Questa ricerca è sostenuta dalla Fondazione di Tom Peterson e NIH concedere R01-HL119780 (Jain, MK).

protocollo animale è stato esaminato e approvato dal comitato di uso presso Case Western Reserve University School of Medicine e istituzionali Animal Care ed è stato condotto in conformità con la guida del National Institutes of Health (NIH) per la Cura e l'uso degli animali da laboratorio (8 ° edizione, 2011). Topi sono stati eutanasizzati dai metodi compatibile con l'associazione medica veterinaria americana (AVMA) linee guida per l'eutanasia degli animali (edizione 2013). C57BL/6J, Maschio, 8-16 settimana vecchi topi sono stati usati in questo protocollo. Topi sono stati alloggiati in un ambiente buio/luce ciclo di 12 h.
 1. preparazione pre-operatoria <ol><li>topi di trasferimento per una gabbia pulita 24 h prima della procedura. Gli animali ammessi libero accesso all'acqua. Sostituire il chow cibo con cibi morbidi (gel integratore alimentare) 4-6 h prima della chirurgia per ridurre il contenuto dell'intestino tenue per chirurgia.</li>
	<li>Sterilizzare tutti gli strumenti chirurgici e preparare i materiali chirurgici sterili monouso. Pulire l'area di chirurgia e il cono di naso anestetico con etanolo al 70%.</li>
	<li>Impostare un microscopio di dissezione, vaporizzatore anestetico isoflurano, strumenti, e di un temperatura controllata piccolo animale tavolo operatorio per manutenzione di temperatura del corpo. Organizzare gli strumenti, suture e siringhe in una posizione per il libero accesso durante l'intervento chirurgico.</li>
	<li>Impostare una fonte di luce per fornire abbastanza luce per l'area chirurgica.</li>
	<li>Preparare soluzione fisiologica 0,9% sterile nella siringa da 5 mL per la pulizia intestinale e addominale.</li>
</ol> 2. Ileectomy e l'anastomosi <ol><li>Anesthetize topi con isoflurano (2-3%) in una camera di incubazione animale piccolo. Determinare adeguate amputate utilizzando la tecnica del pizzico di punta, mentre l'animale è il isoflurane.</li>
	<li>Rimuovere capelli addominali applicando il prodotto di rimozione dei capelli e pulire capelli distanza utilizzando spugne chirurgiche, mantenendo l'anestesia. Posizionare il mouse su un temperatura controllata piccolo animale tavolo chirurgico ( Figura 1 A) per mantenere la temperatura corporea presso l'anestesia Maintain a 37 ° C. con isoflurano (1-2%) attraverso una maschera facciale. Trattare gli occhi del mouse con unguento oculare.</li>
	<li>Pulire la pelle con povidone-iodio e 70% di alcol e coprire l'area chirurgica dell'addome con garza chirurgica sterile ( Figura 1 B).</li>
	<li>Fare un'incisione addominale del midline usando un bisturi chirurgico una volta che l'anestesia è in vigore. Usare un applicatore con punta di cotone per proteggere il muscolo addominale del mouse intestino e tirare con Divaricatori per completamente aprire ed esporre la cavità addominale ( Figura 1 C).</li>
	<li>Individuare l'intestino cieco. A partire dall'intestino cieco, spostare con cautela l'ileo collegato e parte del digiuno dalla cavità addominale ( Figura 1 D). 
	Nota: L'intestino cieco possa essere facilmente identificato grazie alle sue grandi dimensioni anche dopo digiuno.</li>
	<li>Legare il ramo superiore dell'arteria mesenterica superiore con una sutura in seta 7-0 per occludere il sangue fornendo il segmento ileale che deve essere asportato. Il colore ileale cambia dal rosa al viola scuro dopo la legatura. ( Figura 1 -E - F).</li>
	<li>a seconda dello scopo dell'esperimento, usando le forbici, asportare e rimuovere il 50% o 90% dell'ileo. 
	Nota: Per la chirurgia di sham, non eseguire la legatura dell'arteria mesenterica superiore e non rimuovono l'ileo.</li>
	<li>Irrigare il lume di entrambe le estremità ileale con soluzione fisiologica allo 0,9%. 
	Nota: come ileo intatto restante sta ancora ricevendo un rifornimento di anima normale dall'arteria mesenterica superiore, una piccola quantità di sangue verrà svuotata durante il processo. Questo indica anche che l'afflusso di sangue alle estremità ileale è normale e non assicura ischemia durante la procedura di anastomosi ( Figura 1 G).</li>
	<li>Individuare i mesenteries sul lato di entrambe le estremità ileale. Allineare i mesenteries e suturare le estremità ileale insieme usando 8-0 sutura ( Figura 1 G - H).</li>
	<li>Sutura sul lato controlaterale dell'ileo per mantenere l'ileo anastomizzato in maniera naturale ( Figura 1 ho).</li>
	<li>Suturare i lati superiori e inferiori tra le due suture originale a accuratamente unire le due ileale estremità insieme ( Figura 1, J).</li>
	<li>Confermare che non esiste nessuna perdita dal sito anastomosi dopo aver terminato la procedura di suturazione di tre fasi ( Figura 1 G-io). Restituire l'intestino cieco e piccolo intestino nella cavità addominale nella posizione anatomica originale. Lavare l'area operata con soluzione fisiologica 0,9% caldo utilizzando un ago smussato. ( Figura 1 K).</li>
	<li>Chiudere l'incisione dello strato muscolare addominale con sutura 6-0. Allineare l'incisione addominale pelle usando il forcipe e suturare la pelle addominale per facilitare la cicatrizzazione ottimale ( Figura 1 L).</li>
</ol> 3. Cure post-operatorie topi <ol><li>trasferimento post-chirurgia per un'unità di cure intensive per il recupero. Ospitarli in una gabbia di lettiera in carta su un rilievo di riscaldamento a temperatura controllata per continuare il recupero post-operatorio durante la notte. Topi con cibi morbidi, oltre al normale cibo e acqua di alimentazione.</li>
	<li>Amministrare buprenorfina (0,05 - 0,1 mg/kg) con iniezione sottocutanea ogni 8-12 h per analgesia. 
	Nota: Eutanasia topi di CO 2 se gravemente malato.</li>
	<li>Nel punto finale, sacrificare topi utilizzando overdosed isoflurano e raccolto campioni come necessario (sezione 4).</li>
</ol> 4. Valutazione di Overaccumulation di Bile indotta da Ileectomy <ol><li>pesare e sezionare i topi un giorno dopo una resezione di 0% (falsità), 50% o 90% dell'ileo.</li>
	<li>Rimuovere il tratto GI e pesarlo. Calcolare il peso di GI al rapporto di peso del corpo per valutare la severità di malassorbimento di sali biliari e overaccumulation.</li>
	<li>Trasferire i tratti di GI in provette coniche da 15 mL e tagliarli in segmenti più brevi utilizzando le forbici. Dopo il taglio, centrifuga a 3.000 x g per 10 min. trasferire il surnatante (GI fluido contenente sali biliari) in una provetta pulita.</li>
	<li>Misurare il volume totale e il peso del fluido di GI e calcolare il peso di fluido al rapporto di peso del tratto GI per valutare ulteriormente overaccumulation di bile nel tratto GI.</li>
	<li>Determinare la quantità totale di bile nel surnatante dall'analisi degli acidi biliari, come descritto nel riferimento 8.</li>
</ol>

<ol>
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Al fine di eseguire un ileectomy successo, arteria mesenterica superiore deve essere legato in anticipo per bloccare l'afflusso di sangue al segmento resecting. Il segmento ileale ischemico diventerà viola scuro dopo la legatura. Il segmento ileale quindi dovrà essere completamente resecato. Un rifornimento di anima normale deve essere garantito per le estremità mantenute. Questo è essenziale per evitare la rimozione impropria e sanguinamento, che può facilmente causare errori chirurgici a causa di necrosi ischemica...

Nella moderna ricerca biomedica, modelli animali geneticamente manipolati sono ampiamente utilizzati per raccogliere intuizioni in malattie umane. In particolare, tessuto o cellula-specifico funzioni di guadagno e perdita di geni sono state utilizzate per studiare la regolazione molecolare così come gli effetti biologici indotti. Nonostante i progressi nel manipolare geni bersaglio in vivo, non ci sono limitazioni persistente. Primo, molte delle cellule o del tessuto specifiche delezioni interesserà gli organi multipli. Ad esempio, omissione del gene epiteliali eliminerà espressione negli epiteli dei tessuti multipli. Ulteriormente, anche se l'eliminazione è limitato ad un tessuto specifico, controllo spaziale è raramente realizzabile. Per esempio, in un tessuto come l'intestino, segmenti distinti svolgere funzioni molto specifiche che non possono essere manipolate con precisione in vivo. In queste situazioni, resezione dei tessuti contenenti gene è considerata come un approccio più efficiente di studio per determinare il significato meccanicistico e funzionale di comunicazione del tessuto.
Ileectomy è principalmente usato in pazienti con Crohn e malattie infiammatorie che coinvolge l'ileo distale 1,2,3. L'ileo in genere produce diversi ormoni di deposito di energia come fattore di crescita fibroblastico 15/19 (FGF15/19), peptide YY (PYY) e peptide glucagone-simile 1/2 (GLP1/2); questi ormoni svolgono i ruoli importanti locali ed endocrini in molte funzioni biologiche4,5,6. Tra questi ormoni, FGF15 è stato identificato come un robusto endocrino inibitore della sintesi degli acidi biliari nel fegato. Una volta riassorbito nel enterocytes ileale, acidi biliari attivano il recettore di farnesoid X di recettori nucleari (FXR) stimolare l'espressione di Fgf15 , che successivamente conduce ad inibizione di risposte di sintesi di acidi biliari epatici 7. In uno studio recente, abbiamo introdotto il modello di ileectomy di topo per studiare il fattore kruppel-like ileale 15 (KLF15) -Fgf15 segnalazione asse che regola la produzione circadiana dell'acido di bile nel fegato 8. La cosa più importante, abbiamo introdotto una nuova famiglia, i fattori di kruppel-like, specialmente KLF15, in biologia degli acidi biliari. Basato su studi funzionali, compresa la chirurgia ileectomy, abbiamo determinato quella sintesi di acidi biliari sopraregola KLF15 tramite un meccanismo non-epatico indiretto. Infine, KLF15 ileale è anche identificato come il primo regolatore endogeno negativo di Fgf15.
I segmenti intestinali discendente da prossimale a distale regioni sono responsabili di assorbimento dei diverse nutrienti. L'ileo è il segmento principale responsabile degli acidi biliari e della vitamina B12 (VB12) assorbimento 9. Un precedente studio ha impiegato un modello murino di resezione dell'intestino prossimale per studiare la sindrome da intestino corto; vari tipi di sutura, diete e lunghezze di resezione sono stati proposti per mantenere un tasso di sopravvivenza post-operatorio ottimale 10. Inoltre, una revisione più recente indica che la resezione ileale in genere comporta la malattia più severa rispetto altre resezioni di segmento gastrointestinale (GI) a causa della diminuita capacità di adattamento del rimanente tratto 11. Questo argomento ha guadagnato intensivi interessi di gruppi di ricerca clinica e di base, mentre la comprensione del recupero e gli approcci terapeutici efficaci sono ancora limitate.
Acidi biliari diarrea risultati da squilibri nell'omeostasi dell'acido di bile nella circolazione enteroepatica 12,13. Può essere una conseguenza della resezione ileale, malattia gastrointestinale o un risultato di malassorbimento idiopatico dell'acido di bile. Più dell'80% dei pazienti sono stati trovati per presentare con diarrea dopo avere subito la resezione ileale 14. Ileectomy ha il potenziale per essere un modello di intervento chirurgico importante per l'indagine di acidi biliari diarrea. In questo studio, una serie di resezioni ileali forniscono una valutazione gradiente di carenza di FGF15 così come malassorbimento intestinale di sali biliari, overaccumulation e danni tossici.

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	<tbody>
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			<td height="21" style="width:248px;height:21px;">Dissection microscope</td>
			<td style="width:195px;">Olympus</td>
			<td style="width:97px;">SZ61</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Animal temperature controller</td>
			<td style="width:195px;">Physitemp Instruments, Inc.</td>
			<td style="width:97px;">TCAT-2LV</td>
			<td style="width:183px;">For body temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Isoflurane anesthetic vaporizer</td>
			<td style="width:195px;">VetEquip</td>
			<td style="width:97px;">&#160;911104</td>
			<td style="width:183px;">For anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Dissection forceps</td>
			<td style="width:195px;">&#160;Fine Science Tools, Inc.</td>
			<td style="width:97px;">11274-20</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Scissors</td>
			<td style="width:195px;">&#160;Fine Science Tools, Inc.</td>
			<td style="width:97px;">14084-08</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Needle holder</td>
			<td style="width:195px;">&#160;Roboz Surgical Instrument Co.</td>
			<td style="width:97px;">RS-7882</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Micro knives-needle blade</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:97px;">10318-14</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">6-0 monofilament suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">1698G</td>
			<td style="width:183px;">For abdominal skin closure</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">7-0 silk suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">766G</td>
			<td style="width:183px;">For ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">8-0 monofilament suture</td>
			<td style="width:195px;">Ethicon</td>
			<td style="width:97px;">1714G</td>
			<td style="width:183px;">For anastomosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Surgical sponges</td>
			<td style="width:195px;">Dynarex Corp.</td>
			<td style="width:97px;">3333</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Small cotton-tipped applicators&#160;</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:97px;">23-400-118</td>
			<td style="width:183px;">For surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Isoflurane</td>
			<td style="width:195px;">Piramal Healthcare Limited</td>
			<td style="width:97px;">66794-013-25</td>
			<td style="width:183px;">For anasthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Buprenorphine hydrochloride</td>
			<td style="width:195px;">Reckitt-Benckiser Pharmaceuticals</td>
			<td style="width:97px;">12496-0757-1</td>
			<td style="width:183px;">For analgesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">0.9% sodium chloride Injection</td>
			<td style="width:195px;">B. Braun Medical Inc.</td>
			<td style="width:97px;">0264-7800-10</td>
			<td style="width:183px;">For washing/injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Povidone iodine prep solution</td>
			<td style="width:195px;">Dynarex Corp.</td>
			<td style="width:97px;">1413</td>
			<td style="width:183px;">For skin preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Puralube vet ointment</td>
			<td style="width:195px;">Dechra Veterinary Products</td>
			<td style="width:97px;">17033-211-38</td>
			<td style="width:183px;">For eye pretection</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Hair remover lotion</td>
			<td style="width:195px;">Church &#38; Dwight Co., Inc.</td>
			<td style="width:97px;"></td>
			<td style="width:183px;">For skin preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Intensive care unit</td>
			<td style="width:195px;">ThermoCare</td>
			<td style="width:97px;">FW-1</td>
			<td style="width:183px;">For post-surgery recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">DietGel recovery</td>
			<td style="width:195px;">ClearH2O</td>
			<td style="width:97px;">72-06-5022</td>
			<td style="width:183px;">For post-surgery recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Aurum total RNA fatty and fibrous tissue kit</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:97px;">7326830</td>
			<td style="width:183px;">For RNA isolation</td>
		</tr>
		<tr height="31">
			<td height="31" style="width:248px;height:31px;">iScript reverse transcription supermix for RT-qPCR</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:97px;">1708841</td>
			<td style="width:183px;">For reverse transcription assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">TaqMan fast advanced master mix</td>
			<td style="width:195px;">Applied Biosystems/Life Technologies</td>
			<td style="width:97px;">4444965</td>
			<td style="width:183px;">For QPCR analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">Total bile acid assay kit</td>
			<td style="width:195px;">Genzyme Diagnostic</td>
			<td style="width:97px;">DZ042A-K01</td>
			<td style="width:183px;">For bile acid assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:248px;height:21px;">C57BL/6J&#160;</td>
			<td style="width:195px;">The Jackson Laboratory</td>
			<td style="width:97px;"></td>
			<td style="width:183px;"></td>
		</tr>
	</tbody>
</table>

ileectomy-induced bile overaccumulation in mouse intestine

La resezione intestinale è un comune approccio terapeutico per le malattie umane quali l'obesità, le malattie infiammatorie intestinali, morbo di Crohn e tumore del colon che si traduce spesso in effetti avversi di intestino corto grave sindrome-come compreso diarrea degli acidi biliari, disidratazione, alterazioni elettrolitiche e malassorbimento dei nutrienti. Qui vi presentiamo un modello murino di resezione ileale, chiamato ileectomy, per valutare la comunicazione del tessuto e il mantenimento dell'omeostasi sistemica. Dopo resezione ileale, sangue circolante è permanentemente priva il fattore di crescita del fibroblasto ileo-specifico ormone endocrino 15 (FGF15), che rilascia la sua endocrine inibizione della sintesi degli acidi biliari nel fegato. In combinazione con l'aumento della produzione e abolita riassorbimento degli acidi biliari dopo aver rimosso l'ileo, topi che ha subito l'ambulatorio soffrono overaccumulation sale biliare nell'intestino e la diarrea associata, la morbosità e la mortalità. Romanzo l'utilizzo del modello chirurgia introdotto in questo studio può fornire le comprensioni meccanicistiche e funzionale nel controllo ileale di regolazione metabolica sistemica nella fisiologia e nella malattia.

Le procedure per ileectomy sono illustrate nella Figura 1. Il primo passo include preparazione pelle addominale del topo, facendo un'incisione addominale e l'utilizzo di Divaricatori per esporre completamente l'intestino (Figura 1A-C). Successivamente, si trovava l'intestino cieco del mouse (Figura 1D); dato che la sua forma e dimensione lo rendono un punto di riferi...

Watch this Scientific Journal Video about Ileectomy-induced Bile Overaccumulation in Mouse Intestine at JoVE.com

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Riassorbimento degli acidi biliari nell'intestino tenue-dipendente e feedback inibizione della sintesi dell'acido di bile epatica è importante per la salute e l'omeostasi sistemica. In questo studio, descriviamo un modello murino per resezione ileale valutare il malassorbimento indotta da ileectomy bile, overaccumulation e tossicità nell'intestino del mouse.

Overaccumulation ileectomy-indotta della Bile nell'intestino del Mouse

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

ileectomy-induced-bile-overaccumulation-in-mouse-intestine

Research

JoVE Journal

Medicine

9.4K Views.  Case Western Reserve University. Riassorbimento degli acidi biliari nell'intestino tenue-dipendente e feedback inibizione della sintesi dell'acido di bile epatica è importante per la salute e l'omeostasi sistemica. In questo studio, descriviamo un modello murino per resezione ileale valutare il malassorbimento indotta da ileectomy bile, overaccumulation e tossicità nell'intestino del mouse.

Video: Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Iperinsulinemico-euglicemico Morsetti in Cosciente, Mouse sfrenato

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Ricerca

Medicina

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

3-Dimensional Casting resina e imaging di Vena mouse portale o sistema biliare intraepatica Duct

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un metodo semplice di intubazione mouse polmone

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Aspetti tecnici della fistola aorto-cavale mouse

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria intrauterina misurare mouse contrattile Pressione<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Un dispositivo semplice da preparare rapidamente intere Monti della Intestino mouse

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Utilizzando Multi-fluorurati biliari acidi e<em&gt; In Vivo</em&gt; Risonanza Magnetica misurare Bile Acid Trasporti

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

Meccanismi di secrezione dell'ormone intestinale utilizzando l'isolato irrorati intestino tenue del ratto

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Overaccumulation ileectomy-indotta della Bile nell'intestino del Mouse

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