Die Studie wurde mit Unterstützung durch die Schulze-Familienstiftung (zu A.S.B.) durchgeführt.

<ol>
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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=Comparison+of+AAV+serotypes+for+gene+delivery+to+dorsal+root+ganglion+neurons.">Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.</a> Mol Ther. 18 (4), 715-724 (2010).</li><li>Jelicic Kadic, A., Boric, M., Kostic, S., Sapunar, D., Puljak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intraganglionic+injection+of+calcium/calmodulin-dependent+protein+kinase+II+inhibitors+on+pain-related+behavior+in+diabetic+neuropathy.">The effects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy.</a> Neurosci. 256, 302-308 (2014).</li><li>Greenberg, M. 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A model of acute, graded compression of the porcine cauda equina and an analysis of neural and vascular anatomy.</a> Spine. 16 (1), 61-69 (1991).</li><li>Bobo, R. 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=Convection-enhanced+delivery+of+macromolecules+in+the+brain.">Convection-enhanced delivery of macromolecules in the brain.</a> Proc Natl Acad Sci U.S.A. 91 (6), 2076-2080 (1994).</li><li>Lonser, R. R., Sarntinoranont, M., Morrison, P. F., Oldfield, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+to+the+central+nervous+system.">Convection-enhanced delivery to the central nervous system.</a> J Neurosurg. 122 (3), 697-706 (2015).</li><li>Shen, J., Wang, H. Y., Chen, J. Y., Liang, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+analysis+of+normal+human+lumbar+dorsal+root+ganglion+by+3D+MR+imaging.">Morphologic analysis of normal human lumbar dorsal root ganglion by 3D MR imaging.</a> AJNR Am J Neuroradiol. 27 (0195–6108 (Print)), 2098-2103 (2006).</li><li>Pleticha, 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=Minimally+invasive+convection-enhanced+delivery+of+biologics+into+dorsal+root+ganglia:+validation+in+the+pig+model+and+prospective+modeling+in+humans.">Minimally invasive convection-enhanced delivery of biologics into dorsal root ganglia: validation in the pig model and prospective modeling in humans.</a> J Neurosurg. 121 (4), 851-858 (2014).</li></ol>

Keine; die Autoren haben keine Interessenkonflikte im Zusammenhang mit dieser Studie.

Wollten wir beschreiben eine Methode für die chirurgische Exposition der DRG über Laminotomie und intraganglionic Injektion in eine gesunde große Tierarten, insbesondere Schweine. Bei Nagetieren eine ähnliche Methode wurde von detailliertes 12 und verwendet, um konventionelle pharmakologische Agenten 8,10 und Virenvektoren 6,7,9,...

Dorsal Root Ganglien (DRG) sind anatomisch diskrete, neuronale Sammlungen befindet sich entlang der Wirbelsäule. Jede DRG enthält die primären sensorischen Neuronen, die kodieren und Relais periphere Stimuli auf das zentrale Nervensystem (ZNS) von bestimmten Körperregionen. Zum Beispiel beginnt der Schmerz von Osteoarthritis Schmerz-Rezeptoren liegt etwa eine gemeinsame Schmerzreize wahrnehmen. Dieser Vorgang wird Nozizeption bezeichnet. Langfristigen Sensibilisierung für Schmerzreize führt zu chronischen Schmerzen 1.
Chronische Schmerzen sind ein häufiges Thema der präklinischen Studie 2 , wo ein Ziel ist es, nützliche Methoden für die gezielte Gabe von Schmerzmitteln, DRG, z. B. intraganglionic Injektion 3zu entwickeln. Allerdings DRG sind schwer zugänglich, da sie innerhalb der boney Grenzen des intervertebralen Foramina 4befinden. Mehrere Gruppen haben dieses Hindernis durch den Einsatz von Wirbelsäulen-Chirurgie in Nagetieren 5,6,7,8,9,10erfolgreich überwinden.
In der Klinik Laminektomie ist eine gemeinsame Operation der Wirbelsäule und bezieht sich auf chirurgische Entfernung der Wirbelkörper Lamina, dadurch den Wirbelkanal 11Konvektion. Einbeziehung der Operationstechniken, DRG-Direktzugang zu leisten war erfolgreich in Nagetieren 5,12, Übersetzung kann jedoch unrealistisch angesichts Unterschiede in der Größe von relevanten Strukturen und Einflüsse, die Pharmakokinetik oder technische Machbarkeit 13,14. Beispielsweise ermittelt eine Studie den transversalen Rückenmark Durchmesser an T10 3.0, 7.0 und 8,2 mm für Ratte, Schwein und Mensch, bzw. 15sein. So Modelle großes Tier besser ungefähre menschlichen Dimensionen des nervösen Strukturen.
Bei Schweinen verwendet Raore Et Al. mehrstufige Laminektomie Zugang zu der zervikalen Rückenmark für mehrere intraspinal Injektionen 16. Das Verfahren wurde gut vertragen und führte zu einer Phase ich klinische Studie wo vergleichbare chirurgische Ergebnisse dokumentiert 17waren. Diese Ergebnisse ermutigen Weiternutzung von präklinischen große Tiermodellen als Prädiktoren der technischen Machbarkeit und Sicherheit beim Menschen.
Bislang existiert keine detaillierte Methodik für chirurgische Zugang und Injektion der DRG in einem großen Tierarten. Um dieser translationale Kluft zu verringern, berichten wir über ein Protokoll für die DRG-Belichtung und Injektion über Laminotomie bei Schweinen. Standard-neurochirurgische Techniken, Instrumente und Materialien wurden verwendet und die Methode wurde entwickelt, um die modernen chirurgischen Praxis zu imitieren. Wir zeigen intraganglionic Injektion mit einer wässrigen Lösung für Lendenwirbelsäule DRG und bestätigen Sie die erfolgreiche Lieferung per Histologie nach postoperativen Tag 21.

<table border="0" cellpadding="0" cellspacing="0" width="1311">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Large humane animal sling</td>
			<td style="width:167px;">Britz &#38; Company</td>
			<td style="width:119px;">002539</td>
			<td style="width:484px;">Modified to include abdominal aperture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Adhesive patient return electrode - 9 inch</td>
			<td style="width:167px;">Medtronic</td>
			<td style="width:119px;">E7506</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Ranger blood &#38; fluid warming system</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">24500</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Lactated Ringer&#39;s fluid</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:119px;">0409-7953-09</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Force air warming device</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">77500</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:543px;">Duraprep solution with applicator, 26 ml (0.7% iodine povacrylex, 74% isopropyl alcohol</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">8630</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Sterile disposable surgical towels</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDT2168286</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Ioban 2 incise drape</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">6651EZSB</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Disposable suction canister and tubing</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">DYND44703H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Button switch electrosurgical monopolar pencil</td>
			<td style="width:167px;">Medtronic</td>
			<td style="width:119px;">E2450H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fine smooth straight bipolar electrosurgical forceps, 4 1/2 inch</td>
			<td style="width:167px;">Bovie</td>
			<td style="width:119px;">A826</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">#15 blade</td>
			<td style="width:167px;">Miltex</td>
			<td style="width:119px;">4-315</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">#11 blade</td>
			<td style="width:167px;">Miltex</td>
			<td style="width:119px;">4-311</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">4x4 surgical gauze</td>
			<td style="width:167px;">Dynarex</td>
			<td style="width:119px;">3262</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Weitlaner self-retaining retractor, 8 inch</td>
			<td style="width:167px;">Miltex</td>
			<td style="width:119px;">11-618</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Meyerding self-retaining retractor, 1x2 3/8 inch</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">42-2078</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Gelpi self-retaining retractor, 7 inch</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">60-6570</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Freer elevator, 5 mm</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS4641518F</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Bone wax</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">W31G</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Spurling intervertebral disc rongeur, 3 mm</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">42-2852</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Spurling 45-degree, up-biting Kerrison rongeur, 2 mm</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS4052802</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Leksell angled rongeur, 2 mm</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">40-4097</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Gelfoam, size 50</td>
			<td style="width:167px;">Pfizer</td>
			<td style="width:119px;">AZL32301</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Cottonoid patty</td>
			<td style="width:167px;">Medtronic</td>
			<td style="width:119px;">8004007</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Frazier suction tip, 6 Fr</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">50-2006</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Frazier suction tip, 10 Fr</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">50-2010</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Dandy blunt right angle nerve hook</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS4005220</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nylon suture, 6-0</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">697G</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Castroviejo smooth micro needle holder</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDG2428614</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">22 gauge Quinke point spinal needle</td>
			<td style="width:167px;">Halyard Health</td>
			<td style="width:119px;">18397</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:543px;">32 gauge CED needle with locking Luer hub</td>
			<td style="width:167px;">See comments</td>
			<td style="width:119px;">n/a</td>
			<td style="width:484px;">As in: Pleticha, J., Maus, T.P., Christner, J.A., Marsh, M.P., Lee, K.H., Hooten, W.M., Beutler, A.S. Minimally invasive convection-enhanced delivery of biologics into dorsal root ganglia: validation in the pig model and prospective modeling in humans. Technical note. J Neurosurg. 121(4), 851-8 (2014).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Polyethylene tubing, 5 feet</td>
			<td style="width:167px;">Scientific Commodities</td>
			<td style="width:119px;">BB31695-PE/05</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Monoject syringe, 3 ml</td>
			<td style="width:167px;">Kendall</td>
			<td style="width:119px;">SY15352</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">NanoJet syringe pump</td>
			<td style="width:167px;">Chemyx</td>
			<td style="width:119px;">10050</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">DAPI</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fast Green FCF</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7252</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Bulb irrigation syringe</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">DYND20125</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fine-toothed Adson forceps</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS1000212</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Vicryl suture, 0</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">J603H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Vicryl suture, 2-0</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">J317H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Needle counter</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">NC20FBRGS</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Steri-strip skin closure, 1/2x4 inch</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">R1547</td>
			<td style="width:484px;">-</td>
		</tr>
	</tbody>
</table>

laminotomy for lumbar dorsal root ganglion access and injection in swine

Dorsal Root Ganglien (DRG) sind anatomisch gut definierte Strukturen, alle primäre sensorische Neuronen unter dem Kopf enthalten. Diese Tatsache macht DRG attraktive Ziele für die Injektion von neuer Therapeutika zur Behandlung von chronischen Schmerzen. In kleiner Tiermodelle wurde Laminektomie zur DRG-Injektion zu erleichtern, weil es chirurgische Entfernung der vertebralen Knochen rund um jede DRG beinhaltet. Wir zeigen eine Technik für die intraganglionic Injektion von lumbalen DRG in eine große Tierarten, nämlich Schweine. Laminotomie wird durchgeführt, um direkten Zugriff auf DRG mit standard neurochirurgische Techniken, Instrumente und Materialien. Verglichen mit umfangreicher Knochenentfernung über Laminektomie, implementieren wir Laminotomie um Wirbelsäule Anatomie zu sparen, während ausreichenden DRG Zugang zu erreichen. Intraoperative Fortschritt der DRG-Injektion wird unter Verwendung eines ungiftigen Farbstoffs überwacht. Nach Euthanasie am postoperativen Tag 21 bestimmt der Erfolg der Injektion Histologie für den intraganglionic Vertrieb von 4', 6-Diamidino-2-Phenylindole (DAPI). Wir injizieren eine biologisch inaktive Lösung um das Protokoll zu demonstrieren. Diese Methode könnte angewandte in Zukunft präklinischen Studien zum Ziel therapeutischen Lösungen für DRG. Unsere Methodik sollte erleichtern testen die Übersetzbarkeit von intraganglionic kleine Tier Paradigmen in einem großen Tierarten. Darüber hinaus kann dieses Protokoll als eine wichtige Ressource für die Planung von präklinischen Studien der DRG-Injektion bei Schweinen dienen.

Histologische Beurteilung der Injectate Ausbreitung Erfolgreiche Durchführung der Injectate, DRG richtet sich nach histologischen Beurteilung des DAPI verbreiten. Bei dieser Technik wird die Nadelspitze in der dreidimensionalen Mitte der DRG Positionierung. Daher wird erfolgreiche Zustellung bestimmt durch das Ausmaß der DAPI-Färbung von histologischen Abschnitten beide in der Nähe (zentrale DRG Abschnitte) und fernen (periphere DRG Abschnitte) an der Spitze der Nadel auswerten. <strong class="xfi...

Watch this Scientific Journal Video about Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine at JoVE.com

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Wir beschreiben eine Methode zur Laminotomie bei Schweinen, die Zugang zu LWS Dorsal Root Ganglien (DRG) für die intraganglionic Injektion. Intraoperativ und histologisch bestätigt bis zu 21 Tage nach der Operation Injektion Fortschritte überwacht. Dieses Protokoll kann für zukünftige präklinischen Studien mit DRG-Injektion verwendet werden.

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 ...

laminotomy-for-lumbar-dorsal-root-ganglion-access-injection

Research

JoVE Journal

Neuroscience

12.6K Aufrufe.  Mayo Clinic, Translational Science Track, Mayo Graduate School. Wir beschreiben eine Methode zur Laminotomie bei Schweinen, die Zugang zu LWS Dorsal Root Ganglien (DRG) für die intraganglionic Injektion. Intraoperativ und histologisch bestätigt bis zu 21 Tage nach der Operation Injektion Fortschritte überwacht. Dieses Protokoll kann für zukünftige präklinischen Studien mit DRG-Injektion verwendet werden.

Serie: Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Chromatin Immunopräzipitation aus Spinalganglien Tissue folgende axonale Schädigung

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Forschung

Neurowissenschaften

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. ...

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 ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Mess Spinal Präsynaptische Hemmung bei Mäusen durch Spinalgan Mögliche Aufnahme<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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 ...

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.

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 ...

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 ...

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 ...