose af RLN. To måneder efter operationen, evaluerede vi transplanterede dyr af supplerende analyser baseret på videolaryngoscopy, elektromyografi (EMG) og histologiske undersøgelser. Først videolaryngoscopy tilladt os at vurdere larynx-funktioner, især muskuløse cocontractions fænomener. Derefter EMG analyser demonstreret rigdom og synkronisering af muskulære aktiviteter. Endelig histologiske undersøgelser baseret på toluidinblå farvning tillod kvantificering af antallet og profilen af myelinerede fibre. Alle sammen, vi beskriver her, hvordan man isolere, kultur, identify og transplantation OECS fra OM og OB efter RLN sektion-anastomose, og hvordan man vurdere og analysere effektiviteten af ​​disse transplanterede celler på axonale genvækst og larynx-funktioner.

Illustrationer med kontrol og reinnervated (afsnit / anastomoseres) dyr er blevet valgt som resultater kan variere afhængigt af de cellulære transplantationer (OM, OB eller OM + OB). Cellekultur Cellerne klæbe hurtigt til plastoverfladen og blev opdelt i parallelle skår af celler, der er aflang eller tilspidsende, trekantet, multipolær eller tenformet (figur 1A og 1B). Efter 8 dage in vitro flowcytometri analyse viser hastigheden ...

Watch this Scientific Journal Video about Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury at JoVE.com

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

Olfaktoriske ensheathing celler (OECS) er neurale kamceller der tillader væksten af ​​de primære olfaktoriske neuroner. Denne særlige egenskab kan anvendes til cellulær transplantation. Vi præsenterer her en model af cellulær transplantation baseret på anvendelse af OECS i en larynx nerve skade model.

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 ...

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