Gli autori desiderano ringraziare Eric Vasco per la sua assistenza nella preparazione dei substrati PDMS, Dr. Shiyong Wang nel laboratorio del Dr. Marina Mata presso l&#39;Università del Michigan per i suggerimenti e la formazione del isolamento DRG utile, e il dottor Mark Tuszynski e il Dr . W. Marie Campana a UC San Diego per i suggerimenti utili e di protocollo per l&#39;isolamento SC. Questo studio è stato sostenuto in parte dalla National Science Foundation (CBET 0.941.055 e 1.510.895 CBET), il National Institute of Health (R21CA176854, R01GM089866, e R01EB014986).

Tutte le procedure per l&#39;isolamento delle cellule sono stati approvati dal Comitato Istituzionale cura degli animali e Usa presso la Michigan State University. 1. Preparazione di pre-allungato anisotropico Surface <ol><li> Mescolare un 10: 1 soluzione di base e catalizzatore e versare il composto in un piatto di coltura di tessuti (12 cm di diametro). Utilizzare 4.900 mg base e 490 mg di catalizzatore per la miscela totale reticolazione. </li><li> Mantenere la miscela gel sotto vuoto per 20 minuti per rimuovere le bolle d&#39;aria. </li><li> Mettere il composto gel nel forno per polimerizzazione durante la notte a 60 ° C. </li><li> Dopo le cure membrana PDMS, trattare la superficie con plasma di ossigeno utilizzando un sistema di pulizia / attacco in plasma per 3 min a 165 mTorr e 65 sccm flusso di O 2. </li><li> Tagliare un pezzo di membrana rettangolare (5 × 3,5 cm) dal piatto, pur essendo consapevoli che parte è l&#39;ossigeno-trattati, assicurarsi che il lato di ossigeno trattato rivolto verso l&#39;alto e fissare su un telaio di stretching. Posizionare il telaio su una<html> allungamento fase e uniformemente allungare ruotando la manopola sulla scena fino a raggiungere il 10% di allungamento (o qualche altro tratto predeterminato, l&#39;allungamento può essere letta direttamente dalla fase stretching) in asse maggiore 9. Fissare il tratto serrando le viti sul telaio. </li><li> Rimuovere il telaio dal palco. Assicurarsi che la superficie della membrana è asciutto e privo di polvere, quindi posizionare una camera di silicone sulla membrana che permette la parte adesiva della camera per fissare saldamente alla membrana. Nota: La camera di silicone fornisce un pozzo per trattenere il mezzo cellulare sulla membrana PDMS. Il design del dispositivo è mostrato in Figura 1. </li><li> Sterilizzare la superficie con UV per 10 min. </li><li> Aggiungere 1 ml Poly-L-lisina (PLL) (0,01% in tampone fosfato isotonico) alla superficie PDMS all&#39;interno della camera, entro 6 ore di trattamento al plasma e incubare per 2 ore a 37 ° C prima della semina neuroni DRG. Questo migliora l&#39;adesione cellulare. </li></ol>Le &quot;&gt; 2. Isolamento di DRG <ol><li> Preparare terreno di coltura standard per i neuroni DRG. </li><li> Prima l&#39;isolamento, l&#39;apparecchiatura autoclave isolamento e reagenti filtranti. Aggiungere 5 ml di tampone isolamento in un pozzetto di un 6-pozzetti e posizionare la piastra su ghiaccio. Preparare 10 ml di terreno di dissociazione aggiungendo 1 ml di collagenasi A (500 U / ml) in 9 ml di 0,05% tripsina-EDTA (1 mg / ml), filtrare le soluzioni con un filtro da 0,22 um e posto sul ghiaccio. </li><li> Utilizzare dieci a dodici 5 - 7 ratti Sprague-Dawley giorni &#39;per l&#39;isolamento. Spruzzare i cuccioli con il 75% di isopropanolo, e il sacrificio per decapitazione. </li><li> Tagliare via la pelle sovrastante il midollo spinale dal retro e rimuovere il tessuto in eccesso intorno alla spina dorsale. Sotto una lente di ingrandimento chirurgica con i faretti chirurgici su, fare la prima incisione dal collo e poi un taglio lungo la colonna vertebrale su entrambi i lati con le forbici. Rimuovere il rachide dal corpo dei cuccioli e rimuovere i muscoli eccesso. Quindi, usare le forbici per tagliare lungo la long asse della colonna vertebrale e di uso delle pinzette per aprire la colonna vertebrale completamente ed estrarre il midollo cord.Remove la punta da una pipetta per creare una maggiore apertura per il trasferimento dei DRG con tampone di isolamento in una provetta sterile 15 ml. </li><li> Usando un bel paio di pinzette, rimuovere il ganglio dalla tasca ossea e raccogliere circa 10 - 16 DRG da entrambi i lati. Tagliare le radici nervose e trasferire gangli al buffer isolamento ghiacciato. </li><li> Rimuovere la punta da una pipetta per creare una maggiore apertura per il trasferimento dei DRG con tampone di isolamento in una provetta sterile 15 ml. Dopo la dissociazione chimica, centrifugare i gangli a 900 xg e 4 ° C per 5 min. </li><li> Lasciate che i tessuti si depositano sul fondo della provetta e rimuovere delicatamente il buffer di isolamento sulla parte superiore e aggiungere 10 ml di terreno di dissociazione nel tubo. </li><li> Incubare i tessuti sezionati nel mezzo di dissociazione in un bagno d&#39;acqua a 37 ° C per 1 ora agitando il tubo ogni 5 - 10 min. </li><li> Dopola dissociazione chimica, centrifugare i gangli a 900 xg e 4 ° C per 5 min. </li><li> Rimuovere il surnatante e risospendere il pellet in 10 ml di terreni di crescita standard e vortice. </li><li> Centrifugare le cellule dissociate come indicato nel paragrafo 2.9, ancora una volta. </li><li> Rimuovere il surnatante, risospendere il pellet in 12 ml di mezzi di crescita standard e vortice. </li></ol> 3. Cultura di DRG sulla superficie Pre-allungato <ol><li> Prima di semina delle cellule, rimuovere le soluzioni PLL dalla camera e risciacquare la superficie PDMS con acqua sterile e asciugare all&#39;aria. </li><li> Dopo ri-sospensione le cellule, lasciare che il set sospensione per 1 - 2 min per permettere i detriti di depositarsi sul fondo della provetta. Aggiungere 1,5 ml di sospensione cellulare in ciascuna camera elasticizzato e incubare a 37 ° C in 5% CO 2. </li><li> Per eliminare le cellule gliali, a 1 giorno di vitro (DIV), aggiungere 10 ml (o 15 ml) miscela di fluoro-2-deossi uridina e uridina (FDU-U) stock solutisu (Vedi Tabella dei Materiali) in ciascun pozzetto. Dopo 7 ore, sostituire questo mezzo con mezzo di crescita standard di fresco e posizionare il dispositivo di pre-stirato cultura in un incubatore a 37 ° C con 5% di CO 2. </li><li> Durante il periodo di coltura cellulare (2 - 3 settimane), cambiare i media ogni due giorni, sostituendo la metà del mezzo trascorso con terreno fresco. Nota: Dopo coltura sulle superfici tese e non deformato per 2 settimane, SC purificati vengono aggiunti alla camera e coltivate per un&#39;altra settimana (passo 4.7). </li></ol> 4. La co-coltura di cellule di Schwann (SCS) con DRG neuroni sulla superficie Pre-allungato <ol><li> Isolare SC come descritto dal gruppo del Dr. Campana in precedenza 15. <ol><li> In breve, isolare la SC da un 1 giorno di età cucciolo. Raccogliere e dissociare i nervi sciatico sia chimicamente (tripsina-EDTA e Collagenasi A) e meccanicamente (18 gauge / siringa da 10 ml) 15. </li><li> Seme le cellule dissociate in poli-D-lisina (PDL) copallone ated T25 e la cultura a 37 ° C al 5% di CO 2. Il giorno 5, purificare le cellule attraverso la selezione di anticorpi con Anti-thy 1.1 anticorpi e complemento di coniglio e sottocultura le cellule purificate al passaggio 2 - 4. mezzo di Utilizzo di coltura contenente Dulbecco Modified Eagle Medium (DMEM), il 10% di siero fetale bovino (FBS) , 1% penicillina / streptomicina (Penn / Strep), 21 mg / ml Bovine Pituitary Extract (BPE), e 4 micron Forskolin. </li></ol></li><li> Rimuovere il terreno di coltura dalla SC, e aggiungere 5 ml 0,05% tripsina-EDTA nel pallone. Incubare le cellule a 37 ° C al 5% di CO 2 per 2 - 3 min. </li><li> Controllare cellule sotto il microscopio ottico utilizzando un obiettivo 10X per vedere se essi sollevano dal pallone, quindi aggiungere 5 ml di terreno di coltura e mescolare con le cellule nel pallone. </li><li> Aggiungere la sospensione cellulare in una provetta da centrifuga da 15 ml e centrifugare a 200 xg e 20 ° C per 5 min. </li><li> Rimuovere il surnatante e risospendere il pellet in 7 ml di DRG serie med crescitaia. </li><li> Prendere 10 ml di sospensione cellulare in una provetta da 0,5 ml, mescolare con 10 ml di trypan blu, e poi contare il numero di cellule utilizzando un emocitometro al microscopio ottico con un obiettivo 10X. Diluire la sospensione cellulare a 5.000 cellule per ml aggiungendo terreni di crescita DRG. </li><li> Rimuovere 0,5 ml di terreno dalla coltura DRG nella camera allungata e aggiungere 0,5 ml di sospensione SC alla cultura DRG. </li><li> Coltivare le cellule per 1 settimana a 37 ° C e 5% di CO 2. Modificare i media ogni due giorni, sostituendo la metà del mezzo trascorso con terreno di coltura standard di fresco per DRG. Dopo 1 settimana co-coltura, le cellule processo di immunoistochimica 10. </li></ol>

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The authors do not have any conflict of interest to disclose.

Per indurre l&#39;allineamento assone sulla superficie pre-stirato, ci sono due passaggi critici: 1) la membrana PDMS deve essere piana e di spessore omogeneo; e 2) cellule gliali devono essere rimossi dal DRG. Dopo la miscelazione il PDMS e reticolante e cottura in forno, il gel PDMS reticolato dovrebbe essere mantenuto su un banco piana e maneggiato con cura per evitare qualsiasi inclinazione. Il trattamento al plasma di ossigeno della membrana PDMS dovrebbe essere seguita entro 6 ore mediante rivestimento PLL, poich?...

In lesioni nervose, i monconi prossimali e distali del nervo sono spesso impedito di riallineamento diretta di fascicoli nervosi a causa del sito della lesione 1-2. Normalmente, tratti di assoni sono composti da fasci altamente ordinate e allineate di assoni, che formano reti complesse di connettività. Tuttavia, la rigenerazione dei nervi è un processo caotico a causa di allineamento degli assoni mal organizzato 3-4. Pertanto, per generare un numero sufficiente di rigenerare assoni che colmano il sito della lesione, è necessario per indurre ben organizzato allineamento assonale. Inoltre, demielinizzazione accompagna lesioni nervose a causa di morte delle cellule mielinizzanti presso il luogo di ferita. Dal momento che demielinizzazione compromette fortemente la capacità conduttiva di sopravvivere assoni, i trattamenti di targeting demielinizzazione o che promuovono la rimielinizzazione sono significativi per il recupero funzionale dopo lesione del nervo 5. Così l&#39;obiettivo di questo protocollo è quello di illustrare un approccio ingegneristico che affronta questi due problemidi rigenerazione nervosa. Anisotropia di superficie, che è definito come la differenza, quando misurata lungo assi diversi, in proprietà fisiche e meccaniche di un materiale, è stato applicato per influenzare l&#39;allineamento cellulare, la crescita e la migrazione 6-7. Oltre alla topografia, ci sono altri metodi per indurre anisotropia. In precedenza, abbiamo studiato anisotropia superficie indotta dalla meccanica statica pre-tratto di poli-dimetil-silossano (PDMS) membrana. La teoria del &quot;piccolo super-deformazione imposta su grandi&quot;, prevede che la rigidezza efficace le cellule percepiscono nella direzione allungata differisce dalla direzione perpendicolare, e questa differenza di rigidità efficace è dovuto alla superficie anisotropia 8. Cellule staminali mesenchimali (MSC) coltivate su una membrana pre-stirato PDMS sono in grado di percepire l&#39;anisotropia tirando attivamente la superficie e, di conseguenza, allineare in direzione prestirato 9. Analogamente, una superficie ar anisotropoising da una superficie pre-stirato meccanico colpisce l&#39;allineamento, così come la crescita e la mielinizzazione dei gangli delle radici dorsali (DRG) assoni 10. Qui forniamo un protocollo per indurre anisotropia di superficie su un substrato di PDMS pre-stirato statico per migliorare la rigenerazione degli assoni 10. Per suscitare l&#39;allineamento degli assoni, le caratteristiche topologiche con i modelli desiderati, segnalato per fornire una guida contatto attraverso fibre allineate e canali 6,11-12, sono stati dimostrato di facilitare l&#39;allineamento degli assoni 11,13. Tuttavia, le tecniche per indurre l&#39;allineamento assone attraverso le caratteristiche topologiche, come fibre, canali e patterning riportato, erano in grado di allungarsi e aumentare lo spessore degli assoni. Al contrario, graduale stiramento meccanico portato all&#39;allineamento assone nella direzione di stiro con assoni più lunghi e più spessi che aumentano con la grandezza del tratto 14. Tuttavia, incorporante un dispositivo motore alimentato in vivo non èfattibile. Al contrario, statica anisotropia indotta prestirato è meno complicato e può essere più facilmente incorporato in futuri disegni ponteggi per applicazioni in vivo. In questo protocollo, un sistema di coltura cellulare prestirato statico viene utilizzato per indurre anisotropia superficie senza caratteristiche topologiche. Il sistema di coltura prestirato è composto da una membrana PDMS, un telaio estensibile e una fase di stretching, dopo di che la membrana è fissata sul telaio ed una grandezza tratto predeterminato viene applicato sulla scena stretching. Appena isolate neuroni DRG coltivate sulla superficie prestirato che fino a 21 giorni sono monitorati per l&#39;allineamento assone e spessore. Successivamente, le cellule di Schwann (SC) co-coltura con gli assoni allineati vengono monitorati per mielinizzazione. Incorporando pre-stiro indotta anisotropia superficie siamo stati in grado di migliorare l&#39;allineamento delle cellule-differenziazione e assone allineamento crescita delle cellule staminali mesenchimali e neuroni DRG 9-10, rispettivamente.

<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.

Il sistema di coltura pre-stirato cellule promosso DRG allineamento assone 10. neuroni DRG sono stati coltivati ​​su superfici pre-stirato e non deformato per 12 giorni. Gli assoni sono state colorate per β-III-tubulina per dimostrare la loro allineamento. La figura 2 confronta orientamento assone sulle PDMS pre-stirato e non deformato substrati dopo 12 giorni di coltura. Gli assoni DRG allineati parallelamente alla direzione allungata, che hanno mostrato...

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.

Un approccio per migliorare l&#39;allineamento e la mielinizzazione dorsale Root Ganglion neuroni

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

an-approach-to-enhance-alignment-myelination-dorsal-root-ganglion

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JoVE Journal

Neuroscience

8.4K Views.  Michigan State University. 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.

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

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Imaging in diretta degli assoni spinali dopo rizotomia

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

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Neuroscienze

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

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Studio genetico della rigenerazione assonale con colture di neuroni adulti Root dorsali gangliari

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

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

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 study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

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

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Gangli neuroni e cellule staminali derivate da tessuto adiposo differenziata: An<em&gt; In Vitro</em&gt; Co-coltura modello per studiare Peripheral Nerve Regeneration

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

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 neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

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

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modellazione di cancerose Neural Invasion: La dorsale della radice Ganglio Modello

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

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale Axon Regeneration

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

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomia per accesso del ganglio di radice dorsale lombare e l'iniezione nei suini

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

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Analisi delle cellule immuni in singolo nervo sciatico e del ganglio della radice dorsale da un singolo Mouse mediante citometria a flusso

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 root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Isolamento dei gangli di radice dorsale e colture primarie per lo studio del rilascio di neurotrasmettitore

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

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

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

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Inquirenti rigenerazione dell'assone dei mammiferi: In Vivo elettroporazione del ganglio di radice dorsale del topo adulto

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

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Uso della registrazione in fibra singola in vivo e della radice dorsale intatta Ganglion con il nervo Sciatico attaccato per esaminare il meccanismo del fallimento della conduzione

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