Tutti gli autori hanno contribuito alla creazione e la validazione del test presentato e al progetto del manoscritto.

Tutte le procedure sperimentali animali nel protocollo seguono le linee guida per la cura degli animali della Technische Universität München, Germania. 1. Media / Estratto di preparazione <ol><li> Tissue omogeneizzazione La qualità di omogeneizzazione tessuto è fondamentale per la successiva rivelazione di alterazioni neuroplastici nei neuroni in coltura. Qui, un omogeneizzatore è raccomandato che consente dissociazione tessuto senza grande aumento della temperatura del tessuto. <ol><li> Trasferire 5 x 5 mm x 5 mm cubi di tessuto pancreatico direttamente da -80 ° C in azoto liquido e posto in una fase solida nel homogenator tessuto. Il homogenator ideale sarebbe dissociare il tessuto abbastanza veloce, senza farlo scongelare. </li><li> Subito dopo dissociazione, risospendere il solido, omogenato polverulenti in 300-500 ml di 0.1x tampone fosfato salino (PBS). Qui, non utilizzare alcun tampone di lisi (come RIPA), in quanto puo &#39;lisi i neuroni.</li><li> Centrifugare le omogeneizzati per almeno 15 minuti alla velocità massima del centrifuga da banco (es. 21.130 xg). Raccogliere il surnatante chiaro che rappresenta l&#39;estratto del tessuto. </li></ol></li><li> Supernatanti della linea cellulare <ol><li> Lasciate che le cellule di interesse raggiungono almeno il 70-80% di confluenza nel normale terreno di crescita. </li><li> Di solito, questi mezzi contengono componenti siero e possono esercitare effetti incontrollabili sulla crescita neuronale. Pertanto, dopo aver raggiunto la densità cellulare desiderato, lavare le cellule almeno 3x con la coltura cellulare grade PBS e metterli in mezzo privo di siero (SFM) per un massimo di 48 ore. Nota: Qui, ritengono che alcune cellule (come le cellule stellate pancreatiche) possono avere bisogno di siero nel loro terreni di crescita, al fine di mantenere il loro stato e la struttura normale. In tali casi, eseguire diluizioni seriali di siero nel mezzo della cella di interesse al fine di trovare il possibile contenuto siero minima necessaria per funzio cellula intattan. </li><li> Misurare la concentrazione proteica del tessuto omogenato o surnatanti cellulari tramite Bradford protein assay. Mentre questi valori variano a seconda del tipo di tessuto e di cellule, per gli estratti di tessuto pancreatico, ci si aspetterebbe un intervallo di concentrazione tra 5-12 mg / mL, e per supernatanti della linea cellulare tra 3-8 mg / mL. </li></ol></li></ol> 2. Aliquota Estratti e surnatanti cellulari A seconda della concentrazione da utilizzare nel saggio, la concentrazione finale dell&#39;estratto o surnatante nel mezzo neuronale dovrebbe essere 100 pg / ml 5,8. Nota: Per un saggio con i neuroni crescono in 500 microlitri di media in ciascun pozzetto di una piastra a 24 pozzetti, uno ha bisogno di 50 mg di proteina da ogni estratto o surnatante per pozzetto. In una tipica concentrazione estratto di circa 10 mg / mL, uno avrebbe bisogno 5 ml di estratto / surnatante per ciascun bene. In ogni Setting esibito come triplice copia, questo corrisponderebbe a 15 ml di estratto / surnatante. Per i diversi tipi di tessuti o cellule, eseguire diluizioni seriali dell&#39;estratto finale o la concentrazione surnatante e confrontare gli effetti neurotrofici osservati tra le diverse concentrazioni di estratto / surnatante. 3. Isolamento di neuroni Dopo la raccolta estratto / supernatante è finito, proseguire con isolamento di neuroni. <ol><li> Per neuroni DRG, raccogliere la cervicale lombare DRG di ratto appena nati tra il giorno postnatale (P) 2-12 dopo la decapitazione e la dissezione stereomicroscopio di DRG (Figura 1A). Al fine di avere i neuroni DRG sufficienti per una piastra a 24 pozzetti, raccogliere tutte cervicale per lombare DRG di un ratto neonato (pari a 52 DRG) per piastra. <ol><li> Tagliare la periferica (neurale) e le proiezioni centrali (radici) di DRG per mezzo di microscissors. Lasciando le proiezioni in luogo impedisce triturazione e aumenta tegli contaminazione rischio di cultura da parte dei fibroblasti. </li></ol></li><li> Per neuroni MP, tagliare via il mesentere dal piccolo intestino e manualmente e togliere accuratamente lo strato seromuscular del piccolo intestino (per un protocollo dettagliato sull&#39;isolamento MP, consultare Schäfer et al. 9, Figura 1B). Per MP, raccogliere il plesso di due ratti per 24 pozzetti. Nota: Cercate di essere i più dolci possibili al fine di evitare strappi nel livello seromuscular in quanto impediscono la sua separazione con successo dal piccolo intestino. </li><li> Raccogliere il DRG e lo strato seromuscular in terreno minimo essenziale ghiacciata (MEM) fornito con gentamicina (20 mg in mezzo 500ml) e metronidazolo (2,5 mg in 500 ml di mezzo). </li><li> Dopo la raccolta di DRG, incubare in soluzione salina bilanciata di Hank (HBSS) fornito con collagenasi di tipo II per 20-30 min. Per l&#39;isolamento MP, incubare in collagenasi tipo fra 1-3 ore, a seconda dell&#39;etàdell&#39;animale (Figura 1C). </li><li> Per MP, raccogliere i MP pezzi netti come sotto stereomicroscopio e trasferimento MEM ghiacciata. </li><li> Poi triturare il DRG e MP attraverso siringhe con diametro decrescente. Nota: triturazione eccessiva può distruggere i neuroni, ma al netto delle cellule gliali. </li><li> Una volta che il terreno contenente il DRG o MP è diventata torbida, centrifugare la sospensione a 93,9 xg per 5 min, eliminare il mezzo e risospendere in Neurobasal medio (integrato con 100 U / ml di penicillina, 100 mg / ml di streptomicina, L-glutamina 0,5 mm e 2% B-27). </li><li> Contare il numero totale di cellule (cioè neuroni e glia) mediante un emocitometro. Nota: Il numero di cellule necessarie dipende dal parametro di misura. Per la quantificazione della densità neurite, bisogna culture più dense e quindi un maggior numero di cellule che per la misura della crescita dei neuriti, dimensione perikaryonale la ramificazione modello di singoli neuroni. Per le misurazioni della densità dei neuriti, utilizzare ad esempio 10.000 cellule (neuroni + glia) e / o di 1.500 neuroni / e. Per la morfometria su singoli neuroni, breve (1 minuto a lungo) trypsination delle cellule e semina di cellule 2.500 / e o 400 neuroni / bene è raccomandato. La resa tipica di cellule ottenute da un ratto è di circa 300.000-500.000 cellule. </li><li> Seed le cellule su 13 vetrini mm che sono state rivestite la sera prima e l&#39;alto i pozzetti con Neurobasal medio (integrato con 100 U / ml di penicillina, 100 mg / ml di streptomicina, 0.5 mM L-glutammina e il 2% B-27) (Figura 1D). Nota: Utilizzare poli-D-lisina (40 mg / m 2) OR-ornitina e scivola copertura laminina rivestite (1 mg / ml ciascuna) rivestito. Cellule attribuiscono alla poli-D-lisina estremamente veloce, rendendo così adatto per esperimenti che richiedono molte cellule (cioè misurazioni di densità neurite). L&#39;attaccamento al laminina è in qualche generalequello più debole, ma laminina è un forte promotore di crescita dei neuriti. Pertanto, per misure su ramificazione neuronale e la lunghezza dei neuriti, preferire ornitina / laminina-coating. </li><li> Consentire alle cellule attribuiscono ai pozzetti durante la notte. Il giorno successivo, preparare il dispositivo estratto supplementato (ad una concentrazione finale estratto / supernatante di 100 pg / ml). </li><li> Aspirare il mezzo semina, ed eseguire, lavaggio molto delicato opzionale con PBS, e poi lentamente pipetta media estrarre / surnatante-integrato (Figura 1E). </li><li> Lasciate che le cellule crescono per 48 ore. Aspirare i media e fissare nel 4% paraformaldeide per immunoistochimica (Figura 1F). </li><li> Eseguire doppia immunofluorescenza utilizzando neurone-specifico (ad esempio beta-tubulina III) e glia-specifica (ad esempio gliale fibriallary proteina acida / GFAP) marcatori (Figura 1G). </li><li> Per morfometria, utilizzare un microscopio ottico invertito dotato di una fotocamera CCD in combinazione with software automatizzato che consente la misurazione della densità dei neuriti (vedi tabella). </li><li> La densità neurite delle culture neuronali viene misurata su 4-5 microfotografie rappresentative a 200x ingrandimenti da 4 diverse regioni di crescita più densa su ogni vetrino sovrapponendo 50 micron x 50 micron griglia e contare la densità delle fibre per metro quadrato misurato in fibre di intersezione. Crescita dei neuriti, il numero di sportelli per neurone dire, la lunghezza del ramo media e la dimensione perikaryonal possono essere misurati da selezionati in modo casuale 30 neuroni solitarie di ogni vetrino contrassegnando le neuriti e perikarya (Figura 1H). </li></ol>

<ol>
	<li>Demir, I. E., Schafer, K. H., Tieftrunk, E., Friess, H., Ceyhan, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+plasticity+in+the+gastrointestinal+tract:+chronic+inflammation,+neurotrophic+signals,+and+hypersensitivity.">Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity.</a> Acta Neuropathol. 125, 491-509 (2013).</li><li>Vasina, V., 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=Enteric+neuroplasticity+evoked+by+inflammation.">Enteric neuroplasticity evoked by inflammation.</a> Auton. Neurosci. 126-127, 264-272 (2006).</li><li>Lomax, A. E., Fernandez, E., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+enteric+nervous+system+during+intestinal+inflammation.">Plasticity of the enteric nervous system during intestinal inflammation.</a> Neurogastroenterol. Motil. 17, 4-15 (2005).</li><li>Demir, I. E., 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=.">.</a> Neural Invasion in Pancreatic Cancer: The Past, Present and Future. 2, 1513-1527 (2010).</li><li>Demir, I. E., 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+microenvironment+in+chronic+pancreatitis+and+pancreatic+cancer+induces+neuronal+plasticity.">The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity.</a> Neurogastroenterol. Motil. 22, 480-490 (2010).</li><li>Schafer, K. H., Mestres, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GDNF-induced+neurite+outgrowth+and+neuronal+survival+in+dissociated+myenteric+plexus+cultures+of+the+rat+small+intestine+decreases+postnatally.">The GDNF-induced neurite outgrowth and neuronal survival in dissociated myenteric plexus cultures of the rat small intestine decreases postnatally.</a> Exp. Brain Res. 125, 447-452 (1999).</li><li>Schafer, K. H., Van Ginneken, C., Copray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+and+neural+stem+cells+in+the+enteric+nervous+system.">Plasticity and neural stem cells in the enteric nervous system.</a> Anat. Rec. 292, 1940-1952 (2009).</li><li>Liebl, F., 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+severity+of+neural+invasion+is+associated+with+shortened+survival+in+colon+cancer.">The severity of neural invasion is associated with shortened survival in colon cancer.</a> Clin. Cancer Res. 19, 50-61 (2012).</li><li>Sch&#228;fer, K. H., Saffrey, M. J., Burnstock, G., Mestres-Ventura, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+isolation+of+myenteric+plexus+from+the+newborn+rat+gastrointestinal+tract.">A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.</a> Brain Res. Brain Res. Protoc. 1, 109-113 (1997).</li><li>Ceyhan, G. 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=Nerve+growth+factor+and+artemin+are+paracrine+mediators+of+pancreatic+neuropathy+in+pancreatic+adenocarcinoma.">Nerve growth factor and artemin are paracrine mediators of pancreatic neuropathy in pancreatic adenocarcinoma.</a> Ann. Surg. 251, 923-931 (2010).</li><li>Demir, I. E., 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=Neuronal+plasticity+in+chronic+pancreatitis+is+mediated+via+the+neurturin/GFRalpha2+axis.">Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRalpha2 axis.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 303, 1017-1028 (2012).</li><li>Joseph, N. M., 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=Enteric+glia+are+multipotent+in+culture+but+primarily+form+glia+in+the+adult+rodent+gut.">Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.</a> J. Clin. Invest. 121, 3398-3411 (2011).</li></ol>

Gli autori non hanno interessi in gioco e senza informazioni finanziarie.

Il presente protocollo ha lo scopo di illustrare la metodologia alla base del pancreas test neuroplasticità in vitro che è stato sviluppato recentemente dal nostro gruppo per studiare i meccanismi di neuroplasticità in PCa e CP 5. Il protocollo prevede una procedura in tre giorni, che può essere facilmente applicato una volta l&#39;artista ha maturato un&#39;esperienza sufficiente per l&#39;isolamento e la cultura del DRG e MP neuroni. Inoltre, esso rappresenta un valido strumento per studiare la...

Alterazioni nei gastrointestinale (GI) morfologia del nervo e la densità hanno catturato l&#39;attenzione di gastroenterologi e patologi per molto tempo, ma la loro rilevanza per la fisiopatologia delle malattie gastrointestinali rimane sconosciuta 1-3. Infatti, alcuni disturbi gastrointestinali molto comuni come la gastrite, esofagite da reflusso, colite, diverticolite e appendicite sono associati ad un aumento della densità innervazione nelle aree di tessuto infiammato 1. Tuttavia, nessuna vera attenzione è stata finora versati ai meccanismi e significato della neuroplasticità nel tratto GI. Sei nervi GI morfologicamente alterati differiscono dalle normali nervi GI, cioè lo stato normale del sistema nervoso enterico, in termini della loro funzione? Quali sono le implicazioni di alterata contenuti neuropeptide / neurotrasmettitore nei nervi enterici di plastica? Fa neuroplasticità periferica comporta sempre la segnalazione alterata al sistema nervoso centrale? E dove sono le proiezioni centrali di Extri plasticaNSIC percorsi neurali GI? Una lunga serie di questioni fondamentali possono essere facilmente generati quando si guarda la scarsità delle nostre conoscenze sugli aspetti funzionali di GI neuroplasticità. Lo studio di GI neuroplasticità a livello funzionale richiede modelli sperimentali validi, riproducibili e ancora facilmente applicabili. In un&#39;epoca di crescente popolarità e l&#39;accettazione di modelli geneticamente condizionato mouse (GECoMM), come nelle impostazioni vivo portano il potenziale per chiarire gli aspetti sconosciuti di GI neuroplasticità in modo realistico 1. Tuttavia, la progettazione e la produzione di GECoMM resta costoso, alta intensità di lavoro e, soprattutto, in termini di tempo. Inoltre, essi richiedono a priori selezione del bersaglio da condizionale modulato nel topo geneticamente alterato (ad es transgenico sovraespressione del fattore di crescita nervoso / NGF in cellule epiteliali enterici). Quindi, per il design di un GECoMM di successo, i ricercatori hanno bisogno di alcuni indicatori (ad esempio, dati sperimentali precedenti) di un obiettivo utile, vale a dire che la molecola di interesse (qui NGF) può almeno aspettare di esercitare alcuni effetti biologicamente rilevanti sui nervi GI. Tali indicatori possono essere facilmente derivate da adeguata in modelli in vitro in cui sottotipi di cellule isolate dal complesso microambiente di un sistema in vivo possono essere selettivamente co-coltivate in maniera eterotipica 4-7. La modulazione di bersagli molecolari in un tale ambiente cultura eterotipica è in media tecnicamente meno ingombrante, più veloce, e può quindi aiutare nella prefiltraggio degli obiettivi validi per la verifica in studi in vivo. Recentemente, abbiamo presentato un test in vitro neuroplasticità in cui è stato progettato per simulare la maggiore densità neurale e ipertrofia delle ne intrapancreaticarves nel cancro del pancreas umano (PCA) e pancreatite cronica (CP) tessuti. Qui, i neuroni derivati ​​da neonato di ratto gangli spinali (DRG) o plesso mioenterico (MP) sono stati esposti a estratti di tessuto da PCa chirurgicamente asportato o CP tessuti esemplari e rispetto a quelle coltivate in normali pancreas umano (NP) estratti di tessuto 5. Invece di estratti di tessuto, si può anche utilizzare supernatanti della linea cellulare di studiare l&#39;impatto di tipi cellulari selezionati TG. Quando combinato con una misurazione morfometrica standardizzato, il saggio neuroplasticità presentata consente di valutare valido e riproducibile di plasticità neuronale in risposta a diversi microambienti pancreatiche. In particolare, esso consente la simulazione di 1) neuroplasticity morfologica, cioè i cambiamenti nella crescita dei neuriti, ramificazione e dimensione neuronale, e 2) neuroplasticity funzionale, cioè alterazioni nella eccitabilità dei neuroni periferici. Inoltre, non solo periferica ( </em&gt; Enterico), ma anche centrale (es. DRG o spinale secondo ordine) neuroni possono essere incluse nella presente saggio per valutare la loro reazione morfologica e funzionale a diversi contenuti di tessuto GI. Nel presente tutorial video, dimostriamo il protocollo tecnico per l&#39;esecuzione di questo saggio e discutere i suoi vantaggi e debolezze. Inoltre, attiriamo l&#39;attenzione sulla applicabilità del concetto di base di questo test allo studio della neuroplasticità in qualsiasi organo GI.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Poly-D-lysine hydrobromide</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:124px;">P1149</td>
			<td style="width:328px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ornithine/laminin</td>
			<td>Sigma-Aldrich</td>
			<td>P2533/L4544</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">13mm coverslips</td>
			<td>Merck</td>
			<td></td>
			<td>For use in 24-well plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dismembranator S</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Beta-III-tubulin antibody</td>
			<td>Millipore</td>
			<td>MAB1637</td>
			<td>1:200 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP-antibody</td>
			<td>DAKO</td>
			<td>M0761</td>
			<td>1:400 concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RIPA buffer + protease inhibitor</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Gibco/Life sciences</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 supplement</td>
			<td>Gibco/Life sciences</td>
			<td>17504044</td>
			<td>Quality of B-27 is known to depend on the lot number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin/Metronidazol</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimal essential medium</td>
			<td>Gibco/Life sciences</td>
			<td>31095-029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life sciences</td>
			<td>24020133</td>
			<td>Improves collagenase activity when containing Ca/Mg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase type II</td>
			<td>Worthington Biochemical</td>
			<td>CLS-2</td>
			<td>Obtain lots with at least 200U/mg activity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA 0,25%</td>
			<td>Gibco/Life sciences</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td>Any supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">analySIS docu software</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay

Neuroplasticità è una caratteristica intrinseca del sistema e gastrointestinale (GI) innervazione nervoso enterico in condizioni patologiche. Tuttavia, il ruolo fisiopatologico delle neuroplasticità nei disturbi gastrointestinali rimane sconosciuto. Nuovi modelli sperimentali che consentono la simulazione e la modulazione di GI neuroplasticità possono consentire maggiore apprezzamento per il contributo della neuroplasticità in particolare le malattie gastrointestinali come il cancro del pancreas (PCA) e pancreatite cronica (CP). Qui vi presentiamo un protocollo per la simulazione della neuroplasticità del pancreas in condizioni in vitro utilizzando neonato gangli delle radici dorsali di ratto (DRG) e plesso mioenterico (MP) neuroni. Questo approccio dual-neurone permette non solo il controllo di entrambi neuroplasticità organo intrinseca ed estrinseca-, ma rappresenta anche un valido strumento per valutare la morfologia neuronale e gliale e di elettrofisiologia. Inoltre, consente la modulazione funzionale dei contenuti del microambiente forniti per studiare la loro IMPACt sulla neuroplasticità. Una volta stabilito, il presente saggio neuroplasticity porta il potenziale di essere applicabile allo studio della neuroplasticità in qualsiasi organo GI.

Neuroplasticità morfologica Nella fascia di età indicata di ratti neonati (P2-12) e le densità di semina, MP e DRG neuroni già costruiscono reti neuronali dense dopo 48 ore (Figura 2A). Confronto di densità neurite tra neuroni coltivati ​​in CaP, CP, e estratti NP rivela maggiore densità neuriti dei neuroni DRG PCA o estratti CP rispetto estratti NP (Figura 2A) 5. Abbiamo particolarmente preferiamo i neuroni MP per misure su sin...

Watch this Scientific Journal Video about Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay at JoVE.com

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Plasticità neuronale è una caratteristica del tratto gastrointestinale (GI) sempre più riconosciuta, ma non sufficientemente compreso. Qui, nell&#39;esempio di disturbi pancreatici umani, presentiamo un neuroplasticity saggio in vitro per lo studio della plasticità neuronale nel tratto GI a livello sia morfologica e funzionale.

Simulazione pancreatica neuroplasticità:<em&gt; In Vitro</em&gt; Dual-neurone Plasticità Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

simulating-pancreatic-neuroplasticity-vitro-dual-neuron-plasticity

Research

JoVE Journal

Medicine

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

9.7K Views.  Technische Universität München.  Plasticità neuronale è una caratteristica del tratto gastrointestinale (GI) sempre più riconosciuta, ma non sufficientemente compreso. Qui, nell'esempio di disturbi pancreatici umani, presentiamo un neuroplasticity saggio in vitro per lo studio della plasticità neuronale nel tratto GI a livello sia morfologica e funzionale. 

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In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro test Clearance mesoteliali che modella i primi passi di metastasi del cancro ovarico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Ricerca

Medicina

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Sviluppo di un audio-based ambiente di gioco virtuale per assistere con doti di navigazione in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

Un modello murino di cervicale lesioni del midollo spinale per studiare post-lesionale respiratoria neuroplasticità

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Multi-elettrodi array registrazioni di umano Epileptic postoperatoria corticale del tessuto

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Valutare l&#39;encefalopatia spongiforme trasmissibile Specie Barriere con un<em&gt; In Vitro</em&gt; Prion Protein Assay Conversion

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A High-contenuti<em&gt; In Vitro</em&gt; La replica della piattaforma Discovery isole pancreatiche β-cellule

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Applicazione dell'anastomosi to-end in robotica Pancreatectomy centrale

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal trapianto di isole pancreatiche nel modello del topo

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...