Gli autori vorrei ringraziare Benjamin J Weigler, D.V.M., pH.d. e Walt Mandeville, D.V.M (risorse animali & Campus frequentando veterinario, University of Nevada, Reno) per i loro servizi animale eccellente fornito per i topi, come pure la loro consiglio su procedure chirurgiche.

Il seguente protocollo è stato approvato dal istituzionale Animal Care e utilizzare Committee (IACUC) presso l'Università del Nevada-Reno (UNR) animale risorse ed è conforme a tutti gli orientamenti etici istituzionali per quanto riguarda l'uso di animali di ricerca.
1. gli animali.
<ol><li>Ottenere topi C57BL/6 maturo (4-6 settimane) peso compreso tra 20-30 g. House la colonia di topi di laboratorio in una struttura centralizzata animale alle risorse animali UNR.</li>
</ol>2. parziale ostruzione chirurgia
Nota: Interventi sono eseguiti in una sala dedicata alle procedure chirurgiche. Tutti gli strumenti chirurgici sono sterilizzati in autoclave prima dell'intervento. Guanti e camici chirurgici sterili devono essere indossati per tutto il personale nella sala chirurgica in ogni momento.
<ol><li>Preparazione per il mouse chirurgico<ol>
<li>Controllare il sistema di consegna anestetico per garantire l'approvvigionamento di ossigeno e isoflurano è adeguata per la procedura. Attivare la fornitura di ossigeno. Accendere il misuratore di portata gas e regolare a 500-1000 mL/min posto l'animale nella camera di induzione e sigillare la parte superiore.</li>
		<li>Accendere il vaporizzatore di isoflurane al 5% e monitorare l'animale fino a quando diventa recumbent. Passare il sistema di consegna anestetico per il cono di naso.</li>
		<li>Lavare la camera di induzione di qualsiasi residuo di gas con l'ossigeno, quindi disattivare la linea di camera di induzione, mantenendo la linea del naso a cono aperto.</li>
		<li>Rimuovere l'animale dalla camera e con attenzione unguento oftalmico sugli occhi dell'animale.</li>
		<li>Inserire il cono di naso su un pad caldo preriscaldato quando gas continua a fluire.</li>
		<li>Modificare il flusso di ossigeno a 100-200 mL/min, con 2-3% isoflurane. Se l'animale comincia a muoversi, gentlyrestrain l'animale con il cono di naso su fino al completamente anestetizzato nuovamente.</li>
		<li>Monitorare la respirazione e la risposta alla stimolazione durante la procedura e regolare la percentuale di isoflurane (2-5%), se necessario. Livello animale amputate viene monitorato dalla mancanza di riflesso pizzico punta prima che l'ambulatorio è effettuato.</li>
		<li>Iniettare la medicina del dolore (buprenorfina, 1 µ g/g di peso corporeo) intraperitonealmente lontano dal sito di incisione.</li>
		<li>Applicare la lozione di rimozione dei capelli sull'addome con un batuffolo di cotone pulito. Lasciare il sit di lozione per 3-5 min sul mouse, quindi rimuovere i capelli utilizzando tamponi di garza e cotone. Ripetere questo passaggio fino a quando tutti i capelli sono stato rimosso dall'addome del mouse.</li>
		<li>Pulire la pelle con etanolo al 70% utilizzando tamponi di garza e cotone. Applicare la soluzione swabsticks o povidone-iodio per pulire l'addome.</li>
	</ol>
</li>
	<li>Chirurgia di ostruzione parziale<ol>
<li>Drappo il sito chirurgico utilizzando carta sterile 25 x 50 cm con un 2,5 x 2,5 cm apertura centrale per l'area di chirurgia. Fissare il telo all'animale inserendo strisce sterile ai confini dell'apertura e della pelle.</li>
		<li>Fare un'incisione addominale di ~3.0 cm longitudinalmente usando un bisturi a lama n ° 15, garantendo che solo la pelle è incisa ed evitando il taglio nello strato musculoperitoneal in questo momento.</li>
		<li>Utilizzo di pinze e forbici chirurgiche, separare con cura la pelle dallo strato musculoperitoneal senza causare qualsiasi incisione al livello musculoperitoneal. Dopo gli strati sono stati sufficientemente separati (circa 1 cm x 4 cm), identificare la linea alba sul layer musculoperitoneal e taglio ~ 2 cm lungo la linea alba per esporre la cavità intraperitoneale utilizzando micro-pinze e le forbici.</li>
		<li>Accuratamente di individuare e identificare l'intestino cieco. Lentamente e delicatamente rimuovere l'intestino cieco dalla cavità intraperitoneale con micro-forcipe, portando colon prossimale e dell'ileo con il cieco all'esterno sul telo sterile. Immediatamente inumidire il tessuto intestinale con garza imbevuta salina sterile 0,9% e mantenere il tessuto esposto inumidito in ogni momento, mentre si trovano all'esterno della cavità addominale.</li>
		<li>Individuare e identificare il mesentere tra l'ileo e del colon prossimale. Fare un'incisione (~ 1 cm) parallela e appena sotto, l'ileo, nel mesentery ed evitare di tagliare qualsiasi sistema vascolare.</li>
		<li>Prendere un anello di silicone in autoclave (6 mm di lunghezza, 4 mm di diametro esterno, diametro interno 3,5 mm). Tagliare longitudinalmente per aprire il tubo e l'anello con micro-forcipe.</li>
		<li>Inserire un'estremità dell'anello aperto attraverso l'incisione nel tessuto del mesentery. Restituire l'anello a forma di anello completato portando un'estremità in contatto con un altro, con l'ileo circondato dall'anello.</li>
		<li>Assicurarsi che l'anello di silicone circonda completamente l'ileo, chiudere l'anello con sutura e posizionare con cura gli intestini indietro nella cavità intraperitoneale.</li>
	</ol>
</li>
	<li>Chiusura di chirurgia<ol>
<li>Eseguire un semplice sutura continua sul livello musculoperitoneal lungo la linea alba per chiudere il musculoperitoneal della ferita con una sutura assorbibile. Dopo la sutura è completa, togliere qualsiasi sanguinamento con 0,9% soluzione salina sterile imbevuto di garza.</li>
		<li>Per chiudere completamente la ferita, con una sutura in nylon separata, è necessario eseguire una semplice sutura continua sulla pelle.</li>
		<li>Al termine di entrambe le suture, pulire la ferita con nuovo swabstick o povidone-iodio.</li>
		<li>Intraperitonealmente iniettare antibiotici (gentamicina, 150 μL per topo basato sul peso corporeo di 20-30 g).</li>
		<li>Dopo il completamento della procedura, spegnere il vaporizzatore di isoflurano e permettere all'animale di respirare solo fluire ossigeno fino a quando iniziare a guadagnare la coscienza.</li>
		<li>Una volta che l'animale è sveglio, metti l'animale in una zona di recupero separato con supporto termico fino a completamente recuperato.</li>
	</ol>
</li>
</ol>3. post-operazione osservazione.
<ol><li>Dopo il completamento della chirurgia, spostare gli animali in un'incubatrice in una stanza di recupero dove temperatura e umidità è regolamentate. Monitorare gli animali postoperatorio ogni 15 min per la prima ora poi ogni 30 minuti per la seconda ora, mentre gli animali sono in incubatrice.</li>
	<li>Appena l'osservazione prescritto è completo, spostare gli animali alla propria gabbia individuale e monitorarli tutti i giorni per indicazioni cliniche di dolore18e per garantire che la ferita chirurgica è la guarigione correttamente senza segni di complicazioni ( deiscenza) presenti.</li>
</ol>

<ol>
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The process is not a form of enteritis but lymphedema contaminated by intestinal contents.</a> Chir Ital. 52, 109-121 (2000).</li><li>Limsrivilai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analytic+Bayesian+model+for+differentiating+intestinal+tuberculosis+from+Crohn's+disease.">Meta-analytic Bayesian model for differentiating intestinal tuberculosis from Crohn's disease.</a> Am J Gastroenterol. 112, 415-427 (2017).</li><li>Dvorak, D., Adamova, Z., Bar, T., Slovacek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+hernia+as+a+cause+of+small+bowel+obstruction.">Internal hernia as a cause of small bowel obstruction.</a> Rozhl Chir. 96, 34-36 (2017).</li><li>Massani, M., Capovilla, G., Ruffolo, C., Bassi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+stromal+tumour+(GIST)+presenting+as+a+strangulated+inguinal+hernia+with+small+bowel+obstruction.">Gastrointestinal stromal tumour (GIST) presenting as a strangulated inguinal hernia with small bowel obstruction.</a> BMJ Case Rep. , (2007).</li><li>Chen, J., Chen, H., Sanders, M., Perrino, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+SRF/CArG-dependent+gene+transcription+during+chronic+partial+obstruction+of+murine+small+intestine.">Regulation of SRF/CArG-dependent gene transcription during chronic partial obstruction of murine small intestine.</a> Neurogastroenterol Motil. 20, 829-842 (2008).</li><li>Chang, I. Y., 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=Loss+of+interstitial+cells+of+Cajal+and+development+of+electrical+dysfunction+in+murine+small+bowel+obstruction.">Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction.</a> J Physiol. 536 (Pt 2), 555-568 (2001).</li><li>Liu, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+dependent+potassium+channel+remodeling+in+murine+intestinal+smooth+muscle+hypertrophy+induced+by+partial+obstruction.">Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.</a> PLoS One. 9 (2), e86109 (2014).</li><li>Guo, X., 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=Down-regulation+of+hydrogen+sulfide+biosynthesis+accompanies+murine+interstitial+cells+of+Cajal+dysfunction+in+partial+ileal+obstruction.">Down-regulation of hydrogen sulfide biosynthesis accompanies murine interstitial cells of Cajal dysfunction in partial ileal obstruction.</a> PLoS One. 7, e48249 (2012).</li><li>Yang, J., Zhao, J., Chen, P., Nakaguchi, T., Grundy, D., Gregersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdependency+between+mechanical+parameters+and+afferent+nerve+discharge+in+hypertrophic+intestine+of+rats.">Interdependency between mechanical parameters and afferent nerve discharge in hypertrophic intestine of rats.</a> Am J Physiol-Gastr L. 310, G376-G386 (2016).</li><li>Zhao, J., Liao, D., Yang, J., Gregersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+remodeling+of+obstructed+guinea+pig+jejunum.">Biomechanical remodeling of obstructed guinea pig jejunum.</a> J Biomech. 43, 1322-1329 (2010).</li><li>Bowen, E. 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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=Transcriptome+analysis+of+PDGFR&#945;++Cells+identifies+T-types+Ca2++channel+CACNA1G+as+a+new+pathological+marker+for+PDGFR&#945;++cell+hyperplasia.">Transcriptome analysis of PDGFR&#945;+ Cells identifies T-types Ca2+ channel CACNA1G as a new pathological marker for PDGFR&#945;+ cell hyperplasia.</a> PLoS One. 12, e0182265 (2017).</li><li>Park, C., 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=Serum+response+factor+is+essential+for+prenatal+gastrointestinal+smooth+muscle+development+and+maintenance+of+differentiated+phenotype.">Serum response factor is essential for prenatal gastrointestinal smooth muscle development and maintenance of differentiated phenotype.</a> J Neurogastroenterol Motil. 21, 589-602 (2015).</li><li>Spencer, N. J., Sanders, K. M., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrating+motor+complexes+do+not+require+electrical+slow+waves+in+the+mouse+small+intestine.">Migrating motor complexes do not require electrical slow waves in the mouse small intestine.</a> J Physiol. 553, 881-893 (2003).</li><li>Langford, D. 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=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nat Methods. 7, 447-449 (2010).</li><li>Terez, S. D., Notari, L., Sun, R., Zhao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+smooth+muscle+responses+to+inflammation.">Mechanisms of smooth muscle responses to inflammation.</a> Neurogastroenterol Motil. 24, 802-811 (2012).</li><li>Chen, W., 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=Smooth+muscle+hyperplasia/hypertrophy+is+the+most+prominent+histological+change+in+Crohn's+fibrostenosing+bowel+strictures:+A+semiquantitative+analysis+by+using+a+novel+histological+grading+scheme.">Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn's fibrostenosing bowel strictures: A semiquantitative analysis by using a novel histological grading scheme.</a> J Crohns Colitis. 11, 92-104 (2017).</li><li>Huizinga, J. D., Chen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+Cells+of+Cajal:+Update+on+Basic+and+Clinical+Science.">Interstitial Cells of Cajal: Update on Basic and Clinical Science.</a> Curr Gastroenterol Rep. 16, 363 (2014).</li><li>Jirkof, P., Touvieille, A., Cinelli, P., Arras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buprenorphine+for+pain+relief+in+mice:+repeated+injections+vs+sustained-release+depot+formulation.">Buprenorphine for pain relief in mice: repeated injections vs sustained-release depot formulation.</a> Lab Animal. 49, 177-187 (2015).</li><li>Spencer, N. J., Dinning, P. J., Brookes, S. 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Abbiamo dimostrato che i topi che ricevono la chirurgia intestinale PO costantemente e riproducibile sviluppano ipertrofia della muscolatura liscia intestinale, che imita l'ostruzione intestinale umano. Ambulatori di ostruzione intestinale sono stati sviluppati per i diversi animali, tra cui7di topi, ratti10, cavie11, cane12 e gatti13. Il modello del mouse dell'ostruzione intestinale ha tempo, costo, di...

Ostruzioni intestinali sono un blocco parziale o totale nell'intestino piccolo o grande che impedisce il movimento attraverso l' intestino1di cibo digerito, fluidi e gas. Dovuto l'ostruzione, il blocco induce le pareti intestinali per diventare ispessito, restringimento del lume2. Ostruzione intestinale può verificarsi a seguito di interventi chirurgici addominali o pelvici che causano la formazione di tessuto di aderenze addominali o da disturbi GI quali diverticolite, volvolo, Ernie, malattie infiammatorie croniche intestinali (morbo di Crohn), stenosi, invaginazione intestinale, stipsi, fecaloma, pseudo-ostruzione, cancri e tumori3,4,5. Ostruzioni intestinali in questi casi spesso portano all'ipertrofia della tunica fibre del intestino6.
PO del lume induce distensione intestinale e aumenta di spessore dello strato di muscolo liscio intorno l'ostruzione in risposta alla necessità di continuare la peristalsi funzionale7,8,9,10, 11,12,13. Modelli animali di PO intestinale sono stati sviluppati per studiare l'ipertrofia del muscolo liscio in topi7, ratti10, cavie11, cani12e gatti13 che costantemente sviluppare ipertrofia simile entro il strati di muscolatura intestinale.
Un modello murino di PO intestinale è il modo più efficace per generare e studiare ostruzioni intestinali in vivo. Ostruzioni del piccolo intestino sono effettuati nei topi utilizzando un anello di silicone inserito chirurgicamente che circondano l'ileo. I topi di PO hanno mostrato un aumento iniziale del numero di cellule (iperplasia) e un aumento in spessore di strato del muscolo (ipertrofia) dopo PO chirurgia8,15. SMC sono le cellule primarie di plastica che stanno crescendo all'interno strati di muscolo liscio in risposta alle condizioni ipertrofica14, ma altre cellule come ICC e le cellule PDGFRα+ che sono strettamente associati con SMC, inoltre vengono ripopolati. Precedentemente abbiamo riferito che i topi PO sviluppano ipertrofia nel piccolo intestino, in cui SMC sono dedifferenziate nelle cellule PDGFRα+ che sono altamente proliferative7,15,16. Al contrario, ICC sono degenerati e perso all'interno degli strati di muscolo liscio ipertrofico durante lo sviluppo di obsruction intestinale7. Un altro vantaggio importante del modello PO è la sua capacità di indurre i cambiamenti nel sistema nervoso enterico e propagare neurogene schemi motori. La propagazione principali modello neurogeno motore nelle piccole viscere del mouse è il complesso motore migrazione (MMC), che è neurogena e non necessita di ICC o elettrico onde lente17. Il modello PO può fornire chiare intuizioni come MMCs e nervi enterici sono stato rimodernati da ostruzione parziale.
Qui, vi proponiamo un protocollo murino per chirurgia intestinale PO utilizzando un anello di silicone. Topi che riceve la chirurgia PO attendibilmente producono ipertrofia in tunica fibre di piccolo intestino. All'interno del muscolo ipertrofico, SMC, ICC, PDGFRα+e cellule neuronali sono notevolmente ristrutturate.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical drape</td>
			<td>Medical and veterinary supplies</td>
			<td>SMS40</td>
			<td>40&#8221;X100 yards</td>
		</tr>
		<tr>
			<td>Underpad, econ, pro plus</td>
			<td>Medical and veterinary supplies</td>
			<td>MSC281224</td>
			<td>17x24&#8221;</td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Braintree scientific, Inc</td>
			<td>SC-i-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Vantage</td>
			<td>V95-304</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont electronic &#38; jeweler tweezers</td>
			<td>Dumont</td>
			<td>98-180-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided absorbable suture</td>
			<td>Covidien polysorb</td>
			<td>SL-5687G</td>
			<td>5-0, polyglactin</td>
		</tr>
		<tr>
			<td>Nylon non-absorbable mono filament</td>
			<td>AD surgical</td>
			<td>S-N618R13</td>
			<td>6-0, nylon</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Dynarex</td>
			<td></td>
			<td>No.15</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Jacobson microvascular</td>
			<td>36-1342TC</td>
			<td>8.5 inch</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Flinn scientific</td>
			<td>AB1049</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissor</td>
			<td>WPI</td>
			<td>503305</td>
			<td></td>
		</tr>
		<tr>
			<td>Petrolatum ophthalmic ointment</td>
			<td>Puralube VET</td>
			<td></td>
			<td>3.5 g</td>
		</tr>
		<tr>
			<td>Fluriso (isoflurane)</td>
			<td>Vetone</td>
			<td>V1 502017</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Steri-strip reinforced skin closure</td>
			<td>3M</td>
			<td>R1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Medline</td>
			<td>MSG2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear loop face mask</td>
			<td>The safety zone</td>
			<td>RS700</td>
			<td></td>
		</tr>
		<tr>
			<td>Avant gauze non-woven sponges</td>
			<td>Caring</td>
			<td>PRM25444</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical cup</td>
			<td>Admiral&#160; craft OYC-2</td>
			<td>725-A42</td>
			<td>2.5 oz</td>
		</tr>
		<tr>
			<td>Swabstick</td>
			<td>ChloraPrep</td>
			<td>260103</td>
			<td>2% w/v Chlorhexidine&#160; Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)</td>
		</tr>
		<tr>
			<td>Cotton tipped applicator</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Zoo pharm</td>
			<td>BZ8069317</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate</td>
			<td>Vetone</td>
			<td>G-6336-04</td>
			<td>100 mg/ml</td>
		</tr>
		<tr>
			<td>Fast acting gel cream remover</td>
			<td>Veet</td>
			<td>8111002</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>AHS</td>
			<td>AH01T2516</td>
			<td>1 ml with needle</td>
		</tr>
		<tr>
			<td>Silicon ring</td>
			<td>VWR</td>
			<td>60985-720</td>
			<td>6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td>4-6 weeks old</td>
		</tr>
	</tbody>
</table>

a mouse model of intestinal partial obstruction

Ostruzioni intestinali, che ostacolare o bloccano il movimento peristaltico, possono essere causati da aderenze addominali e più gastrointestinale (GI) malattie tra cui escrescenze tumorali. Tuttavia, il cellulare che ritocca meccanismi coinvolti nella, e causato da, ostruzioni intestinali sono capiti male. Sono stati sviluppati diversi modelli animali di ostruzioni intestinali, ma il modello del mouse è più costo/tempo efficace. Il modello del mouse utilizza l'impianto chirurgico di un'ostruzione intestinale parziale (PO) che ha un alto tasso di mortalità se non viene eseguita correttamente. Inoltre, topi che riceve la chirurgia PO non riescono a sviluppare ipertrofia se un adeguato blocco non viene utilizzato o non correttamente inserito. Qui, descriviamo un protocollo dettagliato per la chirurgia di PO che produce ostruzioni intestinali affidabili e riproducibili con un tasso di mortalità molto bassa. Questo protocollo utilizza un anello di silicone chirurgicamente posizionati che circonda l'ileo che blocca parzialmente movimento digestivo nell'intestino tenue. Il blocco parziale rende l'intestino diventa dilatato a causa l'arresto del movimento digestivo. La dilatazione dell'intestino induce l'ipertrofia del muscolo liscio sul lato orale dell'anello che progressivamente si sviluppa più di 2 settimane fino a quando non causa la morte. Il modello di mouse PO chirurgico offre un modello in vivo di ipertrofico tessuto intestinale utile per lo studio delle mutazioni patologiche delle cellule intestinali, compreso le cellule muscolari lisce (SMC), cellule interstiziali di Cajal (ICC), PDGFRα+e di un neurone cellule durante lo sviluppo dell'ostruzione intestinale.

L'ostruzione parziale (PO) è stata chirurgicamente indotta nei topi un mesi di vita inserendo un anello di silicone intorno l'ileo vicino allo sfintere ileocecale. Questo anello creato un blocco parziale nell'ileo. Le operazioni di intervento Sham (modo) inoltre sono stati effettuati senza un anello sul età/sesso abbinato topi e questi topi non hanno mostrato sintomi simili a quelli trovati nei topi del PO. Topi, rapidamente recupero da un intervento chirurgico PO entro poche ore. Essi ...

Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

A Mouse Model of Intestinal Partial Obstruction

Ostruzioni intestinali sono un blocco parziale o totale dell'intestino che può causare dolore addominale, nausea, vomito e impedendo il passaggio delle feci. Questa procedura per la creazione di intestinale parziale ha pilastri in topi è affidabile a studiare i meccanismi alla base di crescita delle cellule patologiche e morte nell'intestino.

Un modello murino di ostruzione intestinale parziale

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

a-mouse-model-of-intestinal-partial-obstruction

Research

JoVE Journal

Biology

20.9K Views.  University of Nevada, Reno School of Medicine. Ostruzioni intestinali sono un blocco parziale o totale dell'intestino che può causare dolore addominale, nausea, vomito e impedendo il passaggio delle feci. Questa procedura per la creazione di intestinale parziale ha pilastri in topi è affidabile a studiare i meccanismi alla base di crescita delle cellule patologiche e morte nell'intestino.

Video: A Mouse Model of Intestinal Partial Obstruction

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Ricerca

Biologia

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Indagare infiammazione intestinale in DSS-indotta Modello di IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Real Time Analisi di profilo metabolico in<em&gt; Ex Vivo</em&gt; Culture del mouse intestinale Cripta organoide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Un protocollo per lentivirali trasduzione e analisi valle di intestinale organoidi

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

Legatura del dotto biliare parziale nel topo: un modello radiocomandato di colestasi ostruttiva localizzata

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Analisi della spettrometria di massa per identificare l'ubiquilazione del CENP-A con tag EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Un modello di topo di instabilità della colonna lombare

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

Monostrato epiteliale derivato da organoidi: un modello in vitro clinicamente rilevante per la funzione di barriera intestinale

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Epatotectomia parziale nel pesce zebra adulto

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...