Name: analysis of schwann-astrocyte interactions using in vitro assays
Uploaded: 2011-01-13T23:59:00
Description: Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Gostaríamos de agradecer a Filipa Warren por sua ajuda espécie.

1. Ensaio de fronteira: <ol><li> Preparação básica: slides Câmara são revestidas com poli-D-lisina lâminas de vidro durante a noite e estéreis são preparados antes de iniciar o experimento. Médio está preparado usando Médio Modified Dulbecco Águia (DMEM), suplementado com 10% de soro fetal bovino (FCS), e 1% penicilina-estreptomicina Fungizon (PSF). Também uma garrafa de cálcio / magnésio livre solução salina equilibrada Hanks (HBSS) é preparado. </li><li> Células de Schwann de rato primários culti...

<ol>
	<li>Assouline, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+astrocytes+and+Schwann+cells+in+culture+synthesize+nerve+growth+factor-like+neurite-promoting+factors.">Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors.</a> Brain Res. 428, 103-118 (1987).</li><li>Taylor, J. S., Bampton, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+secreted+by+Schwann+cells+stimulate+the+regeneration+of+neonatal+retinal+ganglion+cells.">Factors secreted by Schwann cells stimulate the regeneration of neonatal retinal ganglion cells.</a> J Anat. 204, 25-31 (2004).</li><li>Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+and+cell+adhesion+molecules:+neuronal+receptors+that+regulate+axon+growth+on+extracellular+matrices+and+cell+surfaces.">Integrins and cell adhesion molecules: neuronal receptors that regulate axon growth on extracellular matrices and cell surfaces.</a> Dev Neurosci. 11, 332-347 (1989).</li><li>Chiu, A. Y., Monteros, ., Espinosa de los, A., Cole, R. A., Loera, S., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+and+s-laminin+are+produced+and+released+by+astrocytes,+Schwann+cells,+and+schwannomas+in+culture.">Laminin and s-laminin are produced and released by astrocytes, Schwann cells, and schwannomas in culture.</a> Glia. 4, 11-24 (1991).</li><li>Blakemore, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination+of+CNS+axons+by+Schwann+cells+transplanted+from+the+sciatic+nerve.">Remyelination of CNS axons by Schwann cells transplanted from the sciatic nerve.</a> Nature. 266, 68-69 (1977).</li><li>Andrews, M. R., Stelzner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+olfactory+ensheathing+and+schwann+cells+after+implantation+into+a+dorsal+injury+of+adult+rat+spinal+cord.">Evaluation of olfactory ensheathing and schwann cells after implantation into a dorsal injury of adult rat spinal cord.</a> J Neurotrauma. 24, 1773-1792 (2007).</li><li>Iwashita, Y., Fawcett, J. W., Crang, A. J., Franklin, R. J., Blakemore, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+transplanted+into+normal+and+X-irradiated+adult+white+matter+do+not+migrate+extensively+and+show+poor+long-term+survival.">Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival.</a> Exp Neurol. 164, 292-302 (2000).</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>Lakatos, A., Barnett, S. C., Franklin, R. 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+cells+induce+less+host+astrocyte+response+and+chondroitin+sulphate+proteoglycan+expression+than+Schwann+cells+following+transplantation+into+adult+CNS+white+matter.">Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter.</a> Exp Neurol. 184, 237-246 (2003).</li><li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Schwann+cell+interactions+in+culture.">Astrocyte-Schwann cell interactions in culture.</a> Glia. 11, 367-377 (1994).</li><li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+sulfate+proteoglycan+staining+in+astrocyte-Schwann+cell+co-cultures.">Chondroitin sulfate proteoglycan staining in astrocyte-Schwann cell co-cultures.</a> Glia. 14, 145-152 (1995).</li><li>Plant, G. W., Bates, M. L., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+proteoglycan+immunoreactivity+is+higher+at+the+caudal+than+the+rostral+Schwann+cell+graft-transected+spinal+cord+interface.">Inhibitory proteoglycan immunoreactivity is higher at the caudal than the rostral Schwann cell graft-transected spinal cord interface.</a> Mol Cell Neurosci. 17, 471-487 (2001).</li><li>Fishman, P. S., Nilaver, G., Kelly, 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=Astrogliosis+limits+the+integration+of+peripheral+nerve+grafts+into+the+spinal+cord.">Astrogliosis limits the integration of peripheral nerve grafts into the spinal cord.</a> Brain Res. 277, 175-180 (1983).</li><li>Fok-Seang, J., Mathews, G. A., ffrench-Constant, C., Trotter, J., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+oligodendrocyte+precursors+on+astrocytes+and+meningeal+cells.">Migration of oligodendrocyte precursors on astrocytes and meningeal cells.</a> Dev Biol. 171, 1-15 (1995).</li><li>Wilby, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Cadherin+inhibits+Schwann+cell+migration+on+astrocytes.">N-Cadherin inhibits Schwann cell migration on astrocytes.</a> Mol Cell Neurosci. 14, 66-84 (1999).</li><li>Grimpe, B. <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+proteoglycans+in+Schwann+cell/astrocyte+interactions+and+in+regeneration+failure+at+PNS/CNS+interfaces.">The role of proteoglycans in Schwann cell/astrocyte interactions and in regeneration failure at PNS/CNS interfaces.</a> Mol Cell Neurosci. 28, 18-29 (2005).</li><li>Afshari, F. T., Kwok, J. C., White, L., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+migration+is+integrin-dependent+and+inhibited+by+astrocyte-produced.">Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced.</a> Glia. , (2010).</li><li>Santos-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF/heparin+differentially+regulates+Schwann+cell+and+olfactory+ensheathing+cell+interactions+with+astrocytes:+a+role+in+astrocytosis.">FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.</a> J Neurosci. 27, 7154-7167 (2007).</li><li>Afshari, F. T., Kwok, J. C., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-produced+ephrins+inhibit+schwann+cell+migration+via+VAV2+signaling.">Astrocyte-produced ephrins inhibit schwann cell migration via VAV2 signaling.</a> J Neurosci. 30, 4246-4255 (2007).</li></ol>

Os ensaios descritos acima têm sido utilizados em vários estudos demonstrando o papel dos múltiplos fatores envolvidos na formação da fronteira entre o Schwann-astrócitos e migração de células de Schwann limitada em ambiente astrocíticos. Compreensão dos mecanismos subjacentes a estes eventos é essencial, uma vez que permitiria o desenvolvimento de estratégias para otimizar e melhorar a integração de enxertos de células de Schwann após o transplante e com isso facilitar a sa...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
</tr>
<tr>
<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
<td>Goat anti mouse</td>
</tr>
<tr>
<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

analysis of schwann-astrocyte interactions using in vitro assays

Células de Schwann são um dos células normalmente usadas em estratégias de reparo após lesões na medula espinhal. Células de Schwann são capazes de apoiar a regeneração axonal e surgimento de secreção 1,2 fatores de crescimento e fornecer factores de crescimento e moléculas de adesão 3 moléculas de matriz extracelular 4. Além disso, eles mielinizar os axônios desmielinizados no local da lesão 5. No entanto após o transplante, as células de Schwann não migram do local de implante e não se misturam com os astrócitos anfitrião 6,7. Isto resulta na formação de uma fronteira nítida entre as células de Schwann e astrócitos, criando um obstáculo para os axônios em crescimento tentando sair da volta do enxerto no tecido do hospedeiro proximal e distalmente. Astrócitos em contato com células de Schwann também sofrem hipertrofia e up-regular as moléculas inibidoras 8-13. Ensaios in vitro têm sido usados ​​para modelar Schwann células-astrócitos interações e têm sido importantes para a compreensão do mecanismo subjacente ao comportamento celular. Estes ensaios in vitro incluem teste de fronteira, onde a co-cultura é feita através de duas células diferentes, com cada tipo de célula que ocupam diferentes territórios, com apenas uma pequena diferença que separa as duas frentes de células. Como as células se dividem e migrar, as duas frentes de celulares se aproximar uns dos outros e, finalmente colidem. Isso permite que o comportamento das duas populações celulares a serem analisados ​​na fronteira. Outra variação da mesma técnica é misturar as duas populações celulares em cultura e ao longo do tempo os dois tipos de células com as células de Schwann segregar aglutinados como ilhas entre astrócitos juntos criando múltiplas Schwann-astrócito limites. Segundo ensaio utilizado no estudo da interação de dois tipos de células é o ensaio de migração celular, onde o movimento pode ser acompanhado na superfície do tipo de célula outras monocamada 14,15. Este ensaio é comumente conhecido como teste de lamela invertido. Células de Schwann são cultivadas em pequenos fragmentos de vidro e eles são invertidos face para baixo sobre a superfície de monocamadas de astrócitos e migração é avaliada a partir da borda da lamínula. Ambos os ensaios têm sido fundamentais para estudar os mecanismos subjacentes envolvidos na exclusão celular e formação de fronteira. Algumas das moléculas identificadas usando estas técnicas incluem N-caderinas, 15 de proteoglicanos sulfato de condroitina (CSPGs) 16,17, FGF / Heparina 18, Ef / Efrinas 19. Este artigo pretende descrever ensaio limite e ensaio de migração de forma gradual e elucidar os possíveis problemas técnicos que possam ocorrer.

Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Este artigo pretende descrever de forma gradual o comumente usado<em&gt; In vitro</em&gt; Testes usados ​​no estudo de células de Schwann-asrtocyte interações.

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