Ringraziamo il Dott. Alan Tessler per i commenti e aiuto editoriale. Questo lavoro è stato sostenuto da NIH NS062320.

1. Microscopio impostare e preparazione di imaging <ol><li> Il nostro set è composto da immagini di uno stereomicroscopio Leica MZ16 fluorescente con un otturatore veloce e una camera CCD raffreddata controllato dal software Metamorph. </li><li> Preparare una piastra elettrica termostato e regolare uscita a 32,5 ° C per mantenere la temperatura del corpo dell&#39;animale durante e dopo l&#39;intervento. </li><li> Caldo Ringer sterile la soluzione o artificiale liquido cerebrospinale (ACSF) a 32,5 ° C in anticipo per l&#39;irrigazione del midollo spinale durante l&#39;intervento. </li><li> Anestetizzare l&#39;animale con un&#39;iniezione intraperitoneale di xylazina (8 mg / kg) e ketamina (120 mg / kg) cocktail. </li><li> Radere la parte superiore della schiena con tagliaunghie piccoli animali e diffondere una piccola goccia di lozione per capelli rimozione su tutta l&#39;area rasata con tamponi di cotone con punta. Pochi minuti dopo, rimuovere la lozione applicata utilizzando il 70% spugne garza imbevuta di etanolo. </li></ol> 2. Laminectomia e l&#39;esposizione chirurgica della radice L5 dorsale <ol><li> Posto l&#39;animale in una riscaldata (32,5 ° C) pad e disinfettare la pelle con tamponi imbevuti etanolo al 70%. </li><li> Sotto illuminazione in campo chiaro sul stereomicroscopio, eseguire un&#39;incisione mediana (2 - a 3 cm) nella pelle della schiena. Se necessario, utilizzare tamponi di cotone con punta a fermare le emorragie. </li><li> Riflettono la muscolatura della colonna vertebrale per esporre le vertebre lombari sottostante. </li><li> Esporre la L3-S1 segmenti spinali da esterno destro di emi-laminectomia con Rongeurs di piccole dimensioni. Un laminectomia parziale esponendo 4-6 segmenti del midollo lombare e sacrale della colonna vertebrale è creato eliminando la parte destra dorsale delle vertebre L5 a livello della cresta iliaca dell&#39;anca (posizione di DRG L5) rostralmente alle vertebre L2 (2 vertebre caudali alla costola). Perfusione della cavità con soluzione calda di Ringer sterile. </li><li> Posizionare l&#39;animale su un cuscino di supporto (garza di cotone arrotolato) per appiattire la colonna vertebrale. Utilizzare ganci di retrazione per ampliare l&#39;area esposta. </li><li> A questo punto (circa 30 minuti dopo la prima iniezione ip di anestetico, un supplemento (0.5X) deve essere iniettato per via sottocutanea al fine di mantenere l&#39;animale completamente anestetizzato. In alternativa, l&#39;anestesia gas (2-4% isoflurano in ossigeno 0.5L/min ) può essere usato per anestesia ripetuto durante le sessioni di imaging più di 1 ora. </li></ol> 3. Rizotomia / schiacciare radici dorsali <ol><li> Passa alla eccitazione di fluorescenza per visualizzare YFP etichettato (+) assoni. </li><li> Utilizzando la punta di un Sub-Q (26ga.) ago, eseguire una piccola incisione della dura sovrastante la radice L5 dorsale (DR). Profumato ripetutamente con soluzione di Ringer e pulire delicatamente con la punta tamponi di cotone. </li><li> Identificare il sito per essere schiacciato e inserire un lato di una pinza sottile (Dumont # 5) subdurally. </li><li> Chiudere la pinza delicatamente ma con fermezza, tenendo la porzione mediale della radice L5 per 10 secondi, e poi delicatamente rilasciare il forcipe. </li><li> Lavare ripetutamente con soluzione fisiologica e pulire delicatamente con la punta tamponi di cotone. </li></ol> 4. Acquisizione di immagini e post-op procedure <ol><li> Ottenere più immagini di tutta l&#39;area esposta tra cui il sito e schiacciare il DREZ prima e subito dopo la calca sia a basso ingrandimento e alta. </li><li> Le immagini sono acquisite sia come singolo o come istantanee più flussi da 10 a 20 fotogrammi acquisiti entro 30 - 40 ms tempo di esposizione. A fuoco le immagini vengono poi selezionati, e un montaggio panoramica viene creato successivamente con Photoshop. </li><li> Per minimizzare la formazione della cicatrice, strettamente applicare un pezzo di sottile membrana matrice sintetica (Biobrane), seguita da artificiale dura, sulla parte superiore del midollo spinale esposti. Assicuratevi di tagliare pezzi per adattarsi precisamente nella finestra esposta midollo spinale in modo che siano aderenti al midollo spinale. </li><li> Chiudere la muscolatura con 5-0 punti di sutura sterile e chiudere l&#39;incisione sulla linea mediana con clip ferita. </li><li> Iniettare la soluzione di Ringer (0,3 a 0,5 ml per via sottocutanea) e amministrare Buprenorfina come analgesia postoperatoria (0,05 mg / kg) per via sottocutanea ogni 12 ore per 2 giorni. </li><li> Tenere l&#39;animale in una piastra elettrica (34 ° -35 ° C) fino recuperato. </li></ol> 5. Immagini ripetute <ol><li> Anestetizzare l&#39;animale e rimuovere le clip delle ferite e suture. </li><li> Rimuovere artificiale dura e sottile membrana sintetica patch matrice e tenerli in un tubo sterile contenente soluzione di Ringer per un successivo riutilizzo. </li><li> Rimuovere delicatamente ogni accumulato cicatrice tessuto connettivo con la punta piegata di un Sub-Q ago e pinza sottile. Profumato frequentemente con soluzione di Ringer calde. </li><li> Ri-esporre il campo operatorio, compreso il sito schiacciare e DREZ, riposizionare YFP + assoni ripreso nelle sedute precedenti, e ripetere le procedure descritte nella sezione 4. </li></ol> 6. Rappresentante dei risultati: Abbiamo osservato che, mentre essi non rigenerarsi dopo una lesione transezione, quasi tutti gli assoni + YFP cresciuto attraverso il sito dilesioni da 3 giorni dopo schiacciamento (Figura 1) 11. Tipicamente, il giorno dopo schiacciare, abbiamo osservato morire-back degenerazione degli assoni moncone prossimale e la frammentazione / degenerazione degli assoni stessi distale la calca, che ha confermato che gli assoni erano stati opportunamente danneggiati (ad esempio, figura 1, Giorno 3 e 5) . Diversi criteri aggiuntivi vengono applicati per distinguere in modo inequivocabile gli assoni rigeneranti da assoni che sono stati risparmiati o aveva recuperato dall&#39;infortunio. Tra queste vi sono: (1) assoni rigeneranti mostrano un&#39;espansione della non fluorescente porzione di YFP + assoni nel sito schiacciare a causa della degenerazione prossimale e distale (in contrasto con riduzione del divario senza etichetta a causa di citoplasma fluorescenti riempimento del sito schiacciare se assoni sopravvissuti alla lesione), (2) assoni rigeneranti sono molto più sottili, meno intensamente fluorescenti, e più ondulato di assoni sopravvissuti alla lesione; (3) neuriti rigenerante sono più sottili e più debolmente fluorescenti che i frammenti degenerando fluorescenti degli assoni attraverso il quale hanno esteso; (4) in contrasto con gli assoni sopravvissuti o risparmiata, gli assoni rigeneranti sosta al DREZ, e (5) al contrario di sopravvivere o assoni risparmiati, gli assoni rigeneranti non presentano i nodi di Ranvier. La figura 1 mostra quattro superficiale assoni YFP + subito dopo schiacciare (A1; frecce colorate). Tre giorni dopo la calca, tutti e quattro gli assoni estendere un singolo neurite che si sviluppa attraverso il sito schiacciare (A2). Cinque giorni dopo schiacciare, neuriti rimangono stabili e non c&#39;è crescita supplementare da assoni questi o altri prossimale (A3). I neuriti rigenerante che hanno attraversato il sito schiacciare allungata attraverso molto più spessa e più luminoso fluorescente frammenti di una degenerazione degli assoni (ovvero, tubi endoneurale), e arrivare al DREZ già 4 giorni dopo la calca (circa 3 mm / 2 giorni) 11. Immagine ripetuta di questi assoni e le loro punte ogni due o tre giorni per due settimane (Figura 2) ha rivelato che non sono cresciuti in avanti o ritrattare, ma è rimasto immobile. L&#39;unico cambiamento è stato notevole gonfiore delle punte ed alberi di alcuni assoni. Queste osservazioni dimostrano quindi immobilizzazione incredibilmente veloce e croniche degli assoni rigeneranti al DREZ. <img src="/files/ftp_upload/3126/3126fig1.jpg" alt="Figura 1"/> Figura 1: immagine ripetuta di L5 + DR YFP assoni nel sito di schiacciare spinali su 5 giorni. La porzione mediale della radice L5 è stato schiacciato (freccia rossa) e ripreso nei giorni 0, 3, e 5 dopo la calca. L&#39;area della cotta è amplificato nei pannelli di destra (A1-A3). <img src="/files/ftp_upload/3126/3126fig2.jpg" alt="Figura 2"/> Figura 2:. Immagini ripetute di assoni arrivati ​​al DREZ oltre 20 giorni dopo schiacciare radice L5 4 ° giorno, tre assoni (frecce colorate) arrivato al DREZ. Le punte di questi assoni rimangono nella stessa posizione e hanno un aspetto simile a sessioni successive di imaging nei giorni 7, 9, 13, 15 e 20. Posizioni di una punta dell&#39;assone rispetto alle punte degli assoni e altri punti di riferimento (asterischi) sono stati utilizzati per determinare la motilità degli assoni tra le sessioni di imaging.

<ol>
	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+the+neuromuscular+junction.">Watching the neuromuscular junction.</a> J Neurocytol. 32, 767-775 (2003).</li><li>Bishop, D. L., Misgeld, T., Walsh, M. K., Gan, W. B., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+branch+removal+at+developing+synapses+by+axosome+shedding.">Axon branch removal at developing synapses by axosome shedding.</a> Neuron. 44, 651-661 (2004).</li><li>Balice-Gordon, R. J., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+visualization+of+the+growth+of+pre-+and+postsynaptic+elements+of+neuromuscular+junctions+in+the+mouse.">in vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse.</a> J Neurosci. 10, 894-908 (1990).</li><li>Trachtenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Pan, F., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+dendritic+spine+development+in+the+mouse+cortex.">Two-photon imaging of dendritic spine development in the mouse cortex.</a> Dev Neurobiol. 68, 771-778 (2008).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">in vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Misgeld, T., Nikic, I., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+single+axons+in+the+mouse+spinal+cord.">in vivo imaging of single axons in the mouse spinal cord.</a> Nat Protoc. 2, 263-268 (2007).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+in+vivo+imaging+of+densely+populated+glia,+axons+and+blood+vessels+in+the+mouse+spinal+cord+using+two-photon+microscopy.">Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.</a> J Neurosci Methods. 169, 1-7 (2008).</li><li>Maio, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+dorsal+root+regeneration:+Rapid+immobilization+and+presynaptic+differentiation+at+the+CNS/PNS+border.">in vivo imaging of dorsal root regeneration: Rapid immobilization and presynaptic differentiation at the CNS/PNS border.</a> Journal of Neuroscience. 31, 4569-4582 (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

Imaging DR rigenerazione direttamente in topi vivi è particolarmente impegnativa perché richiede una notevole laminectomia dorsale di monitorare la crescita degli assoni su una vasta area seguito da più interventi chirurgici invasivi e anestesia nelle sessioni successive di imaging. Diverse strategie aiutato a superare queste sfide. In primo luogo, l&#39;imaging di successo richiede la riduzione della mortalità del mouse (circa il 25%), riducendo al minimo la durata dell&#39;anestesia e sanguinamento, e da una metic...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Linea H Thy1-YFP (2-4 mesi di età, entrambi i sessi) </td><td> Jackson Laboratory (Bar Harbor, ME) </td><td> 003782 </td><td></td></tr><tr><td> Xylazina (iniezione AnaSed, soluzione sterile) </td><td> Laboratori Lloyd, (Shenandoah, LA) </td><td> 4811 </td><td> 8 mg / kg </td></tr><tr><td> La ketamina (iniezione ketamina cloridrato, USP) </td><td> Hospira, Inc. (Lake Forest, Illinois) </td><td> 2051 </td><td> 120 mg / kg </td></tr><tr><td> Buprenorfina (iniettabile Buprenex) (0,05 mg / kg) </td><td> Reckitt Benckiser Pharmaceuticals Inc. (Richmond, VA) </td><td> 7571 </td><td></td></tr><tr><td> Piccoli animali Tagliacapelli </td><td> Oster Professionale, (McMinnville, TN) </td><td> 76059-030 </td><td></td></tr><tr><td> Depilazione lozione </td><td> Church &amp; Dwight Co. (Princeton, NJ) </td><td> NAIR con Baby Olio </td><td></td></tr><tr><td> Garza spugne </td><td> Fisher Scientific, (Pittsburgh, PA) </td><td> 22-362-173 </td><td></td></tr><tr><td> Tamponi di cotone con punta </td><td> Fisher Scientific, (Pittsburgh, PA) </td><td> 14-960-3Q </td><td></td></tr><tr><td> Siringhe da 1 ml </td><td> Becton, Dickson e Company Franklin Lakes, NJ) </td><td> 309602 </td><td></td></tr><tr><td> Sottocutanea (Sub-Q) aghi, 26ga. </td><td> Becton, Dickson e Società (Franklin Lakes, NJ) </td><td> 305115 </td><td></td></tr><tr><td> Forbici e pinze a molla </td><td> Strumenti Scienza fine, (Foster City, CA) </td><td></td><td></td></tr><tr><td> Rongeurs curve da 2,5 mm </td><td> Strumenti Scienza fine, (Foster City, CA) </td><td> 16221-14 </td><td></td></tr><tr><td> Iniezione Ringer lattato USP </td><td> B. Braun Medical Inc., (Irvine, CA) </td><td> BBR-L7502 </td><td></td></tr><tr><td> Soluzione salina sterile </td><td> APP Pharmaceuticals, (Schaumburg, IL) </td><td> 918610 </td><td></td></tr><tr><td> Sottile membrana matrice sintetica (Biobrane) </td><td> Bertek Pharmaceuticals, (Morgantown, WV) </td><td> 62794-096-251 </td><td></td></tr><tr><td> Dura artificiale </td><td> Gore Impedire MVP Sostituto Dura, WL Gore and Associates, (Flagstaff, AZ) </td><td> 1MVP40 </td><td></td></tr><tr><td> 5-0 punti di sutura di seta </td><td> Ethicon, Inc. (Somerville, NJ) </td><td> K-580 </td><td></td></tr><tr><td> Ferita clip </td><td> Perfetto - Ets Bruneau, (Burnea, Francia) </td><td> A75 </td><td></td></tr><tr><td> Stereomicroscopio a fluorescenza </td><td> Leica Microsystems, (Wetzlar, Germania) </td><td> MZ16 </td><td></td></tr><tr><td> CCD </td><td> Hamamatsu, (Bridgewater, NJ) </td><td> ORCA-Rx2 </td><td></td></tr><tr><td> Controllore di temperatura </td><td> Mondo Strumenti di precisione (Sarasota, FL) </td><td> ATC 1000 </td><td></td></tr><tr><td> Metamorph software </td><td> I dispositivi molecolari, (Sunnyvale, CA) </td><td></td><td></td></tr><tr><td> Photoshop </td><td> Adobe Systems, San Jose, CA </td><td></td><td></td></tr></tbody></table>

live imaging of dorsal root axons after rhizotomy

Gli assoni sensoriali primarie ferito da lesioni radice spinale non riescono a rigenerarsi nel midollo spinale, con conseguente dolore cronico e permanente perdita di sensibilità. Rigenerazione della radice dorsale (DR) assoni nel midollo spinale è impedito l&#39;ingresso in zona dorsale principale (DREZ), l&#39;interfaccia tra il SNC e SNP. La nostra comprensione degli eventi molecolari e cellulari che impediscono la rigenerazione a DREZ è incompleta, in parte perché i cambiamenti complessi associati a danno del nervo sono stati dedotti dalle analisi post-mortem. Dinamica dei processi cellulari, come la rigenerazione degli assoni, sono i migliori studiati con tecniche che catturano eventi in tempo reale con le osservazioni più di ogni animale vivente. La nostra capacità di monitorare i neuroni in serie in vivo, è aumentato drammaticamente a causa di innovazioni rivoluzionarie nel campo dell&#39;ottica e mouse transgenici. Diverse linee di Thy1-GFP topi transgenici, nei quali sottopopolazioni di neuroni sono geneticamente etichettati in distinti colori fluorescenti, consentono singoli neuroni di essere ripreso in vivo 1. Questi topi sono stati ampiamente utilizzati per imaging in vivo dei muscoli e del cervello 2-4 5-7, e hanno fornito indicazioni romanzo in meccanismi fisiologici che le analisi statiche non poteva essere risolto. Studi di imaging di neuroni nel midollo spinale di vita hanno cominciato solo di recente. Lichtman ei suoi colleghi hanno dimostrato la loro fattibilità per il monitoraggio feriti colonna dorsale (DC) assoni con grande campo microscopia 8,9. Multi-fotone in imaging in vivo di profondamente posizionati assoni DC, microglia e dei vasi sanguigni è stato anche compiuto 10. Nel corso degli ultimi anni, siamo stati i primi ad applicare imaging in vivo di monitorare la rigenerazione degli assoni DR utilizzando la microscopia a largo campo e la linea H del Thy1-YFP topi. Questi studi ci hanno portato ad una nuova ipotesi sul perché gli assoni DR viene impedito di rigenerazione nel midollo spinale 11. In linea H del Thy1-YFP topi, distinto YFP assoni + sono posizionati superficialmente, che consente assoni diversi da monitorare contemporaneamente. Abbiamo imparato che gli assoni DR arrivare a DREZ sono meglio ripreso in lombare che nel midollo spinale cervicale. Nella presente relazione si descrivono diverse strategie che abbiamo trovato utile per assicurare il successo di imaging a lungo termine e ripetuti di rigenerare gli assoni DR. Tra questi metodi che eliminano l&#39;intubazione ripetute e interruzione delle vie respiratorie, ridurre al minimo la chirurgia associata a stress e la formazione di cicatrici, e di acquisire immagini stabili ad alta risoluzione senza fototossicità.

Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Live Imaging of Dorsal Root Axons after Rhizotomy

Un<em&gt; In vivo</em&gt; Immagini di protocollo per monitor principale assoni sensoriali seguenti schiacciare spinali è descritto. Le procedure di utilizzo ad ampio campo di microscopia a fluorescenza e topi transgenici Thy1-YFP, e permettere l&#39;imaging ripetuta di rigenerazione degli assoni più di 4 cm nel SNP e le interazioni degli assoni con l&#39;interfaccia del sistema nervoso centrale.

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. ...

live-imaging-of-dorsal-root-axons-after-rhizotomy

Research

JoVE Journal

Neuroscience

13.8K Views.  Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology. Un In vivo Immagini di protocollo per monitor principale assoni sensoriali seguenti schiacciare spinali è descritto. Le procedure di utilizzo ad ampio campo di microscopia a fluorescenza e topi transgenici Thy1-YFP, e permettere l'imaging ripetuta di rigenerazione degli assoni più di 4 cm nel SNP e le interazioni degli assoni con l'interfaccia del sistema nervoso centrale.

Video: Live Imaging of Dorsal Root Axons after Rhizotomy

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.

Immunoprecipitazione della cromatina dal tessuto dei gangli dorsali seguenti danno assonale

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 ...

Ricerca

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 ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Immagini dal vivo di nicotina indotta segnalazione Calcio e neurotrasmettitori uscita Lungo ventrali Hippocampal assoni

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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 ...

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 ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Microscopia spettrale riflettometrica su assoni Myelinated In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Isolamento rapido dei macrofagi di Ganglion dorsale

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

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

Mielinizzazione in vitro di assoni periferici in una cocoltura di espianti di ganglio di radice dorsale di ratto e cellule di Schwann

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...