We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

Tutti i protocolli sperimentali su animali sono stati approvati dal comitato di cura degli animali e uso istituzionale presso l&#39;Istituto di Scienze dell&#39;Alimentazione, Istituti di Shanghai per Scienze Biologiche, Accademia Cinese delle Scienze (Shanghai, Cina). 1. DIO modello murino <ol><li> alimentazione HFHS <ol><li> Feed the otto settimane di età maschile C57BL / 6 topi con una HFHS che contiene 40 kcal% di grassi e 40 kcal% di saccarosio. loro casa a 12 h Condizioni ciclo dark-light. </li><li> Mantenere i topi in HFHS condizione di dieta al seno per quattro a sedici settimane. Controllare il settimanale del peso corporeo. </li></ol></li><li> Analisi della composizione corporea e analisi delle immagini pseudo-color <ol><li> Sottoporre i topi a una analisi della composizione corporea analizzatore. <ol><li> Fissare cosciente, topi non anestetizzati in un tubo di plastica nel. Misurare la massa grassa, massa magra, e fluidi che utilizzano un sistema di NMR, come descritto in precedenza 2. </li></ol></li><li> Eseguire la scansione di tutto il corpo di ogni mouse utilizzando un sistema di risonanza magnetica. <ol><li> Anestetizzare i topi con 1% avertin tramite iniezione intraperitoneale (25 ml / g) ed eseguire l&#39;analisi di imaging in tubi di vetro specializzati. NOTA: Il processo di formazione immagine ha una durata di circa 0,5 ore per ogni mouse. Quando la misurazione è completata, restituire i topi per gabbia. </li></ol></li><li> Eseguire la scansione l&#39;intero corpo dei topi in dorsale centrale, e gli strati ventrale; scandire il fegato in direzione verticale. Utilizzare un magnete 0,55 T e dei seguenti parametri strumentali: spazio K = 192 x 256 micron, lo spessore della sezione = 3 mm, TE = 14,8 ms, TR = 400 ms, FOVRead = 100 mm, FOVPhase = 100 mm, e il numero di scansioni = 8. scansione utilizzando la stessa condizione magnetica per i tutti i gruppi di topi. </li><li> Mappa scala di grigio dell&#39;immagine una pseudo-colore sul secondo l&#39;intensità del segnale 9. </li></ol></li><li> l&#39;eutanasia del mouse <ol><li> sterilizzare dissectistrumenti NG, come pinze e forbici. </li><li> Versare isoflurano sufficienti su un tovagliolo di carta e lasciare che il isoflurano volatilizza in un barattolo di vetro. Assicurarsi che i topi sono eutanasia dal isoflurano. </li><li> Fissare i topi su un tavolo operatorio. Spruzzare ogni addome con 75% etanolo, fare un&#39;incisione nel derma del processo xifoideo con le forbici, e strappare il derma longitudinalmente attraverso la linea mediana con le mani per esporre il peritoneo. </li><li> Bloccare il processo xifoideo con una pinza e tagliare il peritoneo in larghezza attraverso il fondo delle nervature per esporre le viscere. </li><li> Prelevare il sangue in provette EDTA rivestite mediante puntura cardiaca. </li><li> Tagliare il diaframma lungo il fegato e rimuovere con attenzione il fegato dai enterocoelia con le forbici sottili. Tagliare tre pezzi di tessuto epatico per le seguenti misure lipidi: a) 6 x 3 x 3 mm per H &amp; E colorazione, b) 6 x 3 x 3 mm per Oil Red O colorazione, e c) 20-40 mg di fegato per il cloroformio / l&#39;estrazione metanolo. </li></ol></li><li> Colorazione H &amp; E <ol><li> Fissare il fegato in 4% tampone fosfato di formalina acetato a 4 ° C durante la notte e poi incorporare in paraffina. </li><li> Tagliare sezioni di paraffina (5 micron) e montare su vetrini per colorazione H &amp; E, come precedentemente descritto 3, 10. </li></ol></li><li> Oil Red O colorazione <ol><li> preparazione Regent <ol><li> Preparare la soluzione di lavoro Oil Red O con formalina al 10%, il 60% di isopropanolo, ematossilina, e gelatina glicerolo. Sciogliere 0.5 g di Oil Red O polvere con 100 ml di isopropanolo e riscaldare in un bagno a 60 ° C per ottenere una soluzione stock di 0,5%. </li><li> Diluire con acqua bidistillata (DDH 2 O) in un rapporto 3: 2 (3 ml di soluzione madre e 2 ml di DDH 2 O). Filtrare la soluzione di lavoro e lasciate riposare per almeno 10 minuti a temperatura ambiente. </li></ol></li><li> Incorpora il fegato di temperatura di taglio ottimale (OCT) composto in apscatola incasso lastic. Congelarlo a -20 ° C per 30 min. Tagliare in sezioni di 8 micron di un microtomo di congelamento, montare il tessuto su un vetrino, e metterlo a temperatura ambiente per 30 min. </li><li> Posizionare un tovagliolo di carta sul fondo di una vasca di colorazione e versare una piccola quantità di formalina al 10% di bagnare l&#39;asciugamano di carta. Posizionare il vetrino nella colorazione vasca e fissare le sezioni congelate in vapori di formaldeide per 5 minuti a 4 ° C. </li><li> Immergere il vetrino nel 60% isopropanolo nella vasca colorazione per 3 s; ripetere una volta. </li><li> Aggiungere poche gocce di soluzione di lavoro Oil Red O alla diapositiva per coprire tutto il tessuto e incubare per 15 min. Proteggere i tessuti dalla luce a temperatura ambiente. </li><li> Lavare il vetrino in nuova isopropanolo 60% nella vasca colorazione per 3 s; ripetere due volte. </li><li> Lavare delicatamente il vetrino con acqua distillata due volte e rimuovere l&#39;acqua in tutto il tessuto. Assicurarsi di non asciugare il tessuto. </li><li> Posizionare il vetrino in ematossilina per 1 min e risciacquare delicatamente con acqua corrente per 5-10min. Lavare delicatamente il vetrino in acqua distillata due volte e mantenere l&#39;in acqua distillata fino al momento di mettere sul vetrino. </li><li> Riscaldare la gelatina glicerolo nel microonde e mettere alcune gocce sulla parte superiore del tessuto. Posizionare delicatamente il coprioggetto sulla parte superiore e fare attenzione a non formare bolle. </li><li> Osservare la macchia sotto un microscopio e catturare immagini caratteristici utilizzando 20X e 40X obiettivi. </li></ol></li><li> Dosaggio dei lipidi del fegato <ol><li> Posizionare il tessuto epatico sezionato in una nuova provetta da centrifuga 2 mL di plastica. Omogeneizzare in 1 mL di PBS con un omogeneizzatore e trasferire il lisato in un tubo di vetro da 15 ml. </li><li> Aggiungere 5 ml di cloroformio / metanolo (2: 1, v / v), vortex la miscela vigorosamente per 1 minuto, e lasciate riposare per 2 ore sul ghiaccio. </li><li> Centrifugare a 1.650 xg per 10 min a 4 ° C; la miscela deve separare in 3 strati: lo strato superiore metanolo, il disco proteine ​​centrale, e il cloroformio inferiore e lipidi. </li><li> Tran<html> SFER la fase di fondo in un nuovo tubo di vetro con una lente di Pasteur pipetta; non vi è alcuna necessità di ottenere ogni goccia della frazione inferiore. Impostare il tubo da parte su ghiaccio. </li><li> Aggiungere 600 ml di 4 mM MgCl 2 e 1,5 mL di cloroformio alla frazione supernatante sinistra nel tubo precedente e agitare vigorosamente. Incubare in ghiaccio per 30 minuti e centrifugare a 1.650 xg per 10 min a 4 ° C. </li><li> Unire la fase di fondo con il primo tubo fase di fondo utilizzando Pasteur pipetta. Eliminare gli strati superiori e medie. </li><li> Si evapora il cloroformio sotto azoto in un bagno a 37 ° C. Lavare i lati dei tubi di vetro a fondo con 200 ml di 1% Triton X-100 / cloroformio (v / v). Far evaporare sotto azoto. Sciogliere il lipidico con 200 ml di DDH 2 O e vortice bene a fare l&#39;emulsione finale. </li><li> Misurare la trigliceridi totali (TG) e colesterolo (TC) livelli utilizzando un trigliceride o colesterolo kit secondo il protocollo del produttorexref &quot;&gt; 3. NOTA: I valori lipidici sono normalizzati a quelli delle concentrazioni di proteine in omogenato iniziale, come determinato dalla proteina quantificazione 3. </li></ol></li></ol> Modello 2. principale del mouse epatociti <ol><li> L&#39;isolamento di epatociti primari di topo <ol><li> Anestetizzare ogni mouse con il 6% cloralio idrato (10 ml / g per via intraperitoneale). </li><li> Isolare epatociti primari, come descritto in precedenza 10. Crescere le cellule su vetrini collagene rivestite in un 6-pozzetti per Oil Red O colorazione. Per l&#39;estrazione dei lipidi, seminare le cellule su un 10-cm coltura cellulare piatto 10. </li></ol></li><li> Trattamento con cellule e Oil Red O colorazione <ol><li> Sostituire il mezzo con 2 mL di terreno fame e incubare a 37 ° C per 24 h. Trattare con 30 mM glucosio e di insulina 100 nM. Incubare a 37 ° C cultura incubatore per 24 h. </li><li> Aspirare il mezzo. Aggiungere 2 ml di PBS e miscelare delicatamente per sciacquare il medium; ripetere una volta. </li><li> Fissare il vetrino alla slitta con un elastico. Esporre le cellule aderenti alla superficie esterna. Eseguire la colorazione Oil Red O come descritto sopra (punto 1.5). NOTA: I livelli basali di accumulo di lipidi vengono stimolati con un elevato glucosio più trattamento insulina dopo deprivazione di siero. Le gocce lipidiche vengono visualizzati con Oil Red O colorazione. </li></ol></li><li> Dosaggio dei lipidi <ol><li> Trattare gli epatociti con alte concentrazioni di glucosio più insulina nella stessa condizione (punto 2.2.1). Aggiungere 1 ml di soluzione salina in ogni 10 cm piatto di coltura cellulare. Raschiare le cellule e li raccolgono in nuovi tubi di vetro da 15 ml, da cui aliquote di 100 microlitri vengono trasferiti ai tubi freschi 1.5 mL per la quantificazione delle proteine. </li><li> Aggiungere 4 mL di cloroformio / metanolo (2: 1, v / v) al residuo 900 ml di cellule. Con ultrasuoni per circa il 20 emette un segnale acustico e poi vortice vigorosamente per liberarei lipidi nelle cellule. Incubare la miscela in ghiaccio per 2 ore. Eseguire l&#39;estrazione dei lipidi (passo 1,6). </li></ol></li></ol>

<ol>
	<li>Angulo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+progress+-+Nonalcoholic+fatty+liver+disease.">Medical progress - Nonalcoholic fatty liver disease.</a> New England Journal of Medicine. 346 (16), 1221-1231 (2002).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+ATF6+Increases+Fatty+Acid+Oxidation+to+Attenuate+Hepatic+Steatosis+in+Mice+through+Peroxisome+Proliferator-Activated+Receptor+Alpha.">Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice through Peroxisome Proliferator-Activated Receptor Alpha.</a> Diabetes. 65 (7), 1904-1915 (2016).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+phosphorylates+and+inhibits+SREBP+activity+to+attenuate+hepatic+steatosis+and+atherosclerosis+in+diet-induced+insulin-resistant+mice.">AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.</a> Cell Metab. 13 (4), 376-388 (2011).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+SIRT1+attenuates+hepatic+steatosis+and+controls+energy+balance+in+mice+by+inducing+fibroblast+growth+factor+21.">Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21.</a> Gastroenterology. 146 (2), 539-549 (2014).</li><li>Esau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-122+regulation+of+lipid+metabolism+revealed+by+in+vivo+antisense+targeting.">miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.</a> Cell Metabolism. 3 (2), 87-98 (2006).</li><li>Kanda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCP-1+contributes+to+macrophage+infiltration+into+adipose+tissue,+insulin+resistance,+and+hepatic+steatosis+in+obesity.">MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity.</a> Journal of Clinical Investigation. 116 (6), 1494-1505 (2006).</li><li>Cohen, J. C., Horton, J. D., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fatty+liver+disease:+old+questions+and+new+insights.">Human fatty liver disease: old questions and new insights.</a> Science. 332 (6037), 1519-1523 (2011).</li><li>Hebbard, L., George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+nonalcoholic+fatty+liver+disease.">Animal models of nonalcoholic fatty liver disease.</a> Nat Rev Gastroenterol Hepatol. 8 (1), 35-44 (2011).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+effective+inhibition+of+lung+cancer+growth+and+metastasis+by+systemic+delivery+of+siRNA+via+multimodal+mesoporous+silica-based+nanocarrier.">Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier.</a> Biomaterials. 35 (38), 10058-10069 (2014).</li><li>Gong, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+21+improves+hepatic+insulin+sensitivity+by+inhibiting+mammalian+target+of+rapamycin+complex+1+in+mice.">Fibroblast growth factor 21 improves hepatic insulin sensitivity by inhibiting mammalian target of rapamycin complex 1 in mice.</a> Hepatology. 64 (2), 425-438 (2016).</li><li>Anstee, Q. M., Targher, G., Day, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+NAFLD+to+diabetes+mellitus,+cardiovascular+disease+or+cirrhosis.">Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis.</a> Nat Rev Gastroenterol Hepatol. 10 (6), 330-344 (2013).</li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti. Finanziamento: Questo lavoro è stato sostenuto da sovvenzioni dal National Science Foundation naturale della Cina (n ° 81.270.930, 31.471.129, e 31.671.224) e le cento talenti del programma della Accademia Cinese delle Scienze (2013OHTP04) a YL

NAFLD è una serie di malattie epatiche progressive che è associato con la sindrome metabolica, l&#39;obesità, insulino-resistenza, o diabete mellito tipo 2 (DM2) 11. Il segno distintivo di NAFLD è steatosi, l&#39;accumulo di lipidi negli epatociti. Qui, uno spettro di metodi è presentata per caratterizzare i fenotipi e parametri di steatosi epatica utilizzando topi DIO e epatociti del mouse primarie. Questa procedura potrebbe essere utile per chiarire i meccanismi molecolari di NAFLD e altre...

L&#39;obesità è un problema di salute in rapida crescita nei paesi sviluppati e in via di sviluppo. E &#39;stato segnalato per essere una delle condizioni coesistenti frequentemente associati con steatosi epatica non alcolica (NAFLD), con una prevalenza che varia tra il 30 e il 100 per cento in pazienti con NAFLD 1. A causa della forte correlazione tra il fegato grasso e l&#39;obesità, obesi (DIO) modelli murini di dieta-indotta sono ampiamente utilizzati per studiare i meccanismi molecolari complessi associati con lo sviluppo di NAFLD 2, 3, 4, 5, 6. Steatosi epatica è il primo stadio della NAFLD, e può progredire a steatoepatite non alcolica (NASH), cirrosi, e in ultima analisi, il cancro del fegato 7. Pertanto, l&#39;obiettivo generale di questo metodo è quello di generare modelli animali e cellulari di condizioni steatosico epatiche e al PrOvide protocolli dettagliati per la misurazione dei lipidi efficiente ed accurato. Questi modelli e misure sono utili anche per la ricerca di altri disordini metabolici, come la resistenza all&#39;insulina e diabete di tipo 2. Come l&#39;obesità è identificato come uno dei principali fattori di rischio per la NAFLD, un alto contenuto di grassi, ad alto saccarosio dieta (HFHS) che imita la dieta ricca di grassi di tipo occidentale è usato per indurre l&#39;obesità nei topi. Successivamente, il grado di steatosi epatica può essere valutata con metodi diversi. In primo luogo, il peso corporeo e analisi della composizione corporea con risonanza magnetica nucleare (NMR) mostrano l&#39;accumulo di lipidi durante il tempo di alimentazione. La massa grassa e massa magra possono essere quantificati in modo non invasivo e in tempo reale. Inoltre, la risonanza magnetica (MRI) viene utilizzata sia per il corpo intero e la distribuzione fegato grasso. Il segnale scala di grigi dell&#39;analisi MRI può essere convertito un&#39;immagine pseudo-colori leggibile in, e l&#39;intensità dellala scala di grigi e colore è emi-quantificabile. Questa tecnologia offre vantaggi unici per la misurazione di accumulo di lipidi di animali vivi. In secondo luogo, l&#39;analisi istologica del fegato è il metodo più comunemente utilizzato per determinare steatosi epatica. Ematossilina ed eosina (H &amp; E) colorazione istologica fornisce informazioni, quali epatociti morfologia e infiltrazione di macrofagi, mentre Oil Red O colorazione mostra la dimensione e la posizione delle goccioline lipidiche in epatociti. In terzo luogo, l&#39;analisi del contenuto di lipidi utilizzando estrazione con cloroformio / metanolo è una misurazione accurata e quantitativa dei lipidi epatici. Totale livelli di trigliceridi e di colesterolo possono essere misurati con metodi biochimici. È importante sottolineare che l&#39;analisi estrazione dei lipidi e Oil Red O colorazione possono essere utilizzati anche in geneticamente manipolato o trattato farmaceuticamente epatociti. Il vantaggio del presente metodo è la sua utilizzazione di approcci multipli ottimizzati per generare modelli steatosico epatichee caratterizzare completo fenotipi sia in vivo che in vitro. I modelli DIO mouse possono ricapitolare i patologia e metaboliche fenotipi di malattia del fegato grasso umano. Altri parametri metabolici negli esseri umani possono essere replicati in questo modello, come pure 8. La generazione del modello epatociti steatosico in risposta ad alta glucosio più insulina è efficiente, utile, e supera la limitazione del lavoro costoso e richiede tempo mouse. Nel loro insieme, questi metodi sono sufficienti ed essenziali per lo studio della disfunzione epatica lipidi e insulino-resistenza durante sovraccarico di nutrienti.

<table border="0" cellpadding="0" cellspacing="0" width="728">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">O.C.T compound</td>
			<td style="width:183px;">SAKURA</td>
			<td style="width:163px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Oil Red O</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">O0625-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Triglycerides kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR22421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Cholesterol kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR13421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Collagen type I, Rat tail</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:163px;">08-115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DMEM (low glucose)</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">11885-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Penicillin / Streptomycin</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">FBS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">10099141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PBS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">R21-040-CVR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HBSS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Insulin</td>
			<td style="width:183px;">TOCRIS Bioscience</td>
			<td style="width:163px;">3435</td>
			<td>dissolve in PBS, 1mM for stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">G8270-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:163px;">BX53</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Peristaltic pump</td>
			<td style="width:183px;">Longerpump</td>
			<td style="width:163px;">BT100-2J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">10cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">420167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">6-well-plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">3516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BCA assay</td>
			<td style="width:183px;">Beyotime</td>
			<td style="width:163px;">P0010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nuclear Magnetic Resonance</td>
			<td style="width:183px;">Niumag technology</td>
			<td style="width:163px;">MiniQMR23-060H-I</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">High fat high surcose diet(HFHS)</td>
			<td style="width:183px;">Research Diets</td>
			<td style="width:163px;">D12327</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized analysis of in vivo and in vitro hepatic steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Come mostrato in figura 1A, il peso del corpo del mouse è stato aumentato a 45 ± 1,2 g dopo 16 settimane di HFHS alimentazione, che è circa 1,5 volte superiore rispetto al gruppo di alimentazione dieta chow. La composizione corporea NMR analisi mostra la massa grassa e massa magra di topi sono indicati (Figura 1B). Le distribuzioni di grasso del corpo e del fegato sono state determinate mediante MRI, e immagini rappresentative pseudo-colori in vivo, t...

Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Analisi ottimizzata delle<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; Epatica steatosi

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

optimized-analysis-of-in-vivo-and-in-vitro-hepatic-steatosis

Research

JoVE Journal

Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

15.8K Views.  Chinese Academy of Sciences. Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described. 

Video: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualizzazione e analisi del flusso ematico e consumo di ossigeno nella microcircolazione epatica: Applicazione di un modello di epatite acuta

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Ricerca

Medicina

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

Monitoraggio della sistemici e epatica parametri emodinamici nei topi

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Valutare l&#39;efficacia delle Cancer Drug Sensibilizzazione<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

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. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo rilevamento di Mitophagy in cellule umane, C. Eleganse topi

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Preparazione di Gushukang (GSK) Granuli per in vivo e in vitro

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Scratch Migration Ssay and Dorsal Skinfold Chamber for In Vitro e In Vivo Analisi della Guarigione delle Ferite

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...