Die Autoren möchten sich Eric Vasco für seine Unterstützung bei der Vorbereitung der PDMS-Substrate, Dr. Shiyong Wang in Dr. Marina Mata Labor an der University of Michigan für hilfreiche Anregungen und Ausbildung des DRG-Isolation und Dr. Mark Tuszynski und Dr. danken W. Marie Campana an der UC San Diego. für hilfreiche Anregungen und das Protokoll für die SC-Isolation. Diese Studie wurde teilweise von der National Science Foundation (CBET 0.941.055 und CBET 1.510.895), das National Institute of Health (R21CA176854, R01GM089866 und R01EB014986) unterstützt.

Alle Verfahren zur Isolierung der Zellen wurden von der Institutional Animal Care und Use Committee an der Michigan State University genehmigt. 1. Herstellung von vorgespannter anisotroper Oberfläche <ol><li> Mischen Sie eine 10: 1-Lösung von Base und Härter und gießen Sie die Mischung in eine Gewebekulturschale (12 cm Durchmesser). Verwenden Sie 4.900 mg Base und 490 mg Mittel für die gesamte Vernetzungs Mischung härtet. </li><li> Halten Sie die Gelmischung unter Vakuum für 20 min Luftblasen zu entfernen. </li><li> Legen Sie das Gel-Gemisch in dem Ofen über Nacht Aushärtung bei 60 ° C. </li><li> Nachdem die Membran aushärtet PDMS, Behandlung der Oberfläche mit Sauerstoffplasma eine Plasmareinigung / Ätzsystem für 3 min bei 165 mTorr und 65 sccm Fluss von O 2 verwendet wird . </li><li> Schneiden Sie ein Stück von rechteckigen Membran (5 × 3,5 cm) von der Schale, während bewusst zu sein, welche Seite mit Sauerstoff behandelt wird, stellen Sie sicher, dass der Sauerstoff behandelte Seite nach oben zeigt und fixieren auf einem Spannrahmen. Platzieren Sie den Rahmen auf ein<html> durch Drehen des Knopfes auf der Bühne Streckstufe und gleichmäßig dehnen , bis sie 10% Dehnung erreicht (oder eine andere vorgegebene Strecke, kann die Dehnung direkt von der Streckstufe gelesen werden) in der längeren Achse 9. Befestigen Sie die Strecke durch die Schrauben am Rahmen befestigen. </li><li> Entfernen Sie den Rahmen von der Bühne. Sicherstellen, dass die Membran-Oberfläche ist trocken und frei von Staub, legen Sie dann eine Silikonkammer auf die Membran der klebrigen Seite der Kammer können an die Membran zu befestigen fest. Hinweis: Die Silikon-Kammer bietet eine gut zum Halten des Zellmedium auf der PDMS-Membran. Die Vorrichtung ist in Abbildung 1 dargestellt. </li><li> Sterilisieren der Oberfläche mit UV für 10 min. </li><li> 1 ml Poly-L-lysin (PLL) (0,01% in phosphatgepufferter Salzlösung) mit der PDMS-Oberfläche innerhalb der Kammer, innerhalb von 6 Stunden der Plasmabehandlung und Inkubation für 2 h bei 37 ° C, bevor die DRG Neurone Animpfen. Dies fördert die Zellhaftung. </li></ol>le &quot;&gt; 2. Die Isolierung von DRG <ol><li> Bereiten Sie Standard-Wachstumsmedium für die DRG-Neuronen. </li><li> Vor der Isolierung, autoklaviert die Isolations Ausrüstung und Filter Reagenzien. 5 ml Isolationspuffer in eine Vertiefung einer 6-Well-Platte, und legen Sie die Platte auf Eis. Herstellung von 10 ml der Dissoziation Medium durch 1 ml Kollagenase A Zugabe (500 U / ml) zu 9 ml von 0,05% Trypsin-EDTA (1 mg / ml), filtriert die Lösung mit einem 0,22 um-Filter und auf Eis. </li><li> Verwenden Sie zehn bis zwölf fähig 5 - 7 Tage alten Sprague-Dawley Ratten für die Isolierung. Sprühen Sie die Welpen mit 75% Isopropanol und opfern durch Enthauptung. </li><li> Schneiden Sie die Haut über dem Rückenmark von hinten weg und entfernen Sie das überschüssige Gewebe rund um die Wirbelsäule. Unter einem chirurgischen Lupe mit den chirurgischen Scheinwerfer auf, machen den ersten Schnitt aus dem Hals und dann ein Schnitt entlang der Wirbelsäule auf beiden Seiten mit einer Schere. Nehmen Sie die Wirbelsäule aus dem Körper der Welpen und die überschüssigen Muskeln zu entfernen. Dann mit einer Schere entlang der lon zu schneideng Achse der Wirbelsäule und mit Hilfe einer Pinzette die Wirbelsäule vollständig zu öffnen und das Rücken cord.Remove die Spitze aus einer Pipette extrahieren, um eine größere Öffnung für die Übertragung der DRGs mit Isolationspuffer in ein steriles 15 ml Tube. </li><li> Mit einem feinen Pinzette, entfernen Sie das Ganglion aus dem Knochentasche und sammeln etwa 10-16 DRG von beiden Seiten. Schneiden Sie die Nervenwurzeln und übertragen die Ganglien auf dem eiskalten Isolationspuffer. </li><li> Die Spitze aus einer Pipette eine größere Öffnung für die Übertragung der DRGs mit Isolationspuffer in ein steriles 15 ml Tube. Nach der chemischen Dissoziation Zentrifuge die ganglia bei 900 xg und 4 ° C für 5 min. </li><li> Lassen Sie das Gewebe an der Unterseite des Rohres absetzen und sanft den Isolationspuffer oben entfernen und 10 ml Dissoziation Medium in das Röhrchen hinzufügen. </li><li> Inkubieren Sie die sezierten Gewebe im Dissoziation Medium in einem Wasserbad 37 ° C 1 Stunde für während der Schlauch alle 5 Schütteln - mindestens 10. </li><li> Nachdie chemische Dissoziation, zentrifugieren Sie das ganglia bei 900 xg und 4 ° C für 5 min. </li><li> Entfernen Sie den Überstand und resuspendieren Sie das Pellet in 10 ml Standardwachstumsmedien und Wirbel. </li><li> Zentrifugieren Sie die dissoziierten Zellen, wie in Abschnitt 2.9 noch einmal angezeigt. </li><li> Entfernen Sie den Überstand, resuspendieren Sie das Pellet in 12 ml Standardwachstumsmedien und Wirbel. </li></ol> 3. Kultur der DRG auf vorgespannter Fläche <ol><li> Vor der Zellen Aussaat, die PLL-Lösungen aus der Kammer zu entfernen und die PDMS-Oberfläche mit sterilem Wasser und der Luft trocknen zu spülen. </li><li> Nachdem die Zellen wieder suspendiert, lassen Sie die Suspension Set mit 1 - 2 Minuten die Trümmer zu ermöglichen, auf den Boden des Röhrchens zu begleichen. 1,5 ml der Zellsuspension in jede Streckkammer und Inkubieren bei 37 ° C bei 5% CO 2. </li><li> Gliazellen, die am 1. Tag in vitro (DIV), mit 10 &amp; mgr; l (oder 15 &amp; mgr; l) Mischung aus Fluor-2-deoxy Uridin und Uridin (FDU-U) Lager soluti Zur Beseitigungauf (siehe Tabelle der Materialien) zu gut jeder. Nach 7 Stunden, ersetzen Sie dieses Medium mit frischem Standardwachstumsmedium und legen Sie die vorgestreckten Kulturvorrichtung in einem 37 ° C Inkubator mit 5% CO 2. </li><li> Während der Zellkulturperiode (2 - 3 Wochen), ändern Sie die Medien alle zwei Tage um die Hälfte des verbrauchten Medium durch frisches Medium ersetzt. Anmerkung: Nach Kultivierung auf den gedehnten und ungedehnten Flächen für 2 Wochen, gereinigt SCs werden zu der Kammer und eine weitere Woche kultiviert (Schritt 4.7). </li></ol> 4. Co-Kultur von Schwann-Zellen (SC) mit DRG Neurone auf vorgespannter Fläche <ol><li> Isolieren SCs wie von Dr. Campana Gruppe zuvor 15 beschrieben. <ol><li> Kurz gesagt, die SCs aus einem 1 Tage alten Welpen zu isolieren. Sammeln und die Ischiasnerven distanzieren sowohl chemisch (Trypsin-EDTA und Kollagenase A) und mechanisch (18 - Gauge - Nadel / 10 - ml - Spritze) 15. </li><li> Seed die dissoziierten Zellen in Poly-D-Lysin (PDL) coated T25 - Kolben und die Kultur bei 37 ° C bei 5% CO 2. Am 5. Tag reinigen die Zellen durch Antikörper Auswahl Anti-thy 1,1 Antikörper und Kaninchen-Complement verwenden und die gereinigten Zellen Passage 2 Subkultur - 4. Verwenden Kulturmedium, das Dulbecco modifiziertem Eagle-Medium (DMEM), 10% fötalem Rinderserum (FBS) , 1% Penicillin / Streptomycin (Penn / Strep), 21 &amp; mgr; g / ml Rinderhypophysenextrakt (BPE) und 4 &amp; mgr; M Forskolin. </li></ol></li><li> Entfernen Sie das Kulturmedium aus der SCs und 5 ml 0,05% Trypsin-EDTA in den Kolben. Inkubieren der Zellen bei 37 ° C bei 5% CO 2 für 2 - 3 min. </li><li> Überprüfen Zellen unter dem Lichtmikroskop ein 10X-Objektiv verwendet, um zu sehen, ob sie aus dem Kolben heben, fügen Sie dann 5 ml Kulturmedium und mischen sich mit den Zellen in den Kolben. </li><li> Fügen Sie die Zellsuspension in ein 15 ml Zentrifugenröhrchen und zentrifugiere bei 200 xg und 20 ° C für 5 min. </li><li> Entfernen Sie den Überstand und das Pellet in 7 ml Standard-DRG Wachstum media. </li><li> Nehmen Sie sich 10 ul Zellsuspension in ein 0,5 ml Mikrozentrifugenröhrchen, mischen mit 10 ul Trypanblau und dann auf die Zellzahl bestimmen eine Hämozytometer unter der optischen Mikroskopie unter Verwendung eines 10X-Objektiv verwendet wird. Verdünne die Zellsuspension zu 5000 Zellen pro ml durch DRG Wachstumsmedium hinzugefügt wird. </li><li> Entfernen Sie 0,5 ml Medium aus der DRG-Kultur in der gestreckten Kammer und mit 0,5 ml SC Suspension auf die DRG-Kultur. </li><li> Kultur die Zellen für 1 Woche bei 37 ° C und 5% CO 2. Ändern Sie die Medien alle zwei Tage nach der DRG die Hälfte des verbrauchten Mediums mit frischem Standardwachstumsmedium ersetzt. Nach 1 Woche Co-Kultur, verarbeiten die Zellen durch Immunhistochemie 10. </li></ol>

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

Zur Induktion Axon Ausrichtung auf vorgedehnte Oberfläche gibt es zwei wichtige Schritte: 1) die PDMS-Membran muss flach sein und homogener Dicke; und 2) Gliazellen müssen aus dem DRG entfernt werden. Nach dem Mischen in einem Ofen die PDMS und Vernetzer und der Härtung sollte das vernetzte PDMS Gel sorgfältig ein Verkanten zu vermeiden, auf einer flachen Tischplatte und behandelt gehalten werden. Die Sauerstoffplasmabehandlung der PDMS Membran sollte innerhalb von 6 h von PLL-Beschichtung eingehalten werden, da die...

In Nervenverletzungen sind die proximalen und distalen Nervenstümpfe oft von der direkten Neuausrichtung Nervenfaszikel verhindert aufgrund der Läsionsstelle 1-2. Normalerweise werden Axon Striche hoch geordnete und ausgerichtete Bündel von Axonen zusammen, die komplexe Netzwerke von Konnektivität bilden. Allerdings ist die Nervenregeneration ein chaotischer Prozess wegen schlecht organisiert Axon Ausrichtung 3-4. Um daher eine ausreichende Anzahl von regenerierenden Axonen zu erzeugen, das der Läsionsstelle brücken, ist es notwendig, gut organisierte axonalen Ausrichtung zu induzieren. Darüber hinaus begleitet Demyelinisierung Nervenverletzungen durch Tod der myelinisierende Zellen an der Verletzungsstelle. Da Demyelinisierung stark beeinträchtigt die leitende Kapazität Axone zu überleben, Behandlungen Targeting Demyelinisierung oder Remyelinisierung fördern sind bedeutsam für die funktionelle Erholung nach einer Nervenverletzung 5. So ist das Ziel dieses Protokolls ist es, ein Engineering-Ansatz zu verdeutlichen, die diese beiden Fragen behandeltder Nervenregeneration. Oberflächenanisotropie, die als Differenz definiert, wenn sie entlang verschiedenen Achsen gemessen, in einem Material der physikalischen oder mechanischen Eigenschaften, wurde angewendet Zellausrichtung zu beeinflussen, das Wachstum und die Migration 6-7. Neben Topographie, gibt es andere Methoden Anisotropie zu induzieren. Bisher untersuchten wir Oberflächenanisotropie durch mechanische statische Vorstreckung von Poly-Dimethyl-Siloxan (PDMS) Membran induziert. Die Theorie der &quot;kleinen super Verformung auf großen auferlegt&quot; vorausgesagt , daß die effektive Steifigkeit der Zellen in der gedehnten Richtung erfassen von der senkrechten Richtung abweicht, und diese Differenz der effektiven Steifigkeit aufgrund Anisotropie 8 an die Oberfläche. Mesenchymale Stammzellen (MSCs) , kultiviert auf einem vorgedehnten PDMS - Membran können die Anisotropie zu erfassen , indem aktiv die Oberfläche gezogen und als Ergebnis, richten in der vorgedehnten Richtung 9. In ähnlicher Weise eine anisotrope Oberflächen arising von einer mechanischen Oberflächenvorgedehnten wirkt sich auf die Ausrichtung, 10 Axone sowie das Wachstum und die Myelinisierung von dorsal root ganglion (DRG). Hier stellen wir ein Protokoll zur Induktion Oberflächenanisotropie auf einer statischen vorgedehnten PDMS Substrat 10 Axonregeneration zu verbessern. Zu axon Ausrichtung, topologische Merkmale mit gewünschten Mustern hervorzurufen, berichtet Kontaktführung durch die ausgerichteten Fasern zu schaffen und Kanäle 6,11-12 wurden nachgewiesen axon Ausrichtung 11,13 erleichtern. Jedoch zum Induzieren axon Ausrichtung durch topologische Merkmale Techniken berichtet, wie Fasern, Kanäle und Strukturieren, waren nicht imstande, zu verlängern und um die Dicke der Axone erhöhen. Im Gegensatz dazu Reckung mechanischen graduellen zu axon Ausrichtung in Dehnungsrichtung geführt mit längeren und dickeren Axone , die mit der Größe der Dehnung 14 erhöht. Jedoch ist eine angetriebene Motor Vorrichtung in vivo nicht enthältmöglich. Im Gegensatz dazu ist statisch vorgedehnten induzierte Anisotropie weniger kompliziert und leichter in Zukunft Gerüst Entwürfe für in vivo - Anwendungen integriert werden können. In diesem Protokoll wird eine statische vorgedehnten Zellkultursystem wird verwendet Oberflächenanisotropie ohne topologische Eigenschaften zu induzieren. Die vorgedehnten Kultursystem besteht aus einem PDMS-Membran zusammengesetzt ist, einem dehnbaren Rahmen und einer Streckstufe, wobei die Membran an dem Rahmen befestigt ist und eine vorbestimmte Strecke Größe auf der Streckstufe zugeführt wird. Frisch isolierte DRG auf der vorgedehnten Oberfläche kultivierten Neuronen für bis zu 21 Tage für axon Ausrichtung und Dicke überwacht. Anschließend Schwann-Zellen (SCs) co-kultiviert mit den ausgerichteten Axone werden Myelinisierung überwacht. Vorstreckung induzierte Oberflächenanisotropie Durch die Integration konnten wir Ausrichtung der Zellen-Differenzierung und Axon Ausrichtung Wachstum von MSCs und DRG - Neuronen 9-10 bzw. zu verbessern.

<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.

Die vorgedehnten Zellkultursystem DRG axon Ausrichtung gefördert 10. DRG-Neuronen wurden auf vorgestreckten und ungereckten Oberflächen für 12 Tage. Die Axone wurden für β-III-Tubulin gefärbt ihre Ausrichtung zu zeigen. 2 vergleicht axon Ausrichtung auf den vorgestreckten und ungestreckte PDMS Substrate nach 12 Tagen Kultur. Die DRG Axone parallel zur Streckrichtung ausgerichtet sind, während sie zufällige Ausrichtung zeigte und bildeten ein miteinande...

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.

Ein Ansatz zur Verbesserung der Ausrichtung und Myelinisierung von Dorsalwurzelganglion Neurons

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

Research

JoVE Journal

Neuroscience

8.4K Aufrufe.  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.

Serie: 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.

Live-Imaging von Spinalganglien Axone nach Rhizotomie

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

Forschung

Neurowissenschaften

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.

Genetic Study of Axonregeneration mit kultivierten adulten Spinalganglienneuronen

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.

Dorsalwurzelganglien Neuronen und differenzierten Fettgewebe gewonnene Stammzellen: Eine<em&gt; In-vitro-</em&gt; Co-Kultur Modell zur Regeneration peripherer Nerven Studieren

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 - Modellierung von Cancerous Neural Invasion: The Dorsalwurzelganglion Modell

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.

Dorsalwurzelganglion Injection und dorsale Wurzel Quetschverletzungen als Modell für Sensory Axonregeneration

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.

Laminotomie LWS Dorsal Root Ganglion-Zugriff und Injektion bei Schweinen

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.

Analyse von Immunzellen in einzelnen Ischias Nerven und Dorsal Root Ganglion aus einem einzigen Mausklick mit Durchflusszytometrie

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.

Dorsal Root Ganglien Isolation und Primärkultur Neurotransmitter-Freisetzung zu studieren

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.

Ermittelnden Säugetier-Axon Regeneration: In Vivo Elektroporation von Erwachsenen Maus 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 ...

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.

Verwendung von In Vivo Single-Faser-Aufnahme und intakten DorsalWurzel Ganglion mit angeschlossenem Ischiasnerv, um den Mechanismus des Leitungsversagens zu untersuchen

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