Questo lavoro è stato sostenuto dal National Institutes of Health (R01DK58096 a Vincent Poitout). Vincent Poitout detiene il Canada Research Chair in diabete pancreatico e funzione delle cellule beta. Bader Zarrouki ricevuto borse di studio post-dottorato da Merck e Eli Lilly. Ghislaine Fontes è stato sostenuto da una borsa di studio post-dottorato della Canadian Diabetes Association.

Sommario: La procedura consiste cateterizzazione della vena giugulare destra e sinistra arteria carotide in anestesia generale; consentendo un periodo di recupero di 7 giorni; collega i cateteri alle pompe utilizzando una girevole e sistema di contrappesi che consente all&#39;animale di muoversi liberamente nella gabbia; e infondendo glucosio e / o Intralipid (un&#39;emulsione di olio di soia che genera una miscela di circa 80% unsaturated/20% di acidi grassi saturi quando infuso con eparina 9) per 72 hr. 1. Canulation della Vena giugulare destra e l&#39;arteria carotide sinistra <ol><li> Sterilizzare gli strumenti chirurgici. Canulation tubazione deve essere sterilizzato a freddo usando un agente sterilizzante liquido (2,6% glutaraldeide) prima della procedura. Immergere il tubo in un contenitore sterilizzato per 16-24 ore. Risciacquare e lavare abbondantemente con acqua distillata sterile per rimuovere tutte le tracce di glutaraldeide prima dell&#39;uso. </li><li> Pesare il ratto per calcolaredosaggi di farmaci: Carprofene 5 mg / kg: diluizione 1/10 = Peso corporeo (g) x 0.001 ml SC (analgesico) Glicopirrolato 0,01 mg / kg: diluizione 1/10 = BW (g) X 0,0012 ml SC (anticolinergici) </li><li> Anestetizzare il ratto utilizzando isoflurano e ossigeno. </li><li> Posizionare il topo sul suo stomaco. Radere la zona dietro le orecchie alla base delle spalle. Posare il ratto sulla sua schiena. Radersi la regione sotto il collo per le zampe anteriori. </li><li> Preparazione del sito chirurgico con clorexidina, alcol e iodio. Somministrare farmaci. </li><li> Trasferire il ratto al campo chirurgico. </li><li> Utilizzando una tecnica asettica, canulate vena giugulare destra e l&#39;arteria carotide sinistra con una cannula PE-50 attaccato ad una siringa da 1 ml riempita con 5 U di soluzione fisiologica eparinizzata. Lavare le cannule con 50 U di soluzione fisiologica con eparina per evitare la coagulazione durante il periodo di recupero. Utilizzare aghi 23G smussati. Chiudere ogni cannula con un perno 23G. </li><li> Dopo l&#39;intervento chirurgico, tagliare circa 2,5 mm dalincisivi inferiori e il luogo del ratto in una giacca di infusione per evitare che le cannule da essere mangiato. </li><li> Fornire ossigeno (1 L / min per 10 minuti) per aiutare evacuare isoflurano. </li><li> Collocare il ratto in una gabbia riscaldata finché non è completamente sveglio. </li><li> Operare quattro ratti di utilizzare uno per condizione infusione (Tabella 1). </li></ol> 2. Assistenza post-operatoria (trattamento post-chirurgico e di collegamento dei cateteri) <ol><li> Pesare i ratti al giorno 1 e giorno 2 post-chirurgico. </li><li> Somministrare glycopyrrolate (BW (g) x 0,00048 ml) SC due volte al giorno 1 post-chirurgico e una volta il giorno 2 post-chirurgico. </li><li> Ulteriori trattamenti di supporto possono essere somministrati se necessario: liquidi, rilievo di riscaldamento, la dieta umida, ossigenoterapia, analgesici, anticolinergici. </li><li> Il giorno 7 post-chirurgia, pesare i topi per calcolare le portate per infusione. </li><li> Collegare ogni ratto di un sistema di infusione con un laccio e girevole montata su un grill da gabbia (Figura 1).</li><li> Lavare le cannule con 5 U di soluzione fisiologica con eparina per rimuovere i coaguli. Lavare le cannule, una volta di più con 50 U di soluzione fisiologica con eparina per prevenire la coagulazione. </li><li> Lasciare i ratti si adatti al laccio e girevole per almeno 24 ore prima di iniziare l&#39;infusione. </li></ol> 3. Infusione <ol><li> Disegnare 0,15 ml di sangue dalla carotide di ciascun ratto e misurare glicemia. Lavare le cannule giugulari. Utilizzare 50 U di soluzione fisiologica con eparina per prevenire la coagulazione sia cannule di ogni ratto. </li><li> Trasferire il campione di sangue di un tubo di raccolta ml 0,5 contenente il 2% di EDTA. Centrifugare a 10.000 rpm per 2 min e congelare il plasma a -20 ° C. </li><li> Riempire due siringhe 60 ml per ciascuna delle condizioni di infusione sotto elencate. Siringa 1 posto sulla posizione anteriore della pompa e siringa 2 posto sulla posizione posteriore della pompa. </li><li> Unisciti a ogni coppia di soluzioni con y-connettori e CO-EX tubi T22 che è stato sterilizzato. Mettere le siringhe su un Harvard 33doppia pompa siringa. </li><li> Modificare il fondo della gabbia e rimuovere tutti gli alimenti alla griglia superiore della gabbia. </li><li> Pesare 150 g di cibo standard e posto sulla griglia superiore della gabbia. </li><li> Collegare le siringhe per la girella sulla griglia della gabbia. Lavare le siringhe correttamente per rimuovere l&#39;aria intrappolata dalle linee. </li><li> Calcolare le portate di infusione utilizzando il peso corporeo che è stata presa prima di collegare il ratto al sistema di infusione. Le tariffe sono calcolate con un file di Microsoft Excel che converte il tasso di infusione di glucosio (GIR) in ml / h. </li><li> Impostare la pompa per amministrare le portate per 60 ml siringhe in base alle impostazioni del produttore. Inserire il tasso di siringa 1 (siringa anteriore) e il tasso per siringa 2 (torna siringa). </li><li> Avviare la pompa. </li></ol> 4. Monitoraggio <ol><li> Dopo aver avviato la pompa, verificare che non vi sia fuoriuscita dalla girevole o dalle cannule e tubi di infusione non sia attorcigliata. Verificare inoltre che non vi siano bolle d&#39;aria nelil tubo. </li><li> Dopo 3 ore di infusione, prelevare un campione di sangue per monitorare la glicemia. Ripetere dopo 6, 24, 48, e 72 ore di infusione. Per precauzione, glicemia viene monitorato anche dopo 30, 34, 54, e 60 ore di infusione. Glucosio nel sangue può essere misurata usando una goccia di sangue intero utilizzando un monitor portatile glucosio. Questo limita la quantità di sangue prelevato durante l&#39;infusione e quindi evita alterare significativamente il volume del sangue e / o ematocrito. </li><li> Il tasso per siringa 1 è modificato sulla base di valori di glicemia a mantenere glicemia a 220-250 mg / dl. Il tasso di siringa 2 non cambia durante la 72 ore di infusione, perché gli acidi grassi liberi sono mantenuti a 1 mmol / L. </li><li> Condizioni di infusione sono accoppiati in modo che il volume ricevuto per gli animali di controllo è equivalente al volume ricevuto per gli animali di prova (Tabella 1). </li><li> Dopo 24 ore di infusione, cambiare il fondo gabbia e pesare il cibo che rimane nella griglia gabbia. Restituire la parte non mangiato tØ la griglia della gabbia. Ripetere dopo 48 ore. </li><li> Refill siringhe al giorno secondo necessità durante la 72 ore di infusione. </li><li> Durante l&#39;infusione, osservare sempre il ratto per eventuali segni di infiammazione o di disagio. L&#39;assistenza appropriata, se necessario. </li></ol> 5. Eutanasia post-infusione <ol><li class="jove_content"> Dopo 72 h di infusione, i ratti vengono anestetizzati per via endovenosa con 0,5 ml di un cocktail di ketamina / xilazina (182 mg / kg di ketamina e 11,6 mg / kg di xilazina diluito 1:2 con 0,9% NaCl). </li><li class="jove_content"> Il riflesso toe-pinch viene utilizzato per verificare il livello di anestesia. Ulteriori anestetico viene somministrato come necessario. </li><li class="jove_content"> Quando il ratto è anestetizzato, la cavità addominale viene aperto con forbici chirurgiche. Il ratto è dissanguato disegnando 10-15 ml di sangue in una siringa dalla aorta o la vena cava. </li></ol>

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antecedent+hyperglycemia,+not+hyperlipidemia,+is+associated+with+increased+islet+triacylglycerol+content+and+decreased+insulin+gene+mRNA+level+in+Zucker+diabetic+fatty+rats.">Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats.</a> Diabetes. 50, 2481-2486 (2001).</li><li>Bachar, E., Ariav, Y., Cerasi, E., Kaiser, N., Leibowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+nitric+oxide+synthase+protects+the+pancreatic+beta+cell+from+glucolipotoxicity-induced+endoplasmic+reticulum+stress+and+apoptosis.">Neuronal nitric oxide synthase protects the pancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis.</a> Diabetologia. 53, 2177-2187 (2010).</li><li>Peyot, M. L., 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=Beta-cell+failure+in+diet-induced+obese+mice+stratified+according+to+body+weight+gain:+secretory+dysfunction+and+altered+islet+lipid+metabolism+without+steatosis+or+reduced+beta-cell+mass.">Beta-cell failure in diet-induced obese mice stratified according to body weight gain: secretory dysfunction and altered islet lipid metabolism without steatosis or reduced beta-cell mass.</a> Diabetes. 59, 2178-2187 (2010).</li><li>Hagman, D. K., 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=Cyclical+and+alternating+infusions+of+glucose+and+intralipid+in+rats+inhibit+insulin+gene+expression+and+Pdx-1+binding+in+islets.">Cyclical and alternating infusions of glucose and intralipid in rats inhibit insulin gene expression and Pdx-1 binding in islets.</a> Diabetes. 57, 424-431 (2008).</li><li>Fontes, G., 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=Glucolipotoxicity+age-dependently+impairs+beta+cell+function+in+rats+despite+a+marked+increase+in+beta+cell+mass.">Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass.</a> Diabetologia. 53, 2369-2379 (2010).</li><li>Stein, D. T., 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=Essentiality+of+circulating+fatty+acids+for+glucose-stimulated+insulin+secretion+in+the+fasted+rat.">Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat.</a> J. Clin. Invest. 97, 2728-2735 (1996).</li><li>Leahy, J. L., Cooper, H. E., Weir, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin+secretion+associated+with+near+normoglycemia.+Study+in+normal+rats+with+96-h+in+vivo+glucose+infusions.">Impaired insulin secretion associated with near normoglycemia. Study in normal rats with 96-h in vivo glucose infusions.</a> Diabetes. 36, 459-464 (1987).</li><li>Hager, S. R., Jochen, A. L., Kalkhoff, R. 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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=High+glucose+stimulates+early+response+gene+c-Myc+expression+in+rat+pancreatic+beta+cells.">High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.</a> The Journal of Biological Chemistry. 276, 35375-35381 (2001).</li><li>Tang, 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=Glucose-induced+beta+cell+dysfunction+in+vivo+in+rats:+link+between+oxidative+stress+and+endoplasmic+reticulum+stress.">Glucose-induced beta cell dysfunction in vivo in rats: link between oxidative stress and endoplasmic reticulum stress.</a> Diabetologia. 55, 1366-1379 (2012).</li><li>Alonso, L. 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=Glucose+infusion+in+mice:+a+new+model+to+induce+beta-cell+replication.">Glucose infusion in mice: a new model to induce beta-cell replication.</a> Diabetes. 56, 1792-1801 (2007).</li><li>Magnan, C., Gilbert, M., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+free+fatty+acid+infusion+in+rats+results+in+insulin+resistance+but+no+alteration+in+insulin-responsive+glucose+transporter+levels+in+skeletal+muscle.">Chronic free fatty acid infusion in rats results in insulin resistance but no alteration in insulin-responsive glucose transporter levels in skeletal muscle.</a> Lipids. 31, 1141-1149 (1996).</li><li>Goh, T. T., 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=Lipid-induced+beta-cell+dysfunction+in+vivo+in+models+of+progressive+beta-cell+failure.">Lipid-induced beta-cell dysfunction in vivo in models of progressive beta-cell failure.</a> Am. J. Physiol. Endocrinol. Metab. 292, 549-560 (2007).</li><li>Pascoe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+fatty+acids+block+glucose-induced+beta-cell+proliferation+in+mice+by+inducing+cell+cycle+inhibitors+p16+and+p18.">Free fatty acids block glucose-induced beta-cell proliferation in mice by inducing cell cycle inhibitors p16 and p18.</a> Diabetes. 61, 632-641 (2012).</li><li>Bell, C. G., Walley, A. J., Froguel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+human+obesity.">The genetics of human obesity.</a> Nature Reviews. Genetics. 6, 221-234 (2005).</li><li>Fontes, G., Hagman, D. K., Latour, M. G., Semache, M., Poitout, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+preservation+of+insulin+gene+expression+by+a+glucagon-like+peptide+1+agonist+or+a+dipeptidyl+peptidase+4+inhibitor+in+an+in+vivo+model+of+glucolipotoxicity.">Lack of preservation of insulin gene expression by a glucagon-like peptide 1 agonist or a dipeptidyl peptidase 4 inhibitor in an in vivo model of glucolipotoxicity.</a> Diabetes Res. Clin. Pract. 87, 322-328 (2010).</li><li>Crawford, P. A., Schaffer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+stress+in+the+myocardium:+Adaptations+of+gene+expression.">Metabolic stress in the myocardium: Adaptations of gene expression.</a> Journal of Molecular and Cellular Cardiology. , (2012).</li><li>Kewalramani, G., Bilan, P. J., Klip, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+insulin+resistance:+assault+by+lipids,+cytokines+and+local+macrophages.">Muscle insulin resistance: assault by lipids, cytokines and local macrophages.</a> Curr. Opin. Clin. Nutr. 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Vincent Poitout è co-fondatore di contratti di ricerca e ha ricevuto da Bêtagenex Inc., un&#39;organizzazione di ricerca a contratto, che offre il modello di infusione presentato in questo articolo come un servizio commerciale.

Sebbene un certo numero di studi precedenti hanno impiegato infusioni cronici di glucosio (ad esempio 10-15) o lipidi (ad esempio 16,17) nei roditori, a nostra conoscenza l&#39;infusione combinata di entrambi i carburanti è stato riportato soltanto nei topi 18. Il modello di infusione cronica qui presentato offre diversi vantaggi per studiare gli effetti sulla eccesso di nutrienti su una varietà di funzioni biologiche nei ratti. In primo luogo, esso non comporta rodit...

Livelli cronicamente elevati di glucosio e lipidi in circolo sono state proposte per contribuire alla patogenesi del diabete di tipo 2 alterando la funzione dei vari organi implicati nel mantenimento dell&#39;omeostasi del glucosio compreso, ma non limitato a, il pancreas cellule beta (recensione in 1). Le ipotesi postula glucotossicità che l&#39;iperglicemia cronica aggrava il difetto delle cellule beta che ha dato origine alla iperglicemia, in primo luogo, creando così un circolo vizioso e contribuendo al deterioramento del controllo glicemico nei pazienti con diabete di tipo 2. Allo stesso modo, l&#39;ipotesi glucolipotoxicity propone che aumenti concomitanti dei livelli di glucosio e di lipidi, come spesso osservati nel diabete di tipo 2, sono dannosi per la cellula beta. Decifrare i meccanismi cellulari e molecolari degli effetti deleteri di nutrienti cronicamente elevati di funzionalità delle cellule beta del pancreas è la chiave per il understanding della patogenesi del diabete di tipo 2 1. A tal fine, un gran numero di studi hanno esaminato i meccanismi della cronica nutrienti in eccesso ex vivo in isole di Langerhans isolate o in vitro in linee cellulari clonali, secernono insulina. Tuttavia, la traduzione dei risultati ottenuti in questi sistemi modello per l&#39;intero organismo è complesso, in particolare perché le concentrazioni di acidi grassi utilizzati in cellule coltivate o isolotti raramente corrispondono i livelli circolanti nelle vicinanze delle cellule beta in vivo 2. D&#39;altra parte, i meccanismi di guasto beta-cellula in risposta ad eccesso di nutrienti sono stati esaminati in modelli di roditori di diabete, come esemplificato dal ratto Zucker Diabetic Fatty 3,4, il gerbillo Psammomys obesus 5 e l&#39;alta percentuale di grassi dieta- fed del mouse 6. Questi modelli, tuttavia, sono caratterizzati da intrinseche anomalie metaboliche e non sono facilmente suscettibili di manipolazioni di glicemiae / o livelli di lipidi in un ambiente più controllato e meno cronica. Per essere in grado di alterare i livelli circolanti di nutrienti in un lasso di tempo di giorni in animali altrimenti normali, abbiamo sviluppato un modello di infusione cronica in ratti normali che ci permette di esaminare gli effetti di lipidi e del glucosio, da soli o in combinazione, su parametri fisiologici e funzionali 7,8.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Saline 0.9%</td>
			<td>BD</td>
			<td>JB1324</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dextrose 70%</td>
			<td>McKesson</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Intralipid 20%</td>
			<td>Fresenius Kabi</td>
			<td>JB6023</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metricide (Glutaraldehyde 2.6%)</td>
			<td>Metrex</td>
			<td>11-1401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Heparin Sodium 10,000 USP u/ml</td>
			<td>PPC</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Carprofen</td>
			<td>Metacam</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycopyrrolate</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chlohexidine 2%</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alcohol 70%</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Iodine</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PE-50</td>
			<td>BD</td>
			<td>427411</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CO-EX T22</td>
			<td>Instech Solomon</td>
			<td>BCOEX-T22</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Connector 22G</td>
			<td>Instech Solomon</td>
			<td>SC22/15</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Swivel 22G</td>
			<td>Instech Solomon</td>
			<td>375/22PS</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Y-Connector 22G</td>
			<td>Instech Solomon</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Counterbalance and arm</td>
			<td>Instech Solomon</td>
			<td>CM375BP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>23 G blunted needles</td>
			<td>Instech Solomon</td>
			<td>LS23</td>
			<td></td>
		 </tr>
		 <tr>
			<td>23 G canulation pins</td>
			<td>Instech Solomon</td>
			<td>SP23/12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tethers (12 inch)</td>
			<td>Lomir</td>
			<td>RT12D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Infusion jackets </td>
			<td>Lomir</td>
			<td>RJ01, RJ02, RJ03, RJ04</td>
			<td></td>
		 </tr>
		 <tr>
			<td>(SM-XL)</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tether attachment piece</td>
			<td>Lomir</td>
			<td>RS T1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>60 ml syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309602</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a model of chronic nutrient infusion in the rat

Esposizione cronica a livelli eccessivi di nutrienti è postulata a pregiudicare la funzione di diversi organi e tessuti e per contribuire allo sviluppo delle molte complicazioni associate con l&#39;obesità e la sindrome metabolica, compreso il diabete di tipo 2. Per studiare i meccanismi con cui livelli eccessivi di glucosio e acidi grassi influenzano la beta pancreatiche cellule e la secrezione di insulina, abbiamo stabilito un modello infusione di sostanze nutrienti cronica nel ratto. La procedura consiste di cateterizzazione della vena giugulare destra e sinistra arteria carotide in anestesia generale; consentendo un periodo di recupero di 7 giorni; collega i cateteri alle pompe utilizzando una girevole e sistema di contrappesi che consente all&#39;animale di muoversi liberamente nella gabbia; ed infondendo glucosio e / o Intralipid (un&#39;emulsione di olio di soia che genera una miscela di circa 80% unsaturated/20% di acidi grassi saturi quando infuso con eparina) per 72 hr. Questo modello offre diversi vantagGES, compresa la possibilità di modulare finemente i livelli target di glucosio circolante e di acidi grassi, la possibilità di co-infusione composti farmacologici, e il lasso di tempo relativamente breve rispetto a modelli alimentari. Può essere usato per esaminare i meccanismi della disfunzione nutrienti indotta in una varietà di organi e testare l&#39;efficacia dei farmaci in questo contesto.

Su una serie di 42 ratti che hanno subito un intervento chirurgico, 5 ratti sono stati persi durante il periodo post-operatorio e 1 ratto è stato perso durante l&#39;infusione, con un tasso complessivo di successo del 86%. Il peso corporeo medio dei 37 ratti che sono stati poi infuse era 608 ± 5 g prima dell&#39;intervento e 588 ± 6 g al di iniziare l&#39;infusione (media ± SE, n = 37, p &lt;0,0001 per paired t-test). I seguenti risultati rappresentativi sono stati ottenuti in due gruppi: infusione. Saline (SAL), e ...

Watch this Scientific Journal Video about A Model of Chronic Nutrient Infusion in the Rat at JoVE.com

A Model of Chronic Nutrient Infusion in the Rat

Un protocollo per infusioni cronici di glucosio e Intralipid nei ratti è descritto. Questo modello può essere utilizzato per studiare l&#39;impatto di un eccesso di nutrienti in funzione degli organi e dei parametri fisiologici.

Un modello del Chronic Infusion nutrienti nel ratto

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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Research

JoVE Journal

Medicine

12.6K Views.  CRCHUM. Un protocollo per infusioni cronici di glucosio e Intralipid nei ratti è descritto. Questo modello può essere utilizzato per studiare l'impatto di un eccesso di nutrienti in funzione degli organi e dei parametri fisiologici.

Video: A Model of Chronic Nutrient Infusion in the Rat

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Trapianto ortotopico aortica: un modello di ratto per studiare lo sviluppo di vasculopatia cronica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Ricerca

Medicina

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Vescica urinaria Distensione Evocati visceromotoria Risposte come un modello per il dolore della vescica nei topi

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Lento rilascio Drug Delivery attraverso Elvax 40W al Rat Retina: Implicazioni per il trattamento di condizioni croniche

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Neurodegenerazione in un modello animale di cronica amiloide-beta Oligomer infusione viene neutralizzata da anticorpi trattamento infuso con pompe osmotiche

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

L&#39;uso di un venoso Central Line per fluidi, farmaci e nutrienti Administration in un modello murino di malattia critica

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Una nuova pratica migliore per la convalida di coda vena iniezioni nel ratto con agenti vicino-infrarosso-Labeled

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

Agopuntura in un modello di ratto di asma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Un modello murino per la pancreatite cronica tramite infusione di TNBS del dotto biliare

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Migliorare la terapia con FSN nel modello di ratto: minimizzare il disagio e massimizzare l'efficienza

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Autore in primo piano: Svelare gli effetti terapeutici del trattamento con FSN – Collegare la ricerca e le applicazioni cliniche nel dolore neuropatico

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Efficacia dell'agugliatura sottocutanea di Fu sul dolore al nervo sciatico: cambiamenti comportamentali ed elettrofisiologici in un modello di ratto con lesione da costrizione cronica

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Methods Article

Un modello del Chronic Infusion nutrienti nel ratto