Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

Quattro a sei settimane di età femminile C57BL / topi CJ (Harlan, Gerusalemme, Israele) sono stati utilizzati nell&#39;esperimento secondo l&#39;Associazione per la valutazione e l&#39;accreditamento delle specifiche Laboratory Animal Care. Tutte le procedure sperimentali sono state fatte secondo Istituzionale cura degli animali e del Comitato uso e il Dipartimento di Agricoltura regolamenti. 1. La raccolta del midollo spinale <ol><li> Euthanize il mouse utilizzando una camera di CO 2. Evitare dislocazione cervicale in quanto potrebbe causare danni alle radici gangliari a causa di forze di taglio. Da qui in poi, eseguire tutte le operazioni in condizioni sterili. </li><li> Bagnare animale verso il basso con il 70% di etanolo. Questo passaggio è importante per la sterilizzazione e per prevenire i capelli da trascinare attraverso gli organi interni. </li><li> Lasciare il mouse per asciugare da etanolo. Evitare qualsiasi contatto di etanolo con gli organi interni a causa di neurotossicità. </li><li> Dopo essiccamento del mouse, posizionate utilizzando perni in posizione prona(faccia in giù). Posizionare i perni su ogni degli arti posteriori e arti anteriori. </li><li> Utilizzando pinze, fare una incisione mediana che si estende cranio-caudale dal collo posteriore alla colonna lombare. Successivamente, sollevare lembi dermici bilateralmente con pinze ed esporre il tessuto sottocutaneo. </li><li> Palpare cranio del mouse fino al bivio craniocervicale. Utilizzando le forbici perpendicolari per l&#39;animale, tagliare i muscoli cervicali e della colonna vertebrale a livello della giunzione cranio-cervicale (il 7 ° vertebre cervicali), con pinze pesanti. Sezionare la colonna vertebrale caudale e accise della colonna vertebrale con una pinza pesanti fino a raggiungere il livello lombare (posteriori livello degli arti). </li><li> Eseguire un transezione caudale completa a livello della colonna lombare (la 5 ° vertebra lombare) con pinze pesanti. </li><li> Arrotolare il mouse sopra (faccia in su). Non vi è alcuna necessità di coprire l&#39;incisione dal DRG viene estratto dai livelli vertebrali inferiori e non cervicale. </li><li> Tagliare sulla linea mediana dal collo all&#39;addome, ritrarre ilpelle lateralmente e poi tagliato per aprire il peritoneo. </li><li> Rimuovere gli organi interni all&#39;interno del peritoneo (fegato, milza, pancreas, stomaco e intestino) in blocco. Quindi, rimuovere gli organi retroperitoneali (vale a dire, i reni e pancreas). Cranialmente, aprire la parete toracica. </li><li> Utilizzando pinze e una lama chirurgica, tagliare le nervature, lasciando 5 mm di costole bilateralmente distanza dalla colonna vertebrale. A questo punto, la colonna vertebrale è separata dal resto del corpo. </li><li> Lavare la colonna vertebrale, dal sangue freddo (4 ° C) fosfato fresco salina tamponata (PBS) due volte. </li></ol> 2. Isolare la gangli dorsali (DRG) <ol><li> Utilizzare uno stereomicroscopio con 4X ingrandimento. </li><li> Posizionare la colonna vertebrale su una piattaforma assorbente non non aderente (Telfa / nylon). Posizionare la faccia colonna vertebrale fino allo stesso orientamento cranio-caudale. </li><li> Rimuovere tutti i muscoli della colonna vertebrale e del tessuto connettivo. Utilizzare le costole come punto di riferimento per i nervi, come hannolasciare la colonna vertebrale. Costole anche proteggono i nervi dagli strumenti chirurgici. Il DRG si trova alle cervicali, toraciche e lombari siti delle vertebre. </li><li> Successivamente, utilizzando forbici tagliare il corpo vertebrale sulla linea mediana ed espongono il midollo spinale e le radici DRG delicatamente retrazione laterale del corpo vertebrale. </li><li> Nello spazio intercostale, seguire il nervo periferico medialmente lungo il laterale costola al DRG. </li><li> Identificare il DRG. Sembra che il tuorlo d&#39; &#39;uovo fritto&#39; sdraiato sul nervo. </li><li> Tagliare il distale nervi intercostali al DRG lasciando 2-3 mm di distale nervo gangli, da utilizzare per la retrazione. Questa &quot;coda&quot; sarà rimosso dopo la raccolta. </li><li> Afferrare il nervo con pinze. Stringendo il corpo DRG stesso causerà danni alle cellule neurali e deve essere evitato. </li><li> Avvicinatevi al DRG prossimalmente lungo il suo attaccamento al midollo spinale, attraverso il suo anteriore e radici posteriori. </li><li> Applicare delicata retrazione al DRG tirando la efferenti nervo (nervo intercostale moncone) lateralmente, e tagliare l&#39;anteriore e radici posteriori vicino al DRG. </li><li> Mantenere il DRG in una Petri piastra da 35 mm riempita fresco, ghiaccio DMEM freddo supplementato con 10% di siero fetale di vitello (FCS), 1% di penicillina-streptomicina, aminoacidi non essenziali 1% e 1% di sodio piruvato. </li></ol> 3. L&#39;impianto nella capsula di Petri <ol><li> Eseguire i prossimi passi sul ghiaccio con punte pre-raffreddato pipette per evitare ECM solidificazione. </li><li> Sotto uno stereomicroscopio a 2-4x ingrandimento, mettere da 35 mm di vetro di Petri fondo piatto su una griglia di carta. IMPORTANTE: Lavorare sul ghiaccio al fine di mantenere l&#39;ECM allo stato liquido. Creare un punto diametro di 1 mm (circa 20 ml) di fattore di crescita impoverito ECM al centro della griglia. </li><li> Sotto visualizzazione diretta, posizionare il DRG al centro dello spot ECM vicino al fondo del piatto. Nota: Le cellule tumorali utilizzati in questo protocollo sono murini cellule tumorali pancreatiche (KPC) ha istituito ilhed da campioni tumorali appena isolate da topi KPC come descritto altrove 19. </li><li> Celle di raccolta 40.000 tumorali provenienti da culture confluenti, lavarli una volta con PBS, e li risospendere in 40 ml ECM sul ghiaccio. </li><li> Sotto la visualizzazione diretta della griglia utilizzando un micron misura stereomicroscopio 500 in ogni direzione dalla DRG. A questo punto seme lentamente 10.000 cellule / ml 10 ECM. Utilizzare una punta preraffreddato 2-10 ml per iniettare le cellule vicino al fondo piatto, evitando la loro diffusione nella matrice o distacco della matrice. (Figura 1). </li><li> Lasciare il piatto all&#39;interno di una cappa a flusso laminare per 10-15 min a solidificare. Evitare ECM essiccazione. </li><li> Aggiungere lentamente DMEM, preparata come precedentemente accennato, contro la parete laterale della piastra. Aggiungere abbastanza medio per coprire l&#39;ECM (circa 2 ml). </li><li> Mettere il piatto di nuovo per l&#39;incubatore a 37 ° C. </li><li> Sostituire il mezzo con terreno fresco il giorno seguente. </li><li> Sostituire la media ogni 2 giorni. &lt;/ Li&gt; </ol> 4. Acquisizione dati, Time-lapse di videomicroscopia <ol><li> Il giorno 7 dopo l&#39;impianto, prendere il piatto per la microscopia lasso di tempo. Si noti che alcune cellule potrebbero richiedere l&#39;acquisizione in precedenza a causa della migrazione più veloce. </li><li> Durante l&#39;imaging dal vivo, la capsula in un ambiente chiuso ad una temperatura media di 37 ± 0,1 ° C e 5% CO 2. </li><li> Registrare le celle utilizzando un accoppiamento di carica fotocamera dispositivo posto sul microscopio. </li><li> Prendere immagini digitalizzate ogni 10 minuti per un massimo di 72 ore a seguire locomozione cella da tre a sei celle diverse. </li><li> Sostituire media dopo 24 ore. </li><li> Eseguire acquisizione e analisi dei dati semplice utilizzando il software microscopio. </li></ol> Analisi 5. I dati <ol><li> Per applicazioni più avanzate (ad esempio, il monitoraggio delle cellule in 2D o 3D) utilizzare software specializzati (come Imaris 4D). Nota: Questo software determina le coordinate XY di una cella aqualsiasi dato tempo, insieme con la distanza dall&#39;origine, e la velocità di movimento. </li><li> Calcolare la velocità media misurando il percorso fra posizioni successive in intervalli di 10 min. Calcolare la migrazione Index Forward (FMI), che è la distanza della parte anteriore cella dal neuriti e descrive il movimento unidirezionale delle celle verso il DRG. </li></ol>

<ol>
	<li>Carter, R. L., Foster, C. S., Dinsdale, E. A., Pittam, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+spread+by+squamous+carcinomas+of+the+head+and+neck,+a+morphological+study+using+antiaxonal+and+antimyelin+monoclonal+antibodies.">Perineural spread by squamous carcinomas of the head and neck, a morphological study using antiaxonal and antimyelin monoclonal antibodies.</a> J Clin Pathol. 36, 269-275 (1983).</li><li>Beard, C. 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=Perineural+invasion+is+associated+with+increased+relapse+after+external+beam+radiotherapy+for+men+with+low-risk+prostate+cancer+and+may+be+a+marker+for+occult,+high-grade+cancer.">Perineural invasion is associated with increased relapse after external beam radiotherapy for men with low-risk prostate cancer and may be a marker for occult, high-grade cancer.</a> Int J Radiat Oncol Biol Phys. 58, 19-24 (2004).</li><li>Maru, N., Ohori, M., Kattan, M. W., Scardino, P. T., Wheeler, T. 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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotrophic+factor+artemin+promotes+pancreatic+cancer+invasion.">The neurotrophic factor artemin promotes pancreatic cancer invasion.</a> Ann Surg. 244, 274-281 (2006).</li><li>Takahashi, T., 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=Perineural+invasion+by+ductal+adenocarcinoma+of+the+pancreas.">Perineural invasion by ductal adenocarcinoma of the pancreas.</a> J Surg Oncol. 65, 164-170 (1997).</li><li>Zhu, Z., 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=Nerve+growth+factor+expression+correlates+with+perineural+invasion+and+pain+in+human+pancreatic+cancer.">Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer.</a> J Clin Oncol. 17, 2419-2428 (1999).</li><li>Hirai, I., 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=Perineural+invasion+in+pancreatic+cancer.">Perineural invasion in pancreatic cancer.</a> Pancreas. 24, 15-25 (2005).</li><li>Mitchem, J. 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This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

Questo articolo presenta un modello in vitro che ricapitola il microambiente tumorale nella nicchia neurale, il modello DRG. Il video mostra tutte le fasi a partire da riconoscere punti di riferimento anatomici come il DRG nel topo, la sua estrazione, e, infine, la sua coltura in ECM. è presentato anche co-coltura DRG a fianco con le cellule tumorali. Non ci sono altri modelli in vitro per la ricerca invasione perineurale descritti in letteratura rendendo questo modello indispensabile per studiare la nicchia microambiente perineurale in vitro. Il protocollo presentato nel video ha due fasi critiche. In primo luogo, si deve prestare attenzione quando si afferra il nervo adiacente al corpo DRG (passo 2,7 nel protocollo). Cogliere o pizzicare il DRG potrebbe causare irreversibili danni meccanici o ischemico al DRG impedendole di crescere e germogliare quando seminato nella scatola di Petri. Il secondo passo fondamentale è l&#39;impiantodelle cellule tumorali nella capsula di Petri all&#39;interno della ECM. E &#39;importante per seminare le cellule lentamente e con cautela, per evitare la fluttuazione delle cellule e la loro diffusione sul piatto Petri. Lo scopo del fissaggio è di localizzare le cellule in un unico luogo, per facilitare il loro movimento di inseguimento (distanza e direzione). Una volta stabilito il modello microambiente può essere modificato secondo l&#39;ipotesi testato. Per esempio, aggiungendo una terza coltura cellulare per la piastra di Petri (per esempio, le cellule non cancerose) consente al ricercatore di confrontare le capacità di migrazione neurotropic di cellule differenti. Inoltre, il ricercatore può applicare condizioni diverse (ad esempio, temperatura, umidità, fattori solubili, ecc) ed esaminare il loro effetto sulla capacità delle cellule invasione. Il modello DRG consente al ricercatore di studiare l&#39;interazione tra le cellule tumorali e nervi. E &#39;anche usato per esperimenti nel lasso di tempo che morfologicacambiamenti nella nicchia perineurale sono dimostrate in modo temporale. Inoltre, le cellule neuroinvasive possono essere sottoposti a diversi trattamenti e il loro effetto sulla interazione con neuriti dei gangli possono essere valutate. Non ogni linea cellulare è adatto per il modello DRG. Essi dovrebbero essere le cellule tumorali con la capacità intrinseca di invadere attraverso nervi e di degradare ECM. Inoltre, notare che ogni tipo di cellula tumorale ha diverse caratteristiche di invasione e quindi il tempo ci si aspetta le cellule a entrare in contatto con i neuriti DRG varia. Ad esempio, le cellule KPC abbiamo usato necessità circa 7 giorni di invadere attraverso la ECM verso il DRG mentre le cellule MiaPaca (cellule di adenocarcinoma pancreatico umano) solo bisogno di 72-96 ore per fare quel contatto. Una limitazione di questo modello include la possibile incompatibilità con cellule umane. Grazie all&#39;uso delle DRG mouse, l&#39;interazione proteina-proteina topo-umano non può sempre essere dimostrata quando hule cellule tumorali uomo sono utilizzati. Ogni volta che la pianificazione di utilizzare questo modello con le due specie diverse, l&#39;omologia della proteina in esame dovrebbe essere testato e se c&#39;è alta omologia si suppone che il test per essere adatto per valutare le interazioni proteina-proteina. Così, si dovrebbe tener presente che il saggio DRG come proposto in questo protocollo rappresenta il microambiente neurale comprese altre cellule, non solo i neuroni (cioè, cellule di Schwann, fibroblasti, macrofagi, ecc). Quindi, specifiche popolazioni cellulari non possono essere testati specificamente.

tumori solidi diffondere in tre modi principali: invasione diretta, diffusione linfatica e diffusione hematogenic. Tuttavia, c&#39;è un quarto mezzo di diffusione del cancro che viene spesso ignorata, diffusione lungo i nervi. Cancerose invasione neurale (CNI) è un percorso ben noto di diffusione del cancro, in particolare nei tumori della testa e del collo, della prostata 1 2, 3 e del pancreas. 4-8 CNI si verifica in più del 80% degli individui con adenocarcinoma del pancreas, portando per tumore retroperitoneale diffondersi attraverso i nervi celiaca gangliari. Queste cellule tumorali neurotropic hanno una capacità unica di migrare unidirezionalmente lungo i nervi verso il sistema nervoso centrale (CNS). 9 Questa scoperta suggerisce che il microambiente perineurale può essere sfruttato da cellule tumorali, fornendo fattori che favoriscono la crescita maligna. Uno dei pochi modelli in vitro per la ricerca CNI è gangli delle radici dorsali (DRG) / modello di cellula tumorale. Questo modEL è spesso usato per studiare l&#39;interazione paracrino tra cellule tumorali stroma e neurali. 10-18 In questo modello, mouse o linee cellulari tumorali umane sono cresciuti in matrice extracellulare (ECM) adiacente a preparazioni di DRG colta appena dissociato. In questo articolo il video mostra l&#39;applicazione in vitro CNI in adenocarcinoma del dotto pancreatico.

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:417px;">Equipments:</td>
			<td style="width:121px;"></td>
			<td style="width:108px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Operating microscope</td>
			<td>Leica</td>
			<td>M205</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tiime Lapse System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blade</td>
			<td>Sigma-Aldrich</td>
			<td>Z309036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35mm petri dishes, glass bottom</td>
			<td>de groot</td>
			<td>60-627860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol</td>
			<td>sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cold PBS</td>
			<td>Biological industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Biological industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Rhenium</td>
			<td align="right">10108165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and streptomycin</td>
			<td>Biological industries</td>
			<td>01-031-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>Biological industries</td>
			<td>03-042-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Biological industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth factor depleted matrigel</td>
			<td>Trevigen</td>
			<td>3433-005-01</td>
			<td></td>
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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.

<html> Utilizzando immagini di microscopia video, il DRG può essere visto germogliare neuriti 5-7 giorni dopo l&#39;impianto, mentre le cellule tumorali migrare lontano dalle loro colonie verso il DRG. Con il 7 ° giorno dopo l&#39;impianto, le cellule tumorali vengono a contatto con neuriti (Figura 2). L&#39;indice di migrazione Forward delle cellule tumorali pancreatiche utilizzati nel protocollo è di 3-4 volte superiore a quella di altre linee cellulari (QLL2, B16F) (Figura 3a). Figura 3b presenta una X rappresentativo e coordinata Y grafico raffigurante la migrazione di una cellula tumorale KPC in contatto con il nervo; time-lapse analisi videomicroscopia ha mostrato differenze nel movimento in avanti, ma non la velocità tra le cellule KPC e le cellule non invadere (Figura 3c-d). figura 1 &quot;src =&quot; / files / ftp_upload / 52990 / 52990fig1.jpg &quot;/&gt; Figura 1:. Schematica illustrazione del protocollo Passi <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/52990/52990fig2.jpg" /> Figura 2:. Cancer Cell Invasion Lungo i neuroni del DRG (a) DRG (in alto) e le cellule tumorali (in basso) il giorno 0 dopo la semina (ingrandimento 5X). (B) DRG e il cancro delle cellule il giorno 7 dopo la semina (ingrandimento 5X). Cellule (c), il cancro migrano lungo la DRG neurone (frecce). Tutte le barre rappresentano 50 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Figura 3:. Dorsale della radice gangliari (DRG) Neuroni Indurre CNI (a) indice di Nerve l&#39;invasione delle cellule tumorali, le cellule MiaPaCa2 KPC, QLL2, e le cellule NIH3T3. (B) Una coordinata grafico che rappresenta il percorso di migrazione di una cellula tumorale KPC in contatto con il nervo (rosso) e cellule QLL2 (viola). Le coordinate X e Y sono mostrate. (N = 12-20 in ciascun gruppo). (C) Analisi della distanza dall&#39;origine di migrazione di cellule tumorali QLL2 con contatto assonale e cellule (d) cancro KPC (n = 20). La direzione della migrazione era costantemente verso il ganglio nervoso. I valori P in (a) sono stati calcolati con il test t di Student a due code. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig3large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

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 ...

in-vitro-modeling-cancerous-neural-invasion-dorsal-root-ganglion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

9.0K Views.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

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

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Modellistica neurale segnalazione immunitario di emicrania episodica e cronica utilizzo Depressione Diffusione In Vitro</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

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Neuroscienze

Live Imaging of Dorsal Root Axons after Rhizotomy

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

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

Imaging in diretta degli assoni spinali dopo rizotomia

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

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 ...

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.

Misurare spinale Presinaptiche inibizione nei topi radice dorsale Recording potenziale<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 ...

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

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

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

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

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

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

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.

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

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

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Estrusione idraulica del midollo spinale e isolamento dei gangli dorsali nei roditori

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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

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

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

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

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

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

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

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

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

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

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

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

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

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

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

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

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Modellistica neurale segnalazione immunitario di emicrania episodica e cronica utilizzo Depressione Diffusione In Vitro

Imaging in diretta degli assoni spinali dopo rizotomia

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

Misurare spinale Presinaptiche inibizione nei topi radice dorsale Recording potenziale

Gangli neuroni e cellule staminali derivate da tessuto adiposo differenziata: An

Un approccio per migliorare l'allineamento e la mielinizzazione dorsale Root Ganglion neuroni

Estrusione idraulica del midollo spinale e isolamento dei gangli dorsali nei roditori

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

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

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