Funding from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) is gratefully acknowledged.

The authors have nothing to disclose.

Il modello di ratto mesenterica linfa incannulamento consente la quantificazione diretta della concentrazione e la velocità di trasporto di varie cellule e molecole (come lipidi e farmaci) da l&#39;intestino nella linfa e le modifiche a questi che si verificano in risposta a sfidare di varie sostanze (dieta , antigene, farmaci, formulazioni, ecc) 10,27 e la malattia (cancro, virus, colite, insulino-resistenza, etc.) 5-7. I componenti che sono raccolti nella linfa possono essere u...

La linfa fluisce dal piccolo intestino attraverso un processo unidirezionale che origina a singoli lacteals che sono contenuti all&#39;interno di ogni piccola villi intestinali 1. Lacteals sono relativamente permeabile ai fluidi, macromolecole e cellule e la formazione della linfa quindi inizia con l&#39;ingresso di questi fattori in lacteals. La linfa iniziale nei lacteals scorre successivamente dall&#39;intestino tramite una rete di microvasi linfatici, raccolta (afferente) vasi linfatici, una serie di linfonodi mesenterici e, infine, i post-nodale (efferenti) vasi linfatici. Nei nodi, linfa passa attraverso una serie di seni midollari dove avviene lo scambio con le cellule immunitarie nodo residenti nonché materiale che entrano nodo dal sangue. Tutte linfa scorre dal piccolo intestino eventualmente converge nel dotto efferente linfa mesenterica superiore e successivamente il chyli cisterna. Il chyli cisterna raccoglie anche linfodrenante tessuti periferici caudali, intestinale, regioni epatiche e lombari e si unisce al dotto linfatico toracico con la linfa del mediastino e parti del cranio del corpo. Il dotto linfatico toracico svuota linfa direttamente nel sistema venoso a livello della giunzione delle vene giugulari e succlavia sinistra interna. Il protocollo qui descritto, che consente la raccolta di linfa direttamente dal condotto mesenterico linfatico superiore, facilita quindi l&#39;analisi di vari fattori che transitano direttamente dall&#39;intestino al (generale) circolazione sistemica attraverso il sistema linfatico intestinale. Le principali funzioni fisiologiche assegnate al sistema linfatico intestinale sono di mantenere l&#39;equilibrio dei fluidi, per facilitare l&#39;assorbimento dei lipidi e molecola lipofila, e per consentire opportune risposte immunitarie 1. Le cellule tumorali e virus si propagano anche attraverso i vasi linfatici intestinali 2-4 e principali cambiamenti si verificano all&#39;interno dei vasi linfatici in diverse patologie infiammatorie e metaboliche 5-7. Lattinanulation del condotto linfatico mesenterico per raccogliere linfa nel mesentere consente un&#39;analisi del flusso di fluido di massa attraverso i vasi linfatici intestinali e quantificazione del tasso di concentrazione e trasporto di varie cellule e molecole. Modifiche alla concentrazione o di transito di questi fattori in risposta alle varie sfide (ad esempio, diete, antigeni, farmaci) e in modelli di malattia (ad esempio, colite, HIV, diabete) possono anche essere valutate. Sebbene sia impossibile descrivere ampiamente ogni componente linfa che possono essere analizzati e confrontati qui, linfonodi mesenterici consiste semplicisticamente di acquosa, lipidi e fasi cellulari. I componenti di interesse nella fase acquosa includono peptidi e proteine, come antigeni o tolerogens 8, messaggeri del sistema immunitario come le citochine e mediatori dei mastociti 9, e mediatori metaboliche come incretine 10. La frazione cellulare del mesenterico linfatico post-nodale consiste quasi interamente (oltre il 99%) di lymphocytES 11. Varie cellule immunitarie (cellule dendritiche, mastociti, ecc) entrano nei vasi linfatici mesenterici pre-nodali, ma rimangono all&#39;interno del nodo 12. Se le cellule all&#39;interno afferente linfa sono di interesse, è possibile raccogliere queste cellule mediante la rimozione della linfa mesenterica nodi diversi giorni prima cannulazione del condotto linfatico mesenterico 12. In questo modo i condotti linfatici afferenti ed efferenti sono direttamente collegati e le cellule linfatici afferenti linfa passano direttamente nel condotto linfa mesenterica. Il transito e il fenotipo delle varie cellule immunitarie che passano attraverso i vasi linfatici intestinali possono quindi essere esaminati. Forse il motivo più comune citato per la raccolta della linfa mesenterica fino ad oggi, tuttavia, è quello di studiare la trasformazione intestinale, l&#39;assorbimento e il trasporto dei lipidi alimentari e molecole lipofile 10. Dopo ingestione, lipidi alimentari sono digeriti (ad esempio, da trigliceridi di acidi grassi e monogliceridi, phospholipid di acidi grassi e lysophospholipid e esteri del colesterolo ad acidi grassi e colesterolo, ecc) e dispersi nel lume intestinale in piccola micelle e strutture vescicolari tramite l&#39;aggiunta di anfifili di bile (fosfolipidi, sali biliari e colesterolo) e l&#39;azione di enzimi pancreatici 10,13. Da qui essi vengono assorbiti nel enterociti. Una parte dei componenti sono assorbiti riesterificati per formare trigliceridi, fosfolipidi e gli esteri del colesterolo nelle cellule assorbenti (enterociti). Questi lipidi riesterificati sono costituiti da una combinazione di esogenamente ingerito componenti lipidici e dei componenti lipidici endogeni dalla bile secreta, piscine lipidici mucosa intestinale o fornitura di sangue 13. Da qui i lipidi esterificati o sono memorizzate all&#39;interno di enterociti o assemblati in lipoproteine ​​intestinale (chilomicroni, lipoproteine ​​a bassissima densità (VLDL)) insieme a varie apoproteine ​​e altre molecole lipofile ( <em&gt; per esempio, vitamine) 10,13. Dopo l&#39;uscita enterociti, lipoproteine ​​sono specificamente trasportati dall&#39;intestino nella circolazione sistemica attraverso il sistema linfatico mesenterico le lacteals intestinali sono più permeabili al loro ingresso di capillari sanguigni intestinali. Una parte dei componenti lipidici assorbiti vengono anche trasportati dall&#39;intestino alla circolazione sistemica attraverso i capillari sanguigni e della vena porta, come singolo, non-lipoproteine ​​associati, molecole 14. In generale, tuttavia, la via di trasporto vena porta è solo un operatore significativo l&#39;assorbimento dei lipidi lunghezza della catena corta e media. La raccolta della linfa mesenterica consente quindi la valutazione del trasporto delle lipoproteine ​​e componenti associati (lipidi, molecole lipofile, apo-proteine) dall&#39;intestino. Le lipoproteine ​​possono essere quantizzati e caratterizzati con il vantaggio che lipoproteine ​​linfonodi mesenterici, in generale, sono in un nascenStato t poiché non sono stati ampiamente modificati da enzimi sistemici come lipoproteina lipasi 15. Mentre la linfa mesenterica modello di ratto cannulato è forse storicamente più ampiamente descritto per l&#39;analisi del trasporto di lipidi / lipoproteine ​​dall&#39;intestino, una zona di espansione interesse è il ruolo dei linfatici nel trasporto di farmaci lipofili, profarmaci e altri xenobiotici 13,16 che è al centro del modello qui descritto. Farmaci lipofili (generalmente quelli con log P&gt; 5 e solubilità in catena lunga trigliceridi&gt; 50 mg / g, anche se eccezioni sono evidenti) 17,18, profarmaci 19 e altri xenobiotici 13,16 può accedere ai vasi linfatici intestinali passivamente o da integrando attivamente in lipoproteine ​​intestinale vie di trasporto 19. Il ratto linfa mesenterica tecnica incannulamento ha quindi molte applicazioni. Bollman et al. Primo descrisse una technique per cannulare condotto linfa mesenterica nei ratti nel 1948 20. Da allora una serie di variazioni sul modello sono state descritte. Ad esempio, la raccolta si può verificare quando il ratto è anestetizzato con diversi anestetici 21,22, o in stato cosciente, mentre trattenuto 15 o liberamente in movimento 23,24. I ratti possono essere somministrati diverse soluzioni reidratanti e di altre sostanze come i lipidi e le formulazioni di farmaci a prezzi diversi nello stomaco, l&#39;intestino o parenterale (in genere 0-5 ml / h) 25. In alcuni studi dotto linfatico toracico piuttosto che condotto linfatico mesenterico è cannulati stimare trasporto dall&#39;intestino attraverso i vasi linfatici anche se questo può sovrastimare transito dal piccolo intestino, a seconda del fattore di interesse, come il dotto linfatico toracico riceve anche linfatico da altri regioni 22,26. Modelli incannulamento linfonodi sono stati descritti in numerose altre specie, tra cui topi 15,27, mini-pigs 12, pecore 28,29, maiali e cani 30 31. Tuttavia, il modello di ratto è il più ampiamente e costantemente citata. Protocolli dettagliati per l&#39;incannulamento della condotta della linfa mesenterica seguita dalla raccolta di linfa in conscia 25 o anestetizzato 22 ratti e topi 15,27 sono stati pubblicati in precedenza e il lettore interessato è rivolta a questi protocolli. Questo protocollo è il primo a dimostrare la tecnica in un formato visualizzato. La linfa cannulata modello murino ha dei vantaggi rispetto ai modelli animali di maggiori dimensioni in termini di costi, la facilità di chirurgia e le considerazioni etiche. Rispetto al modello di topo, mesenterica chirurgia incannulazione linfatico è anche più facile nel ratto, anche se il modello del mouse consente studi più approfonditi sugli animali transgenici 27. Tuttavia, ci sono alcune limitazioni del modello di ratto, in particolare quelle legate a differenze nella fisiologia, tale limite extrapolatione ad altre situazioni pre-clinici e clinici. Ad esempio, nel flusso di ratto biliare è costante ed indipendente dall&#39;assunzione di cibo che negli alimenti specie superiore o lipidi stimolano flusso biliare 32. Questo crea problemi per ottenere rappresentanza ambienti pre e post-prandiale nel ratto che riflettono ciò che si vede in specie più grandi e gli esseri umani. Per gli studi di somministrazione di farmaci, specie più grandi possono anche essere preferito al momento della valutazione del trasporto linfatico dopo la somministrazione di dosi realistico umano forma 25. In un recente studio, prezzi di trasporto dei lipidi nella linfa mesenterica sono risultati comparabili tra le specie (topo, ratto, cane) dopo la somministrazione di una massa equivalente e il tipo di lipidi che fornisce una certa fiducia in estrapolando i dati di trasporto dei lipidi tra le specie 27. Tuttavia, il trasporto di un modello lipofilo droga, alofantrina, ordinati in ordine di grandezza animale (cioè, cane&gt; rat&gt; mouse). Un fattore di scala può quindi essere richiesto di extrapolate dati sul trasporto di droga linfatici da topo ad altre specie. Una limitazione di modelli linfatico cannulazione, in generale, è che la raccolta della linfa passiva direttamente da un condotto linfatico può modificare il flusso linfatico e trasporto dal vasi linfatici funzionano contro un gradiente di pressione che viene alterata quando il vaso è cannulata 33. Il modello linfa incannulamento può anche essere difficile stabilire in laboratori che non hanno familiarità con la tecnica. Sono stati così descritto modelli alternativi. Per esempio, il transito di fattori attraverso il sistema linfatico intestinale, come lipoproteine ​​e molecole lipofile, è stata studiata indirettamente tramite la raccolta di sangue. Un tale modello prevede confrontando concentrazioni ematiche di lipidi e / o farmaci dopo somministrazione orale in presenza e in assenza di inibitori (ad esempio, colchicina, Pluronic L81, cicloeximide) della produzione lipoproteine ​​intestinali che bloccano il trasporto 34 linfatica. Un vantaggiodi modelli che quantificano il trasporto linfatico indirettamente tramite la raccolta di campioni di sangue è che permette una valutazione del trasporto linfatico negli esseri umani come la chirurgia invasiva, non è richiesto 35. Tuttavia, gli inibitori del trasporto linfatico non sono specifici e fattori che sono trasportate attraverso i vasi linfatici sono diluiti e modificati nella circolazione sistemica che complica tali valutazioni. In vitro alternative sono stati descritti. Ad esempio, le culture Caco-2 cellulari o isolato enterociti sono stati utilizzati per studiare in modo più approfondito la secrezione intestinale di molecole che entrano i vasi linfatici 36-38. Un modello avanzato in vitro che è più rappresentativo del microambiente intestinale umano è stato anche recentemente descritto 39. In questo modello di uno strato di cellule endoteliali linfatico è co-coltura con cellule Caco-2 che consente un&#39;analisi più dettagliata del trasferimento di sostanze dall&#39;intestino nei vasi linfatici. Tuttavia, in vitro sistemi cellulari mancanza di flusso di scambio e di trasferimento, ossia l&#39;interconnessione con un lume intestinale e sangue alla base e la fornitura vascolare linfatico. In un approccio alternativo, Kassis et al. Stabilito un doppio canale (campo chiaro ad alta velocità di video e fluorescenza) in situ sistema di imaging che permette di confronto quantitativo fra contrazione nave, il flusso linfatico e le concentrazioni di lipidi fluorescenti mesenterici vasi linfatici 33. Un vantaggio di questo modello sopra citato in sistemi in vitro è che consente un monitoraggio accurato del passaggio di cellule immunitarie attraverso i vasi linfatici. Misurazioni assolute di lipidi massa (o droga) trasporto sono, tuttavia, non sono ancora stabiliti con metodi di imaging. In vitro e in silico approcci per prevedere in particolare la misura del trasporto di droga lipofila attraverso i vasi linfatici intestinali sono stati anche pubblicati 40-42. Ad esempio, l&#39;ex vivo affinità di vari compounds per chilomicroni plasma è risultato correlare ragionevolmente bene con il loro trasporto linfatico in vivo 41. Successivamente, lo stesso gruppo ha stabilito un modello in silico per prevedere affinità farmaco per chilomicroni basate su più di 40 proprietà fisico-chimiche. Holm et al. Anche stabilito un modello relativamente complessa in silico per prevedere definitiva trasporto linfatico di composti lipofilici sulla base di descrittori molecolari 42. Questi modelli possono fornire un approccio utile prevedere l&#39;ampiezza del trasporto linfatico droghe sconosciute. Validazione dei modelli con una vasta gamma di farmaci e tra i vari laboratori, tuttavia, è necessario per confermare la loro accuratezza e riproducibilità. Incannulazione del condotto mesenterico linfatico rimane pertanto l&#39;unico mezzo per esaminare direttamente il contenuto della linfodrenante tenue e il tasso di transito della complessa serie di fattori (cellule, proteine,peptidi, lipidi, farmaci) in linfa in una situazione in vivo. Qui si descrive un protocollo per incannulazione del dotto linfatico e carotide arteria mesenterica che consente la raccolta di linfa mesenterica e arteriosa sistemica da ratti anestetizzati. Dati rappresentativi dimostrano come il modello può essere utilizzato per esaminare lipidico e trasporto del farmaco dall&#39;intestino attraverso il sistema linfatico mesenterico. Questa è seguita da una discussione di difficoltà che si possono incontrare nella definizione del modello e una guida alla risoluzione. Una volta stabilito il modello è un potente strumento per studiare il trasporto linfatico intestinale.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Sterile saline</td>
			<td>Baxter healthcare</td>
			<td>AHB 1307</td>
			<td>Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50</td>
		</tr>
		<tr >
			<td >70 % ethanol in water</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Chlorhexidine gluconate solution (Microshield 4)</td>
			<td>Livingstone International</td>
			<td>JJ60243L</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=JJ60243L</td>
		</tr>
		<tr >
			<td >Betadine solution</td>
			<td>Livingstone International</td>
			<td>BU0510</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=BU0510</td>
		</tr>
		<tr >
			<td >Ilium Ketamil (Ketamine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >KETA I 1</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Ilium Xylazil (Xylazine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >TRO-3828</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >ACP 10 Injection (Acepromazine 10 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >VTG-DACP010020</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Sodium pentobarbitone</td>
			<td>PROVET VICTORIA&#160;</td>
			<td>24529</td>
			<td>Any brand can be used. Example here is Lethabarb&#174; 325 mg/ml sodium pentobarbitone, Virbac Animal Health. http://www.provet.com.au</td>
		</tr>
		<tr >
			<td >Heparin (35000I.U. in 35 mL)</td>
			<td>Sigma Pharmaceuticals</td>
			<td>337220</td>
			<td>http://sigmaco.com.au/</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td >Sigma-Aldrich</td>
			<td >E1644</td>
			<td>Any brand can be used. Example here is disodium salt of EDTA from Sigma.&#160;</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm</td>
			<td>Microtube extensions</td>
			<td>PE8050</td>
			<td>Any brand can be used. Example here is PE tubing 0.8x0.5 mm, 30 m</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm</td>
			<td>Microtube extensions</td>
			<td>PE9658</td>
			<td>Any brand can be used. Example here is PE tubing 0.96x0.58 mm, 30 m</td>
		</tr>
		<tr >
			<td >Ruler</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Markers</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cigarette lighter</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cyanoacrylate glue</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >23 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN23GX0.75LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT= 
			6&#38;search=DN23GX0.75LV</td>
		</tr>
		<tr >
			<td >25 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN25GX1.0LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search= 
			DN25GX1.0LV</td>
		</tr>
		<tr >
			<td >1 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS01TA</td>
			<td>Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS01TA</td>
		</tr>
		<tr >
			<td >10 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS10SA</td>
			<td>Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS10SA</td>
		</tr>
		<tr >
			<td >Gauze swabs</td>
			<td>Livingstone International</td>
			<td>GSC075</td>
			<td>Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=GSC075</td>
		</tr>
		<tr >
			<td >Cotton buds</td>
			<td>Livingstone International</td>
			<td>CTAST075DP</td>
			<td>Any brand can be used. Example here is Livingstone cotton applicator plastic double tipped. 75MM. 100/PK. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=CTAST075DP</td>
		</tr>
		<tr >
			<td >Heating pad</td>
			<td>Ratek</td>
			<td>WT1</td>
			<td>Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.</td>
		</tr>
		<tr >
			<td >Surgical light</td>
			<td>Harvard Apparatus</td>
			<td>72-0215 with 72-0267</td>
			<td>Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_ 
			-1_HAI_ProductDetail and&#160; http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_ 
			-1_HAI_ProductDetail___</td>
		</tr>
		<tr >
			<td >Surgical microscope</td>
			<td>Zeiss</td>
			<td>495005-0014-000</td>
			<td>Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=e&#38;i=10143</td>
		</tr>
		<tr >
			<td >Silk suture</td>
			<td>Livingstone International</td>
			<td>DTSK163019F4</td>
			<td>Any brand can be used. Example here is *&#160; 
			Email this item to my friend 
			3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=DTSK163019F4</td>
		</tr>
		<tr >
			<td >Scalpel blades</td>
			<td >Fine Science Tools (FST)</td>
			<td >10020-00</td>
			<td>Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191</td>
		</tr>
		<tr >
			<td >Scalpel handle</td>
			<td >Fine Science Tools (FST)</td>
			<td>10004-13</td>
			<td>Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&#38;CategoryId=51</td>
		</tr>
		<tr >
			<td >1 x Small surgical scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>14060-09</td>
			<td>Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=40&#38;CategoryId=17</td>
		</tr>
		<tr >
			<td >2 x Forceps with serrated curved tip</td>
			<td >Fine Science Tools (FST)</td>
			<td >11001-13</td>
			<td>Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=405&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Iridectomy scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>15000-08</td>
			<td>Any brand can be used. Example here is FST Vannas Spring Scissors - 2.5mm Cutting Edge, Straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=17&#38;CategoryId=16&#160;</td>
		</tr>
		<tr >
			<td >1 x Forceps with straight serated tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11650-10</td>
			<td>Any brand can be used. Example here is FST Graefe 10 cm straight with serrated 1 x 0.99 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=390&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth sharp straight fine tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11251-10</td>
			<td>Any brand can be used. Example here is FST Dumont #5 forceps straight 11cm with 0.08 x 0.04mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=335&#38;CategoryId=29</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth fine curved forceps</td>
			<td >Fine Science Tools (FST)</td>
			<td>11063-07</td>
			<td>Any brand can be used. Example here is FST Delicate Forceps 9 cm with smooth 0.4 x 0.3mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=360</td>
		</tr>
		<tr >
			<td >2 x Hemostats</td>
			<td >Fine Science Tools (FST)</td>
			<td >13010-12</td>
			<td>Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&#38;CategoryId=33</td>
		</tr>
		<tr >
			<td >1 x Suture needle holder</td>
			<td >Fine Science Tools (FST)</td>
			<td>12001-13</td>
			<td>Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&#38;CategoryId=70</td>
		</tr>
		<tr >
			<td >1 x Artery clamp</td>
			<td >Fine Science Tools (FST)</td>
			<td>18050-28</td>
			<td>Any brand can be used. Example here is FST Bulldog Serrefines straight, 28 mm long, 9x1.6 mm jaw dimension with medium clamp press. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=270&#38;CategoryId=82</td>
		</tr>
		<tr >
			<td >Oleic acid</td>
			<td >Sigma Aldrich</td>
			<td >O1008</td>
			<td>When required, any brand can be used. Example here is 99% pure oleic acid. http://www.sigmaaldrich.com/catalog/product/sial/o1008?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >14C-oleic acid</td>
			<td >Perkin&#160;</td>
			<td>NEC317050UC&#160;</td>
			<td>Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50&#181;Ci (1.85MBq). http://www.perkinelmer.com/Catalog/Product/ID/NEC317050UC</td>
		</tr>
		<tr >
			<td >Sodium taurocholate</td>
			<td >Sigma Aldrich</td>
			<td >T4009</td>
			<td>Any brand can be used. Example here is taurocholic acid sodium salt hydrate &#8805;95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Halofantrine</td>
			<td >Glaxo Smith Kline</td>
			<td ></td>
			<td>Halofantrine was kindly provided as a gift from Glaxo Smith Kline</td>
		</tr>
		<tr >
			<td >Sodium phosphate monobasic</td>
			<td >Sigma Aldrich</td>
			<td >71507</td>
			<td >Any brand can be used. Example here is sodium phosphate monobasic monohydrate, BioXtra, for molecular biology, &#62;99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Sodium phosphate dibasic</td>
			<td >Sigma Aldrich</td>
			<td >71643</td>
			<td >Any brand can be used. Example here is sodium phosphate dibasic dihydrate, BioUltra, for molecular biology, &#62;99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en&#38;region=AU</td>
		</tr>
	</tbody>
</table>

the mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport

Il sistema linfatico intestinale gioca un ruolo chiave nel trasporto di liquidi, l&#39;assorbimento dei lipidi e la funzione immunitaria. La linfa scorre direttamente dal piccolo intestino attraverso una serie di vasi linfatici e nodi che convergono al dotto linfatico mesenterica superiore. Incannulazione del condotto mesenterico linfatico consente quindi la raccolta della linfa mesenterica scorre dall&#39;intestino. Mesenterici costituito da una frazione cellulare di cellule immunitarie (99% linfociti), frazione acquosa (liquido, peptidi e proteine ​​quali citochine e ormoni intestinali) e frazione lipoproteina (lipidi, molecole lipofile e apo-proteine). Il condotto linfatico modello cannulazione mesenterica può quindi essere utilizzato per misurare la concentrazione ed il tasso di trasporto di una serie di fattori dall&#39;intestino attraverso il sistema linfatico. Modifiche a questi fattori in risposta alle sfide differenti (ad esempio, le diete, gli antigeni, droga) e nella malattia (ad esempio, malattia infiammatoria intestinale, HIV, diabete) può anche be determinato. Un&#39;area di espansione interesse è il ruolo del trasporto linfatico nell&#39;assorbimento dei farmaci lipofili somministrati per via orale e profarmaci che associano con intestinali vie di assorbimento dei lipidi. Qui si descrive, in dettaglio, un modello di topo cannulata dotto linfatico mesenterica che consente di valutare la velocità e il grado di lipidi e di trasporto della droga attraverso il sistema linfatico per diverse ore dopo la consegna intestinale. Il metodo è facilmente adattabile alla misurazione di altri parametri in linfa. Forniamo una descrizione dettagliata delle difficoltà che si possono incontrare quando si stabilisce questo metodo chirurgico complesso, così come i dati rappresentativi di esperimenti falliti e di successo per fornire istruzioni su come per confermare il successo sperimentale e interpretare i dati ottenuti.

I risultati di un esperimento rappresentativo per quantificare la portata cumulativa e tasso di lipidi e di trasporto del farmaco attraverso il sistema linfatico seguente erogazione intestinale utilizzando il modello linfa cannulazione mesenterica sono mostrati in Figura 4 e Figura 5. In questo esperimento, 200 ug del modello lipofilo alofantrina farmaco è stato somministrato nel duodeno di ratti oltre 2 ore in una formulazione contenente 40 mg di acido oleico (compresi 2-5 uCi 14...

Watch this Scientific Journal Video about The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport at JoVE.com

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

La linfa mesenterica Duct Cannulated Rat Modello: Applicazione alla valutazione di intestinale linfatico Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

the-mesenteric-lymph-duct-cannulated-rat-model-application-to

Research

JoVE Journal

Immunology and Infection

28.3K Views.  Monash University (Parkville Campus). Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

Video: The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravitale del linfonodo inguinale

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Ricerca

Immunologia e infezioni

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplificazione e quantificazione di HIV-1 RNA in soggetti affetti da HIV con una carica virale inferiore il limite di rilevamento da parte di Standard saggi clinici

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imaging intravitale del Nodo poplitea Lymph mouse

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non invasiva di imaging ottico del sistema vascolare linfatico di un mouse

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generazione di Linfonodo grassi Pad Chimere per lo studio del linfonodo stromale origine cellulare

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Metodo dell&#39;agitatore magnetico per la rilevazione di<em&gt; Trichinella</em&gt; Le larve in campioni di muscolo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sovrapressione e purificazione dell&#39;uomo<em&gt; Cis</em&gt; -preniltransferasi in<emEscherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

Analisi di basati su reazione a catena della polimerasi quantitativa del Microbiota intestinale murina dopo il trattamento antibiotico orale

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Infezione da Salmonella cronica Fibrosi intestinale indotta

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...