Name: neuron-macrophage co-cultures to activate macrophages secreting molecular factors with neurite outgrowth activity
Uploaded: 2018-03-30T15:00:00
Description: Watch this Scientific Journal Video about Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity at JoVE.com

Denne protokol er støttet af en bevilling NRF-2015R1A2A1A01003410 fra ministeriet for videnskab, IKT og fremtidige planlægning, Republikken Korea.

Alle eksperimenter, der involverer dyr blev godkendt af det institutionelle Animal Care og brug Udvalget af Ajou University School of Medicine.
1. kultur forberedelse af dissocierede voksen DRG Neuron
<ol><li>Før du konfigurerer kulturen, pre pels 6-godt plade med poly-D-Lysin og laminin. Ruger en 6-godt plade med 0,01% poly-D-lysin ved 37° C i 2 timer eller ved 4° C natten over. Derefter vask plade to gange med destilleret vand.</li>
	<li>Inkuber pladen med laminin løsning ved en koncen...

<ol>
	<li>Donnelly, D. J., Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+its+role+in+neuroprotection,+axonal+regeneration+and+functional+recovery+after+spinal+cord+injury.">Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.</a> Exp Neurol. 209 (2), 378-388 (2008).</li><li>Festoff, B. 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=Minocycline+neuroprotects,+reduces+microgliosis,+and+inhibits+caspase+protease+expression+early+after+spinal+cord+injury.">Minocycline neuroprotects, reduces microgliosis, and inhibits caspase protease expression early after spinal cord injury.</a> J Neurochem. 97 (5), 1314-1326 (2006).</li><li>Bowers, C. A., Kundu, B., Hawryluk, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylprednisolone+for+acute+spinal+cord+injury:+an+increasingly+philosophical+debate.">Methylprednisolone for acute spinal cord injury: an increasingly philosophical debate.</a> Neural Regen Res. 11 (6), 882-885 (2016).</li><li>Gensel, J. C., Kigerl, K. A., Mandrekar-Colucci, S. S., Gaudet, A. D., Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+CNS+axon+regeneration+by+manipulating+convergent+neuro-immune+signaling.">Achieving CNS axon regeneration by manipulating convergent neuro-immune signaling.</a> Cell Tissue Res. 349 (1), 201-213 (2012).</li><li>DiSabato, D. J., Quan, N., Godbout, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation:+the+devil+is+in+the+details.">Neuroinflammation: the devil is in the details.</a> J Neurochem. 139 Suppl 2, 136-153 (2016).</li><li>Jin, X., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+central+nervous+system+repair+after+injury.">Microglia in central nervous system repair after injury.</a> J Biochem. 159 (5), 491-496 (2016).</li><li>Yin, 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=Macrophage-derived+factors+stimulate+optic+nerve+regeneration.">Macrophage-derived factors stimulate optic nerve regeneration.</a> J Neurosci. 23 (6), 2284-2293 (2003).</li><li>Yin, 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=Oncomodulin+is+a+macrophage-derived+signal+for+axon+regeneration+in+retinal+ganglion+cells.">Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells.</a> Nat Neurosci. 9 (6), 843-852 (2006).</li><li>Gensel, 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=Macrophages+promote+axon+regeneration+with+concurrent+neurotoxicity.">Macrophages promote axon regeneration with concurrent neurotoxicity.</a> J Neurosci. 29 (12), 3956-3968 (2009).</li><li>Barrette, 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=Requirement+of+myeloid+cells+for+axon+regeneration.">Requirement of myeloid cells for axon regeneration.</a> J Neurosci. 28 (38), 9363-9376 (2008).</li><li>Kwon, M. 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=Contribution+of+macrophages+to+enhanced+regenerative+capacity+of+dorsal+root+ganglia+sensory+neurons+by+conditioning+injury.">Contribution of macrophages to enhanced regenerative capacity of dorsal root ganglia sensory neurons by conditioning injury.</a> J Neurosci. 33 (38), 15095-15108 (2013).</li><li>Niemi, J. 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=A+critical+role+for+macrophages+near+axotomized+neuronal+cell+bodies+in+stimulating+nerve+regeneration.">A critical role for macrophages near axotomized neuronal cell bodies in stimulating nerve regeneration.</a> J Neurosci. 33 (41), 16236-16248 (2013).</li><li>Hannila, S. S., Filbin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cyclic+AMP+signaling+in+promoting+axonal+regeneration+after+spinal+cord+injury.">The role of cyclic AMP signaling in promoting axonal regeneration after spinal cord injury.</a> Exp Neurol. 209 (2), 321-332 (2008).</li><li>Kwon, M. 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=CCL2+Mediates+Neuron-Macrophage+Interactions+to+Drive+Proregenerative+Macrophage+Activation+Following+Preconditioning+Injury.">CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.</a> J Neurosci. 35 (48), 15934-15947 (2015).</li><li>Niemi, J. P., DeFrancesco-Lisowitz, A., Cregg, J. M., Howarth, M., Zigmond, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+monocyte+chemokine+CCL2+in+dorsal+root+ganglion+neurons+causes+a+conditioning-like+increase+in+neurite+outgrowth+and+does+so+via+a+STAT3+dependent+mechanism.">Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism.</a> Exp Neurol. 275 Pt 1, 25-37 (2016).</li><li>Cafferty, W. 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=Conditioning+injury-induced+spinal+axon+regeneration+fails+in+interleukin-6+knock-out+mice.">Conditioning injury-induced spinal axon regeneration fails in interleukin-6 knock-out mice.</a> J Neurosci. 24 (18), 4432-4443 (2004).</li><li>Cai, 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=Neuronal+cyclic+AMP+controls+the+developmental+loss+in+ability+of+axons+to+regenerate.">Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate.</a> J Neurosci. 21 (13), 4731-4739 (2001).</li><li>Neumann, S., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+dorsal+column+fibers+into+and+beyond+the+lesion+site+following+adult+spinal+cord+injury.">Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.</a> Neuron. 23 (1), 83-91 (1999).</li></ol>

Der er flere kritiske trin for generation af dette fælles kultur system. Det er vigtigt at sikre, at musen DRG neuroner og peritoneal makrofager forberedt friske og sunde. Vi har oplevet formindsket neurite udvækst aktivitet af CM når dissektion af alle DRG tog mere end 30 min. Derudover kontaminering af blodkomponenter i peritoneal makrofager også ført til et fald på neurite udvækst aktivitet i CM. For at fremkalde robust neuron-makrofag interaktion af db-cAMP, grundig vask ville også være et afgørende skridt ...

En række undersøgelser har forsøgt at forbedre CNS axon regenerering efter skader på rygmarven eller hjernen. Inflammatoriske reaktioner, uundgåeligt ledsager skader på nervesystemet, er traditionelt tænkt til at deltage i sekundære patologiske processer, der fører til ødelæggende resultater1,2. Methylprednisolon, der kan undertrykke inflammatoriske reaktioner er faktisk den eneste godkendte behandling for akut rygmarv skade3. Nyere undersøgelser har imidlertid fremlagt beviser, makrofager, et repræsentativt inflammatoriske celletype, kan deltage i regenerering eller reparation af tilskadekomne nervesystem4,5,6. For eksempel, infiltrerer makrofager efter en objektiv skade producere pro-regenererende molekyler til at fremme regenerering af retinal ganglion neuroner7,8. Derudover øget transplanterede DRG neuroner axon vækst op regionen hvor makrofager blev aktiveret af zymosan9. Desuden kan makrofager på webstedet læsion skabe et vækst-eftergivende miljø for tilskadekomne perifere nerver10.
Vores arbejde også givet stærke beviser for, at makrofager kan bidrage til kapaciteten af axon regenerering i tilstødende neuroner. Vi har vist, at i aktiveringen af makrofager i dorsalrodsganglier (DRG) var afgørende i den forbedrede regenerative kapacitet af DRG sensoriske neuroner efter en forkonditionering perifere nerve skade11. Lignende forskning blev selvstændigt rapporteret fra et andet laboratorium12. Vi viste også, at intraganglionic injektion af dibutyryl cykliske AMP (db-cAMP), som er en velkendt molekyle til at øge kapaciteten i axon regenerering13, induceret aktiveringen af makrofager. Deaktivering af makrofager afskaffet virkningerne af db-lejren på neurite udvækst aktivitet. Efterfølgende værker identificeret skade-induceret udtryk for CCL2 i neuroner som et signal til at stimulere makrofager med en pro-regenererende fænotype14,15.
Baseret på de ovenstående eksperimentelle resultater, har vi etableret en in vitro- model ligner molekylære begivenheder, der opstår i DRG efter en forkonditionering skade model11,14. I denne model anvendes db-cAMP til neuron-makrofag Co kulturer fremkalde intercellulære signalering, der fører til aktivering af makrofager med en pro-regenererende fænotype. Her, beskriver vi detaljerede protokoller, som vi kan generere makrofager, der udskiller molekylære faktorer fremmer neurite udvækst (fig. 1). Denne eksperimentelle model illustrerer et koncept at makrofager kan stimuleres eller tilskyndet til at støtte axon regenerering efter skaderne på nervesystemet. Vores model vil også være nyttige i at studere mekanismer i intercellulære signalering, der fører til aktivering af pro-regenererende makrofager.

<table>
	<tbody>
		<tr>
			<td>Cell culture insert transparent PET membrane 0.4&#956;m pore size</td>
			<td>Corning,Falcon</td>
			<td>353090</td>
			<td>Transparent PET membrane with 0.4-&#956;m pore size, for 6-well plate</td>
		</tr>
		<tr>
			<td>70-&#956;m nylon cell strainer</td>
			<td>Corning, Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well culture slide</td>
			<td>Biocoat</td>
			<td>354632</td>
			<td>with a uniform application of Poly-D-Lysine</td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Qiagen</td>
			<td>158904</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C9407-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>21103-049</td>
			<td>Containing 1% glutamax and 1% penicilin-streptomycin</td>
		</tr>
		<tr>
			<td>B-27 supplement, serum free</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>17504-044</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine Hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 3&#39;, 5&#39;-cyclic monophosphate, N6,O2&#39;-dibutyryl-, sodium salt</td>
			<td>Merck Millipore Corporation, Calbiochem</td>
			<td>28745</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16210072</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Daejung Chemical and Metal Co</td>
			<td>8566-4405</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti &#946; III tubulin (Tuj-1)</td>
			<td>Promega Corporation</td>
			<td>G7121</td>
			<td>Mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Marienfeld-Superior</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture CO2 incubator</td>
			<td>Panasonic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi DV4</td>
			<td></td>
		</tr>
		<tr>
			<td>Twist shaker</td>
			<td>FINEPCR</td>
			<td>Tw3t</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td>Sorvall</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus America Inc</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>VWR International, Hyclone</td>
			<td>SH30919.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman Pearson Rongeurs</td>
			<td>FST (Fine Science Tools)</td>
			<td>16021-14</td>
			<td>Stainless steel, 14cm, curved, single joint action</td>
		</tr>
		<tr>
			<td>Fine Scissors - Tungsten Carbide &#38; ToughCut</td>
			<td>FST (Fine Science Tools)</td>
			<td>14558-11</td>
			<td>sharp, serrated</td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>FST (Fine Science Tools)</td>
			<td>11272-30</td>
			<td>Dumoxel, 0.07 x 0.04mm, curved</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>FST (Fine Science Tools)</td>
			<td>15000-00</td>
			<td>straight, 3mm cutting-edge, sharp</td>
		</tr>
		<tr>
			<td>Qualitron DW-41 Micro-Centrifuge</td>
			<td>Artisan Technology Group</td>
			<td>DW-41</td>
			<td>Input Voltage: 115VAC</td>
		</tr>
	</tbody>
</table>

neuron-macrophage co-cultures to activate macrophages secreting molecular factors with neurite outgrowth activity

Der er stærke beviser at makrofager kan deltage i regenerering eller reparation af tilskadekomne nervesystem. Her, beskriver vi en protokol, hvori makrofager er tilskyndet til at producere aircondition medium (CM), der fremmer neurite udvækst. Voksen dorsalrods ganglion (DRG) neuroner er akut adskilles og forgyldt. Efter neuronerne er stabilt fastgjort, er peritoneal makrofager Co kulturperler på en celle kultur indsætte overlejret på samme godt. Dibutyryl cykliske AMP (db-cAMP) anvendes på fælles kulturer i 24 timer, hvorefter cellekultur indsætte indeholdende makrofager er flyttet til en anden brønd at indsamle CM til 72 h. CM fra Co kulturer behandlet med db-lejren, når de påføres et separat voksen DRG neuron kultur, udstiller robust neurite udvækst aktivitet. CM fra db-cAMP-behandlede kulturer bestående af enkelt celletype alene, enten DRG neuron eller peritoneal makrofag, ikke udstille neurite udvækst aktivitet. Dette indikerer, at samspillet mellem neuroner og makrofager er uundværlig for aktiveringen af makrofager secernerende molekylære faktorer med neurite udvækst aktivitet i CM. Således, vores fælles kultur paradigme vil også være nyttigt at studere intercellulære signalering i neuron-makrofag interaktion at stimulere makrofager at være begavet med en pro-regenererende fænotype.

Vi beskriver en protokol, der kan generere makrofager i stand til at udskille molekylære faktorer med neurite udvækst aktivitet. Makrofag CM fremstillet af fælles kulturer behandlet med db-cAMP resulterede i robust neurite udvækst når de påføres et separat DRG neuron kultur (figur 2A). I sammenligning, CM fra PBS-behandlede Co kulturer ikke fremkalde neurite udvækst i vores 15-h kultur varighed (figur 2B). Når db-cAMP er...

Watch this Scientific Journal Video about Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity at JoVE.com

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

Neuron-makrofag Co kulturer at aktivere makrofager secernerende molekylære faktorer med Neurite udvækst aktivitet

Den nuværende protokol præsenterer eksperimentelle procedurer for at stimulere kulturperler makrofager at være begavet med evnen til at frigive molekylære faktorer, der fremmer neurite udvækst. Behandling af cAMP til neuron-makrofag Co kulturer inducerer makrofager at producere konditioneret medium, der besidder stærke neurite udvækst aktivitet.

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Research

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Neuroscience

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Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

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Dissektion og Kultur af Chick Statoacoustic Ganglion og rygmarven eksplanteret i Kollagen Gels for neurite udvækst Analyser

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

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Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

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Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

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Undersøgelse Funktionel Regenerering i Organotypiske Spinal Cord Co-kulturer dyrket på Multi-elektrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

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