Name: an approach to enhance alignment and myelination of dorsal root ganglion neurons
Uploaded: 2016-08-24T14:00:00
Description: Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

Yazarlar PDMS alt tabakaların hazırlanmasında yaptığı yardım için Eric Vasco teşekkür etmek istiyorum, yararlı öneriler ve DRG izolasyon eğitimi için Michigan Üniversitesi&#39;nden Dr. Marina Mata&#39;nın laboratuarında Dr. Shiyong Wang ve Dr. Mark Tuszynski ve Dr . UC San Diego W. Marie Campana SC izolasyonu için yararlı öneriler ve protokol için. Bu çalışma, Ulusal Bilim Vakfı (CBET 0941055 ve CBET 1510895), Ulusal Sağlık Enstitüsü (R21CA176854, R01GM089866 ve R01EB014986) tarafından kısmen desteklenmiştir.

hücrelerin izolasyonu için tüm prosedürler, Michigan State Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Önceden gerilmiş Anisotropik Yüzeyin hazırlanması 1. <ol><li> taban ve sertleştirme maddesinin 1 çözeltisi ve bir doku kültürü kabı (12 cm çapında) içine dökün: 10 karıştırın. 4900 mg tabanı ve toplam çapraz bağlama karışımı için sertleştirici 490 mg kullanın. </li><li> Hava kabarcıklarını uzaklaştırmak için 20 dakika süreyle vak...

<ol>
	<li>Liu, 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=.">.</a> Layer-by-Layer Films for Biomedical Applications. , 525-546 (2015).</li><li>Faweett, J. W., Keynes, 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=Peripheral+Nerve+Regeneration.">Peripheral Nerve Regeneration.</a> Annu Rev Neurosci. 13, 43-60 (1990).</li><li>Li, Y., Field, P. M., Raisman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+adult+rat+corticospinal+tract+by+transplants+of+olfactory+ensheathing+cells.">Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells.</a> Science. 277, 2000-2002 (1997).</li><li>Geller, H. M., 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=Building+a+bridge:+Engineering+spinal+cord+repair.">Building a bridge: Engineering spinal cord repair.</a> Exp Neurol. 174, 125-136 (2002).</li><li>Totoiu, M. O., Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury+is+accompanied+by+chronic+progressive+demyelination.">Spinal cord injury is accompanied by chronic progressive demyelination.</a> J Comp Neurol. 486, 373-383 (2005).</li><li>Chua, J. S., 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=Extending+neurites+sense+the+depth+of+the+underlying+topography+during+neuronal+differentiation+and+contact+guidance.">Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance.</a> Biomaterials. 35, 7750-7761 (2014).</li><li>Dowell-Mesfin, N. M., 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=Topographically+modified+surfaces+affect+orientation+and+growth+of+hippocampal+neurons.">Topographically modified surfaces affect orientation and growth of hippocampal neurons.</a> J Neural Eng. 1, 78-90 (2004).</li><li>Baek, S., Gleason, R. L., Rajagopal, K. R., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+small+on+large:+Potential+utility+in+computations+of+fluid-solid+interactions+in+arteries.">Theory of small on large: Potential utility in computations of fluid-solid interactions in arteries.</a> Comput Method Appl M. 196, 3070-3078 (2007).</li><li>Liu, 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=Effect+of+Static+Pre-stretch+Induced+Surface+Anisotropy+on+Orientation+of+Mesenchymal+Stem+Cells.">Effect of Static Pre-stretch Induced Surface Anisotropy on Orientation of Mesenchymal Stem Cells.</a> Cell Mol Bioeng. 7, 106-121 (2014).</li><li>Liu, 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=The+impact+of+pre-stretch+induced+surface+anisotropy+on+axon+regeneration.">The impact of pre-stretch induced surface anisotropy on axon regeneration.</a> Tissue Eng Part C Methods. , (2015).</li><li>Berns, E. 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=Aligned+neurite+outgrowth+and+directed+cell+migration+in+self-assembled+monodomain+gels.">Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels.</a> Biomaterials. 35, 185-195 (2014).</li><li>Kidambi, S., Lee, I., Chan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+neuron/astrocyte+co-culture+on+polyelectrolyte+multilayer+films:+A+template+for+studying+astrocyte-mediated+oxidative+stress+in+neurons.">Primary neuron/astrocyte co-culture on polyelectrolyte multilayer films: A template for studying astrocyte-mediated oxidative stress in neurons.</a> Adv Funct Mater. 18, 294-301 (2008).</li><li>Xia, 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=Directed+neurite+growth+of+rat+dorsal+root+ganglion+neurons+and+increased+colocalization+with+Schwann+cells+on+aligned+poly(methyl+methacrylate)+electrospun+nanofibers.">Directed neurite growth of rat dorsal root ganglion neurons and increased colocalization with Schwann cells on aligned poly(methyl methacrylate) electrospun nanofibers.</a> Brain Research. 1565, 18-27 (2014).</li><li>Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+Extremes+and+exploitations.">Stretch growth of integrated axon tracts: Extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Mantuano, E., Jo, M., Gonias, S. L., Campana, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+Density+Lipoprotein+Receptor-related+Protein+(LRP1)+Regulates+Rac1+and+RhoA+Reciprocally+to+Control+Schwann+Cell+Adhesion+and+Migration.">Low Density Lipoprotein Receptor-related Protein (LRP1) Regulates Rac1 and RhoA Reciprocally to Control Schwann Cell Adhesion and Migration.</a> Journal of Biological Chemistry. 285, 14259-14266 (2010).</li><li>Kim, B., ET, K. P., Papautsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+of+plasma+oxidized+PDMS+surfaces.">Long-term stability of plasma oxidized PDMS surfaces.</a> Conf Proc IEEE Eng Med Biol Soc. 7, 5013-5016 (2004).</li><li>Lopera, S., Mansano, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-Based+Surface+Modification+of+Polydimethylsiloxane+for+PDMS-PDMS+Molding.">Plasma-Based Surface Modification of Polydimethylsiloxane for PDMS-PDMS Molding.</a> ISRN Polymer Science. 2012, (2012).</li><li>Chandra, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organosilicon Materials. , (2013).</li><li>Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+poly(dimethylsiloxane)+surface+modification+to+control+cell+adhesion.">Simple poly(dimethylsiloxane) surface modification to control cell adhesion.</a> Surf Interface Anal. 41, 11-16 (2009).</li><li>Moore, 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=Multiple-channel+scaffolds+to+promote+spinal+cord+axon+regeneration.">Multiple-channel scaffolds to promote spinal cord axon regeneration.</a> Biomaterials. 27, 419-429 (2006).</li><li>Clarke, 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=Micropatterned+methacrylate+polymers+direct+spiral+ganglion+neurite+and+Schwann+cell+growth.">Micropatterned methacrylate polymers direct spiral ganglion neurite and Schwann cell growth.</a> Hearing Res. 278, 96-105 (2011).</li><li>Pfister, B. 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=Development+of+transplantable+nervous+tissue+constructs+comprised+of+stretch-grown+axons.">Development of transplantable nervous tissue constructs comprised of stretch-grown axons.</a> J Neurosci Methods. 153, 95-103 (2006).</li></ol>

önceden gerilmiş bir yüzeye akson hizalama teşvik etmek için, iki kritik adımlar vardır: 1) PDMS zarı ve homojen kalınlıkta düz olmalıdır; ve 2) glia hücreleri DRG uzaklaştırılmalıdır. PDMS ve çapraz bağlayıcı karıştırma ve bir fırında kür sonra, çapraz PDMS jel düz bir tezgah üstünde tutulur ve herhangi bir eğilmesini önlemek için dikkatle ele alınmalıdır. Plazma tedaviden sonra (hücre bağlanması için gerekli olan) sert yüzeye adsorbe zamanla azaltır çünkü PDMS dokunun ok...

Sinir yaralanmalarının ise, yakın ve uzak sinir kütükleri çoğu zaman lezyon yerinde 1-2 sinir liflerinin direkt yeniden düzenlenmesi engellenir. Normalde, akson yolları bağlantı karmaşık ağlar oluşturan aksonların çok düzenli ve uyumlu demetleri, oluşmaktadır. Ancak, sinir rejenerasyonu kötü organize akson hizalama 3-4 dolayı kaotik bir süreçtir. Bu nedenle, lezyon yerinin köprü aksonlar rejenere yeterli bir sayı üretmek için, iyi organize aksonal hizalanma yaratmak için de gereklidir. Ayrıca, demiyelinizasyon yaralanma yerinde myelinating hücrelerin ölümüne bağlı sinir yaralanmaları eşlik eder. Demiyelinizasyon kuvvetle aksonlar hayatta iletken kapasitesini bozar yana, demiyelinizasyon hedefleme veya yeniden miyelin oluşumunu teşvik tedaviler sinir hasarı 5 sonra fonksiyonel iyileşme için önemlidir. Dolayısıyla, bu protokolün amacı, bu iki konuyu ele alan bir mühendislik yaklaşımı göstermektirSinir rejenerasyon. Bir malzemenin fiziksel ve mekanik özellikleri, farklı eksenler boyunca ölçüldüğünde, bir fark olarak tanımlanan yüzey anizotropi, hücre hizalama, büyüme ve taşıma 6-7 etkilemek için uygulanmaktadır. topografya ek olarak, anizotropi indükleme için diğer yöntemler bulunmaktadır. Daha önce, poli-dimetil-siloksan (PDMS) membranın mekanik statik ön streç neden olduğu yüzey anizotropi incelenmiştir. &quot;Büyük uygulanan küçük deformasyon süper&quot; teorisi hücreleri gerilmiş yönde anlamda etkili bir sertliği dik yönde farklıdır tahmin ve etkin sertlikteki bu fark anizotropi 8 yüzey kaynaklanmaktadır. Önceden gerilmiş PDMS zar üzerinde kültürlenmiş mezenkimal kök hücreler (MSC&#39;ler) aktif yüzeyi çekerek ve bunun sonucu olarak anizotropiye duyu önceden gerilmiş yönde 9 hizalamak edebiliyoruz. Benzer şekilde, anizotropik yüzey armekanik ön-gerilmiş yüzeyinden ising sırt kök gangliyonu hizalamasını yanı sıra büyüme ve miyelinasyonun (DRG) 10 aksonlar etkiler. Burada akson rejenerasyonu 10 artırmak için statik bir önceden gerilmiş PDMS substrat yüzey anizotropi uyarılması için bir protokol sağlar. Akson hizalama, istenilen desenleri ile topolojik özellikleri ortaya çıkarmak için, hizalanmış lifleri ve kanallar 6,11-12 ile temas rehberlik sağlamak için rapor, akson hizalama 11,13 kolaylaştırmak için ortaya konmuştur. Ancak, bu tür lifleri, kanallar ve desen olarak topolojik özellikler aracılığıyla akson hizalama uyarılması için teknikler bildirdi uzatmak ve akson kalınlığını artırmak için koyamadık. Tersine, mekanik kademeli bir esneme 14 büyüklüğü arttı daha uzun ve daha kalın akson streç yönünde akson hizalama yol germe. Bununla birlikte, in vivo koşullarında, bir çalışan motorlu cihazı içeren değildirmümkün. Bunun aksine, statik önceden gerilmiş neden anizotropi daha az karmaşık ve daha kolay bir şekilde in vivo uygulamalar için gelecekteki iskele tasarımlar içine dahil edilebilir. Bu protokol, statik önceden gerilmiş hücre kültür sistemi topolojik özellikler olmadan yüzey anizotropi indüklemek için kullanılır. Membran çerçevesi üzerine sabitlenir ve önceden tespit edilmiş bir germe büyüklüğü germe aşaması uygulanır, bunun üzerine önceden gerilmiş kültür sistemi, PDMS zar, bir gerilebilir çerçeve ve bir germe aşamasında oluşmaktadır. 21 güne kadar önceden gerilmiş yüzünde kültür taze izole DRG nöronları akson hizalama ve kalınlık için izlenir. Daha sonra, Schwann hücreleri (SCS) miyelinasyon izlenir hizalanmış aksonlar ile birlikte kültürlendi. Ön streç kaynaklı yüzey anizotropi birleşmeyle biz MSC ve DRG nöronlarının sırasıyla 9-10, hücre hizalama farklılaşma ve akson hizalama büyümeyi artırmak başardık.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane &#65288;PDMS)</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium 1X</td>
			<td>GibcoBRL</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement 50X</td>
			<td>GibcoBRL</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax-I 100X</td>
			<td>GibcoBRL</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax-I</td>
			<td>GibcoBRL</td>
			<td>11020-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor-7S</td>
			<td>Invitrogen</td>
			<td>13290-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>GibcoBRL</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA/1mM EDTA</td>
			<td>GibcoBRL</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Trevigen</td>
			<td><a href="http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/">3438-100-01</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>p-6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-2 deoxy-uridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Isolation Buffer</td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11885</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE</td>
			<td>Clonetics</td>
			<td>CC-4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Calbiochem</td>
			<td>344270</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone chamber</td>
			<td>Greiner bio-one</td>
			<td>FlexiPERM ConA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaning/etching system</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Thy 1.1 antibody</td>
			<td>Sigma- Aldrich</td>
			<td>M7898</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Complement</td>
			<td>Sigma- Aldrich</td>
			<td>S-7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard growth medium</td>
			<td></td>
			<td></td>
			<td>For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &#956;l of NGF-- 7S (50 ug/ml).</td>
		</tr>
		<tr>
			<td>FDU and Uridine stock solution</td>
			<td></td>
			<td></td>
			<td>FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>then divide in 1 ml aliquots and store at -20 &#186;C.</td>
		</tr>
	</tbody>
</table>

an approach to enhance alignment and myelination of dorsal root ganglion neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

Önceden gerilmiş hücre kültür sistemi DRG akson hizalama 10 terfi. DRG nöronları için 12 gün önceden gerilmiş gerek gerilmemiş yüzeylerine kültürlenmiştir. Aksonlar hizalarını göstermek için β-III-tubulin boyandı. 2 Kültür 12 gün sonra önceden gerilmiş gerek gerilmemiş PDMS yüzeylerde akson yönünü karşılaştırır Şekil. rastgele uyum gösterdi ve gerilmemiş PDMS substrat üzerinde birbirine bağlı bir ağ ...

Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Bir Yaklaşım sırt kök ganglion nöronlar hizalama ve myelinizasyondur Artırma

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

The overall goal of this protocol is to enhance axon alignment and myelination of dorsal root ganglion neurons using a static, pre-stretched cell culture system. This method can help answer key question in the nerve tissue engineering field, such as axon alignment and thickness growth. The main advantage of this technique is that this pre-stretched method not only enhances axon alignment but also promotes myelination.To begin this procedure, pour the mixture of base and curing agent into a tissue culture dish. Keep the gel mixture under vacuum for 20 minutes to remove air bubbles. Then place the gel mixture in the oven at 60 degrees Celsius for overnight curing.After the PDMS membrane has cured, treat the surface with oxygen plasma in the plasma cleaning and etching system for three minutes. Subsequently, cut a piece of rectangular membrane from the dish. Fix it onto a stretching frame with the oxygen-treated side facing up.Then place the frame on a stretching stage and evenly stretch it by turning the knob on the stage until it reaches 10%elongation in the longer axis. Next, fix the stretch by tightening the screws on the frame. After that, remove the frame from the stage.Ensure the membrane surface is dry and free of dust before placing a silicone chamber onto the membrane. Allow the sticky side of the chamber to attach tightly to the membrane. Then sterilize the surface with UV for 10 minutes.Afterward, add one milliliter of PLL to the PDMS surface in the chamber within six hours of plasma treatment. Next, incubate it for two hours at 37 degrees Celsius prior to seeding the DRG neurons to enhance cell attachment. In this step, prepare the standard grow medium for the DRG neurons.Prior to isolation, autoclave the isolation equipment and filter reagents. Add five milliliters of isolation buffer into one well of a six-well plate and place the plate on ice. Then prepare 10 milliliters of dissociation medium by adding one milliliter of collagenase A to nine milliliters of 0.05%Trypsin-EDTA.Filter the solution with a 0.22 micrometer filter and place it on ice. Afterward, sacrifice 10 to 12 five to seven days old SD rats and cut away the skin overlying the spinal cord from the back of each one. Remove any excess tissue around the spine.Under a surgical magnifier, make the first incision from the neck then make one cut along the spine on both sides with scissors. Detach the spine from the body of the pup and remove excess muscles. Then cut along the long axis of the spine and use tweezers to open the spine completely to extract the spinal cord.Next, remove the ganglion from the bone pocket. Trim the nerve roots and transfer the ganglia to the ice-cold isolation buffer. Collect approximately 10 to 16 DRGs from both sides.Repeat the dissection procedure for the rest of the pups. Now, transfer the DRGs with isolation buffer into a sterile 15-milliliter tube. Let the tissue settle to the bottom of the tube and gently remove the isolation buffer on top.Then add 10 milliliters of dissociation medium into the tube. Incubate the dissected tissues in the dissociation medium in a 37-degree Celsius water bath for one hour while shaking the tube every five to 10 minutes. After the chemical dissociation, centrifuge the ganglia at four degrees Celsius for five minutes.After five minutes, remove the supernatant, resuspend the pellet in 10 milliliters of standard growth medium and vortex it. Subsequently, centrifuge the dissociated cells once again. Then remove the supernatant, resuspend the pellet in 12 milliliters of standard growth medium and vortex it.Prior to seeding the cells, remove the PLL solution from the chamber. Rinse the PDMS surface with sterile water and air-dry it. After resuspending the cells, let the suspension sit for one to two minutes to allow the debris to settle to the bottom of the tube.Next, add 1.5 milliliters of cell suspension into each stretched chamber and incubate it at 37 degrees Celsius with 5%CO2. To eliminate glial cells at one day in vitro, add 10 microliters or 15 microliters of the FDU-U mixture stock solution to each well. After seven hours, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37-degree Celsius incubator in 5%CO2.During the cell culture period, change the medium every two days by replacing half the spent medium with fresh medium. In this procedure, remove the culture medium from the Schwann cells and add five milliliters of 0.05%Trypsin-EDTA into the flask. Incubate the cells at 37 degrees Celsius in 5%CO2 for two to three minutes.Check the cells under the optical microscope using a 10X objective to see if they lift up from the flask. Then add five milliliters of culture medium to the cells in the flask and mix. Subsequently, add the cell suspension to a 15-milliliter centrifuge tube and centrifuge at 20 degrees Celsius for five minutes.Then remove the supernatant and resuspend the pellet in seven milliliters of standard DRG growth medium. Following this, transfer 10 microliters of the cell suspension into a 0.5-milliliter microcentrifuge tube. Mix it with 10 microliters of Trypan blue and then count the cell number with a hemocytometer using a 10X objective.Add the DRG growth medium to dilute the cell suspension to 5, 000 cells per milliliter. Next, remove 0.5 milliliters of the medium from the DRG culture in the stretched chamber and add 0.5 milliliters of Schwann cell suspension to the DRG culture. Culture the cells for one week at 37 degrees Celsius in 5%CO2.Change the medium every two days by replacing half of the spent medium with fresh standard growth medium. After culturing on the stretched and unstretched surfaces for two weeks, purified Schwann cells were added to the chamber and cultured for another week. Shown here is the beta-III tubulin staining for axons on the stretched surface.And this is the overlay of beta-III tubulin and P0 staining on the stretched surface, suggesting the Schwann cells are attached to and likely myelinating the axons. This image shows the beta-III tubulin staining for axons on the unstretched surface. And the overlay of beta-III tubulin and P0 staining on the unstretched surface suggests that the Schwann cells are not attached to the axons.Once mastered, this technique can be done in three to four hours if it is performed properly. While attempting this procedure, it is important to remember to keep the PDMS membrane flat and of homogeneous thickness. Following this procedure, atomizers like pre-stretched microchannels can be performed in order to answer additional questions like axon lens and Schwann cells migration.After its development, this technique will pave the way for researchers in the field of tissue engineering to explore the role of surface anisotropy on nerve regeneration in spinal cord and sciatic nerve. After watching this video, you should have a good understanding of how to induce axon alignment and myelination using pre-stretched device.

Research

JoVE Journal

Neuroscience

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

Kültür Yetişkin Dorsal Root Ganglion Nöronlar ile Axon Rejenerasyon Genetik Çalışma

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Nörobilim

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Kök Ganglia Nöronlar ve Farklılaştırılmış Yağ-türetilmiş Kök Hücreler Dorsal: bir<em&gt; İn Vitro</em&gt; Co-kültür Modeli Periferik Sinir Rejenerasyon Eğitim

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Sırt kök ganglion Modeli: Kanserli Sinir Invasion Vitro Modelleme

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Sırt kök ganglion Enjeksiyon ve Dorsal Kök Duyusal Axon Rejenerasyon Modeli Olarak Ezilme Yaralanması

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Lomber Dorsal kök gangliyon erişim ve domuz yerleştirmeye laminotomy

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Bağışıklık hücreleri tek siyatik sinirleri ve Dorsal kök gangliyon üzerinden tek bir fare kullanarak Akış Sitometresi Analizi

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal kök gangliyon yalıtım ve birincil kültür nörotransmitter yayın çalışmaya

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Soruşturma memeli Axon rejenerasyon: Vivo Elektroporasyon yetişkin fare Dorsal kök gangliyon

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

İletim Yetmezliği Mekanizmasını İncelemek Için Bağlı Siyatik Sinir ile In Vivo Tek Lifli Kayıt ve Bozulmamış Dorsal Kök Ganglion Kullanımı

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

Dorsal Kök Ganglion Makrofajlarının Hızlı İzolasyon

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...