The authors acknowledge all colleagues who made this work possible; Ann Demarr&#233;, Veerle Laurysens, Jan De Jonge and Erik Quartier for technical assistance. Financial support was from the VUB Research Council, the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Beta Cell Biology Consortium (BCBC), the Fund for Scientific Research Flanders (FWO), Diabetes Onderzoek Nederland (DON) and Interuniversity Attraction Pole (IAP).

Tutte le operazioni seguono le linee guida emanate dalla Convenzione europea per la protezione degli animali vertebrati utilizzati a fini sperimentali o ad altri fini scientifici (STE 123 e il 2010/63 / UE). 1. Preparazione di area di lavoro <ol><li> Fornire una zona di preparazione dedicata, una zona di intervento e una zona di recupero. </li><li> Effettuare l&#39;intera procedura chirurgica in una cappa a flusso laminare per minimizzare contaminanti ambientali. </li><li> Montare le forniture (elencati in Materiali e Metodi) necessari sulla zona di preparazione, chirurgica e di recupero, con una corretta tecnica asettica. </li><li> Assicurarsi che gli strumenti chirurgici sono sterilizzati in autoclave prima dell&#39;intervento. </li><li> Fornire un rilievo di riscaldamento dell&#39;acqua di ricircolo ad una temperatura di 38 ° C per la stabilizzazione della temperatura durante l&#39;intervento chirurgico. Coprire la piastra elettrica con un tampone impermeabile sterile. </li><li> Fornire un microscopio operativo con un ingrandimento di almeno 6.3x. </li><li> Utilizzare un strumento sterilizer, come un tallone sterilizzatrice a caldo, per sterilizzare strumenti tra procedure chirurgiche. </li><li> Preparare una zona di recupero costituito da una grande gabbia, fiancheggiata da biancheria da letto di carta piatta </li></ol> 2. Preparare gli Animali per Chirurgia <ol><li> Per Pdl e chirurgia simulata, utilizzare 8 settimane di età topi maschi, alloggiati in gabbie standard e mantenuti su una 12 ore di luce / 12 ore ciclo buio e nutriti un roditore dieta ad libitum standard. Nota: Qui, usiamo topi BALB / cJrJ ma abbiamo anche utilizzato con successo altri ceppi e vari ceppi transgenici. </li><li> Utilizzare Buprenophine come preemptive analgesia (0,05-0,1 mg / kg) 30 minuti prima dell&#39;intervento. </li><li> Anestetizzare topi mediante iniezione intraperitoneale di 100 mg / kg di ketamina e 5 - 10 mg / kg di xilazina. </li><li> Valutare corretta anestesia osservando graduale perdita di movimenti volontari e rilassamento muscolare. Testare la perdita dei riflessi da pizzicamento punta. </li><li> Applicare una pomata oftalmica a prevsecchezza ent degli occhi mentre sotto anestesia. </li></ol> 3. chirurgico Preparazione del sito <ol><li> Disinfettare torace e addome con soluzione di clorexidina antisettica. </li><li> Radere un&#39;area di 2,5 cm x 1,5 cm dell&#39;addome. </li><li> Disinfettare la zona rasata con una garza imbevuta di soluzione di clorexidina, poi soluzione alcolica e una applicazione finale di soluzione di clorexidina. </li><li> Posizionare l&#39;animale nella zona di intervento in modo che il sito chirurgico preparato viene rivolto verso l&#39;alto il chirurgo. </li><li> Passare il mouse utilizzando un telo chirurgico impermeabile con un&#39;apertura che lascia la regione addominale disinfettata esposta mentre copre il resto del corpo per creare un campo di lavoro sterile. Monitorare il mouse prima della procedura di profondità dell&#39;anestesia. </li></ol> 4. Dotto pancreatico legatura <ol><li> Laparotomia <ol><li> Fare un&#39;incisione mediana superiore nella pelle estende dal processo xifoideo all&#39;ombelico utilizzando una sterilelama chirurgica. Separare il sottostante linea alba e il peritoneo usando forbici sterili al fine di esporre il quadrante addominale superiore. </li><li> Prevenire essiccazione-out degli organi interni per aspersione regolare con sterile allo 0,9% di cloruro di sodio. </li><li> Con una pinzetta sterili o tampone, ritrarre lo stomaco superiormente, esponendo la milza e il lobo milza (la regione coda) del pancreas. </li><li> Retrarre delicatamente duodeno e parte del digiuno superiore al quadrante destro dell&#39;addome superiore per esporre la testa, il collo e la regione di body del pancreas coperte dal peritoneo viscerale. </li></ol></li><li> Legatura <ol><li> Individuare la posizione anatomica del dotto pancreatico principale nella regione del collo del pancreas. </li><li> Incidere il peritoneo viscerale e il legamento gastrocolico, la concessione dell&#39;accesso al retroperitoneo, esponendo la regione del corpo e la coda del pancreas. Al fine di esporre la regione del collo, eseguire una manovra di Kocher. Sollevare il duodeno e testa diil pancreas al largo della retroperitoneo elevandoli dalla vena cava inferiore e l&#39;aorta sotto. </li><li> Posizionare con cura la spatola sotto la regione del collo. Legare il dotto pancreatico con 6-0 Prolene filo sul lato sinistro della vena porta che separa i lobi gastro-duodenale e splenica. </li><li> Eseguire una seconda legatura in prossimità della prima legatura per garantire che i lobi sono adeguatamente separati. </li><li> Legare molto attentamente in modo da non danneggiare i vasi sanguigni sottostanti, cioè l&#39;arteria pancreaticoduodenale superiore, l&#39;arteria pancreaticoduodenale inferiore e la parte pancreatica della arteria splenica. </li><li> Posizionare gli organi nella cavità addominale. </li><li> Chiudere l&#39;incisione con filo filamentosa 4-0 poliglicole in un modello sutura continua per il muscolo / strato peritoneale e in un modello di sutura discontinua per la pelle. </li></ol></li></ol> 5. Sham Chirurgia <ol><li> Eseguire tutti i passi come dedescritto nei passaggi da 1 a 4.1.4. Mentre il pancreas è esposto, non eseguire la legatura del dotto pancreatico. </li><li> Alla fine del punto 4.1.4, posizionare gli organi interni nella cavità addominale. </li><li> Chiudere l&#39;incisione come descritto in 4.2.7. </li></ol> Cura 6. post-operativa e monitoraggio <ol><li> Dopo la procedura chirurgica è completa, posizionare il mouse nella zona di recupero. Questo è costituito da una gabbia collocato su una piastra elettrica e allineato con biancheria carta in orizzontale in modo da mantenere la temperatura corporea normale. </li><li> Fornire supporto nutrizionale per evitare ipoglicemia postoperatoria dal cibo inumidito posto sul fondo della gabbia. A questo scopo, utilizzare pellet roditore dieta standard ammollata in acqua fino a quando si ammorbidiscono. </li><li> Fornire un supporto liquido dal cibo umido e fornire acqua ad libitum. </li><li> Utilizzare Buprenophine come analgesico (0,05-0,1 mg / kg) due volte al giorno per 2 giorni post-operatorio. Durante tutto sperimentale, il follow-up, observe il mouse per comparsa di eventuali segni di infezione, tra cui la secrezione di liquido o pus dalla ferita, o per deterioramento fisico caratterizzato da una riduzione nel governare il comportamento e il livello di attività, più basso appetito e perdita di peso corporeo. </li></ol> 7. Valutazione di successo PDL e Harvest del PDL Tail e Sham coda del pancreas <ol><li> Euthanize topi di dislocazione cervicale. </li><li> Re-radere regione addominale per evitare riporto di pelliccia. </li><li> Aprire lo strato di pelle e muscoli addominali e rimuovere una grande area di pelle e muscoli per ottenere un buon accesso al pancreas. </li><li> Usando pinzette sterili o tampone, lo stomaco è retratto superiormente, esponendo la milza e il lobo splenica (regione coda) del pancreas. </li><li> Estrarre delicatamente il duodeno e parte del digiuno superiore per esporre lobo gastro-duodenale (regione della testa) del pancreas. Nota: La parte legatura del pancreas Pdl ha ora ridotto in termini di dimensioni ed è diventato quasitrasparente in modo che le isole sono visibili come piccoli punti bianchi. La porzione di testa del pancreas è rosa opaco e esocrina distinto lobuli può essere osservato. </li><li> Usando forbici sterili, separare la parte di coda legatura del pancreas dalla milza, tagliando lungo la milza. Tagliare perdere il tessuto connettivo che collega la regione di coda del pancreas agli organi interni. </li><li> Tagliare regione coda PDL proprio di fronte alla legatura, escludendo la legatura e il tessuto immediatamente adiacente ad esso dal tessuto raccolto. </li><li> Per isolare il tessuto coda finzione, seguire i passaggi da 7.1 a 7.5. Utilizzando forbici sterili separano la regione coda del pancreas farsa dalla milza tagliando lungo la milza. Isolare la regione di coda tagliando nella regione del collo del pancreas e taglio perdere il tessuto connettivo che collega il pancreas coda agli organi interni Nota: Sia la coda e regione della testa del pancreas sham sono rosa opaco, per isolare due parti separatamente,tagliare il pancreas nella regione del collo. </li></ol>

<ol>
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The authors have nothing to disclose.

In the present study, we describe in detail the methodology behind PDL, a mouse injury model to study beta cell neogenesis and proliferation and transdifferentiation of pancreatic non-beta cells. Ambiguity in data on PDL among labs stimulates the need for a standardized protocol for PDL surgery.
Critical steps in the PDL protocol include the selection of healthy, young mice for surgery. Preferably male mice should be used since unpublished data from our lab suggest that estrogen receptor signa...

La crescente prevalenza del diabete, che colpisce più di 300 milioni di persone in tutto il mondo 1,2 ha favorito la ricerca di nuove fonti di cellule beta produttrici di insulina, sia in vitro che in vivo, per ricostituire la massa di cellule beta carente. 3 Identificazione chiave meccanismi e fattori che regolano la proliferazione delle cellule beta e beta neogenesi delle cellule, cioè, la differenziazione di cellule beta da una cellula cellula o progenitrici non beta, possono fornire nuovi bersagli per lo sviluppo di terapie rigenerative nel diabete. Nei sviluppo pancreas roditori, tutti i tipi di cellule endocrine differenziare da una popolazione transitoria di cellule progenitrici endocrine, che esprime il fattore di trascrizione Neurogenin3 (Ngn3). 4,5 nel pancreas roditori adulti, in condizioni fisiologiche normali, la massa delle cellule beta è mantenuta ad un numero ottimale per soddisfare esigenze metaboliche. Cambiamenti nella dimensione delle cellule beta,apoptosi e replica costituiscono i principali meccanismi di espansione delle cellule beta e turn-over. 6-8 Mentre il potenziale delle cellule beta di proliferare in condizioni fisiologiche normali è omogenea in tutta la popolazione, 9 il loro tasso di proliferazione è basso e ri-replica è limitato da un periodo di stasi dinamica o periodo refrattario 6,7, influenzato da età e il metabolismo del glucosio. 10 Dato che le cellule progenitrici endocrine non sono finora state identificate nel normale pancreas adulti, neogenesi è pensato di non contribuire alla crescita delle cellule beta adulta normale. 8 Pertanto, l&#39;identificazione di una cellula progenitrice facoltativo endocrine nel pancreas adulto che è espandibile e in grado di produrre nuove cellule beta fornirebbe un romanzo, possibilmente fonte illimitata di cellule beta. Legatura del dotto parziale (PDL) è un modello di lesione animale che è stato descritto per indurre neogenesi delle cellule betas nel pancreas adulti. 11,12 In questo modello, il dotto pancreatico principale svuotamento della coda pancreatica è chirurgicamente legatura. L&#39;ostruzione conseguente drenaggio esocrina induce rimodellamento tissutale principale, accompagnato da infiammazione e atrofia acinare distale alla legatura. 12-14 All&#39;interno di questo ambiente infiammatorio, ri-espressione del marcatore endocrino progenitore Ngn3 è indotta e il volume delle cellule beta aumenta duplice . Questo raddoppio risultati beta di volume delle cellule dalla generazione di nuove cellule beta da un Ngn3 esprimere embrionale-tipo di cellule endocrine progenitore e dalla proliferazione delle cellule beta preesistenti e di nuova costituzione che tendono a ri-duplicazione senza &quot;periodo di quiescenza&quot;. 11,15 Neogenesi delle cellule beta e la replica in modelli di pregiudizio come la pancreatectomia 6,7,16-19 e l&#39;ablazione selettiva delle cellule beta 20 sono stati ampiamente descritti. Tuttavia, il risultato rigenerativa in these modelli è influenzato dalla entità del danno inflitto ed è associata con una diminuzione della beta iniziale massa cellulare 21. PDL è un modello chirurgica in cui la massa di cellule beta iniziale non è interessato e neogenesi e la proliferazione delle cellule beta sono robustamente attivati. Infatti, nel pancreas di topi sottoposti a PDL, cellule che esprimono Ngn3 sono identificati vicino al rivestimento epiteliale del condotto. Queste cellule possono essere isolate dal pancreas legatura di Ngn3-GFP topi transgenici utilizzando la fluorescenza delle cellule attivate (FACS) e sono in grado di differenziare verso le cellule beta funzionali seguente attecchimento in e ex vivo della cultura del pancreas di E12.5 Ngn3 - / - . topi 11 Allo stesso modo, in Ngn3 CreERT;. topi R26 YFP in cui le cellule che attivano il gene Ngn3 sono etichettati in modo permanente dopo l&#39;iniezione tamoxifene, Ngn3 cellule beta cellule derivate label-positivi vengono rilevati dopo Pdl 15 Inoltre, le cellule beta di nuova formazione diluire pre -existing cellule beta e preferenzialmente localizzare in piccoli isolotti entro cui le cellule beta mostrano un alto potenziale di proliferazione. 15 Ngn3 è importante per l&#39;espansione delle cellule beta dopo PDL da ridotta espressione Ngn3 utilizzando RNA breve tornante specifici target diminuisce significativamente la massa delle cellule beta e la proliferazione delle cellule beta dopo PDL. 11 In particolare, la frazione di cellule beta cellule derivate Ngn3 e la massa delle cellule beta dopo PDL dipende criticamente dal livello di Ngn3 induzione 15. Ciò è in accordo con l&#39;osservazione che alto livello di espressione Ngn3 è un passaggio fondamentale per l&#39;impegno endocrino da progenitori pancreatici multipotenti durante lo sviluppo del pancreas 22 Inoltre, ablazione selettiva di Ngn3 cellule che esprimono di amministrazione di tossina difterica per Ngn3 CreERT;. R26 IDTR topi risultati in contenuto di insulina diminuita e ridotta proliferazione delle cellule beta, soprattutto nei piccoli isolotti. 15 <pclass = &quot;jove_content&quot;&gt; Anche se l&#39;induzione dell&#39;espressione Ngn3 in cellule del dotto dopo Pdl è stata confermata da molti 11,15,16,23,24, espressione Ngn3 nelle cellule isolotti 24,25 e discrepanze in esito del Pdl sfidato nostre osservazioni iniziali di una maggiore massa di cellule beta 26,27, comparsa di Ngn3 esprimere condotto di derivazione progenitori endocrini 24,26,28,29, e un aumento della proliferazione delle cellule beta 27 dopo Pdl. 30 Questi risultati contrastanti potrebbero, almeno in parte, essere attribuito ad una combinazione di fattori, tra variazioni di punti di tempo post-chirurgiche di analisi, peso corporeo, il sesso e l&#39;età dei topi, condizioni fisiologiche e ambientali post-operative e, soprattutto, differenze di tecnica chirurgica. 30 Nelle nostre mani, la proliferazione delle cellule beta, il contenuto di insulina, il volume delle cellule beta e il numero di piccoli isolotti sono costantemente aumentati dopo Pdl. AncheNgn3 mRNA aumenta costantemente, ma ci sono grandi differenze nell&#39;espressione Ngn3 mRNA tra pancreas coda PDL, per i quali non abbiamo alcuna spiegazione diretta. Abbiamo ipotizzato che il livello di mRNA Ngn3 potrebbe correlare con il grado di neogenesi delle cellule beta da cellule non-beta 15, ma questo necessita di ulteriori fondatezza. Anche se non rimuove tutte le varianti sperimentali, un metodo standard per l&#39;esecuzione di un intervento chirurgico PDL consente una migliore uniformità dei risultati e apre nuove strade per lo studio della proliferazione delle cellule beta e neogenesi.

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			<td>Supplies for preparation area&#160;</td>
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			<td></td>
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			<td>Hibiscrub&#160;</td>
			<td>Regent Medical</td>
			<td>5601IE5F11</td>
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			<td>Ketamin</td>
			<td>Ceva</td>
			<td>BE-V202526</td>
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			<td>Rompun(2%) (Xylazin)</td>
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			<td>Alcon</td>
			<td>34335-8</td>
			<td>ointment for eyes</td>
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			<td>Leica Operating microscope</td>
			<td>Leica</td>
			<td>10446320</td>
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			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools (FST)</td>
			<td>18000-45</td>
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			<td>autoclaved surgical instruments</td>
			<td>Fine Science Tools (FST)</td>
			<td></td>
			<td></td>
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			<td>Recirclulating water heating pad</td>
			<td>Gaymar Industries, Inc.</td>
			<td>TP702</td>
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			<td>Adhesive OP-towel</td>
			<td>BARRIER</td>
			<td>706500-07</td>
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			<td>Stella 3/5 Compresse de gaze (Sterile)</td>
			<td>Lohmann&#38;Rauscher International</td>
			<td>35968</td>
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			<td>Mini Plasco 0,9% NaCl solution</td>
			<td>B.BRAUN</td>
			<td>3521680</td>
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			<td>6-0 prolene suture</td>
			<td>Ethicon</td>
			<td>8706H</td>
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			<td>4-0 polyglycol suture</td>
			<td>Ethicon</td>
			<td>SL-607</td>
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			<td>Paper bedding (paper towel)</td>
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			<td>BE-V342955</td>
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			<td>Materials for analysis</td>
			<td></td>
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			<td></td>
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			<td>Taqman Ngn3 primer</td>
			<td>Integrated DNA technologies</td>
			<td>Mm.PT.56a.33574796.gs</td>
			<td></td>
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		<tr>
			<td>Taqman CycloA primer</td>
			<td>Integrated DNA technologies</td>
			<td>Mm.PT.39a.2.gs</td>
			<td></td>
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			<td>anti-Rabbit Cleaved Caspase 3 antibody (D175)</td>
			<td>Cell Signaling</td>
			<td>5A1E</td>
			<td></td>
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			<td>anti-mouse/rat Ki67 antibody</td>
			<td>eBioscience</td>
			<td>14-5698-82</td>
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		<tr>
			<td>monoclonal anti-actin alpha-Smooth muscle-Cy3 (mouse)</td>
			<td>Sigma</td>
			<td>085K4889</td>
			<td></td>
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			<td>anti-rat Cytokeratin 19&#160;</td>
			<td>DSHB</td>
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			<td>monoclonal Mouse anti-BrdU</td>
			<td>Dako</td>
			<td>M0744</td>
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			<td>Hoechst</td>
			<td>Sigma</td>
			<td>33342</td>
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surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation

L&#39;espansione delle cellule beta pancreatiche in vivo o ex vivo, o la generazione di cellule beta per il differenziamento di una cellula staminale embrionale o adulto, in grado di fornire nuove fonti espandibili di cellule beta per alleviare la scarsità dei donatori nel trapianto di isole umana come terapia per il diabete. Nonostante i recenti progressi sono stati compiuti verso questo obiettivo, i meccanismi che regolano la crescita delle cellule beta e la differenziazione da cellule staminali / progenitrici devono ancora essere caratterizzato. Qui, descriviamo un protocollo per un modello di ferita nel topo pancreas adulto che può funzionare come strumento per studiare i meccanismi di rimodellamento del tessuto e la proliferazione delle cellule beta e la differenziazione. Legatura del dotto parziale (PDL) è una lesione indotta sperimentalmente del pancreas roditore coinvolge legatura chirurgica del dotto pancreatico principale con conseguente ostruzione del drenaggio di prodotti esocrine dalla regione coda del pancreas. Il danno inflitto induce atrofia acinare, infilt cellule immunitarierazione e severo rimodellamento tissutale. Abbiamo già riferito l&#39;attivazione di neurogenina (NGN) 3 esprimono cellule progenitrici simili endogene e un aumento della proliferazione delle cellule beta, dopo Pdl. Pertanto, PDL fornisce una base per studiare segnali coinvolti nella dinamica cellule beta e le proprietà di un progenitore endocrina pancreatica adulta. Dal momento che, resta ancora in gran parte poco chiaro, quali fattori e percorsi contribuiscono alla neogenesi delle cellule beta e la proliferazione in PDL, un protocollo standardizzato per PDL permetterà confronto tra laboratori.

PDL induce atrofia acinare e infiammazione, ma non influenza il peso corporeo e la glicemia In otto settimane vecchio topi maschi BALB / c, la conduttura di scarico gli enzimi esocrine della coda del pancreas è legatura mentre la testa del organo, che si trova adiacente allo stomaco e del duodeno, rimane inalterata. L&#39;età, il sesso e topi maschi BALB / c peso di pari subiscono chirurgia farsa ricapitolando tutte le fasi di un intervento chirurgico condotto legatura par...

Watch this Scientific Journal Video about Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation at JoVE.co

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Lesioni chirurgica al pancreas mouse attraverso la legatura del Dotto pancreatico come un modello per Endocrine e Esocrine riprogrammazione e la proliferazione

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

surgical-injury-to-mouse-pancreas-through-ligation-pancreatic-duct-as

Research

JoVE Journal

Medicine

16.0K Views.  Vrije Universiteit Brussel. This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation. 

Video: Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Un modello murino di ischemia miocardica e riperfusione lesioni da legatura della arteria discendente anteriore sinistra

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Ricerca

Medicina

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Bile Duct Ligation in topi: Induzione infiammatoria epatica Lesioni e fibrosi da colestasi ostruttiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Infusione del dotto pancreatico: un metodo efficace e selettivo di somministrazione di farmaci e virus

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissezione del Pancreas del Mouse per l'analisi istologica e Profiling metabolico

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Pancreaticojejutostomia laterale robotica per la pancreatite cronica

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Laparoscopica Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Un modello di trapianto di polmone di ratto di ischemia calda / danno da riperfusione: ottimizzazioni per migliorare i risultati

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Visualizzazione longitudinale stabilizzata a livello cellulare in vivo del pancreas in un modello murino con una finestra di imaging intravitale pancreatico

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

Creazione di un modello di pancreatite acuta grave del topo mediante iniezione retrograda di taurocolato di sodio nel dotto biliopancreatico

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Legatura parziale del nervo sciatico: un modello murino di dolore neuropatico cronico per studiare l'effetto antinocicettivo di nuove terapie

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...