Questo lavoro è stato sostenuto dal russo Science Foundation, concessione n. 14-50-00060 e LLC RUSCHEMBIO. Questo lavoro è stato sostenuto anche dal Crohn e Colite Foundation of America concedere 294.083 (a AVT), e dal NIH concedere RO1 DK47700 (CJ).

Gli studi sugli animali sono stati eseguiti in accordo con l&#39;Istituto Nazionale di Salute e linee guida sono state approvate dalla cura degli animali Istituzionale e utilizzare Comitato dell&#39;Istituto Trudeau. 8-12 settimane di età topi C57BL / 6 sono stati utilizzati per lo studio. 1. Preparazione per la chirurgia <ol><li> Preparare e sterilizzare gli strumenti chirurgici. </li><li> Preparare il sistema di anestesia isoflurano-based con cono, e pad riscaldata. Assicurarsi pad riscaldata non è surriscaldato (&lt;39 ° C). </li><li> Assicurarsi che il isoflurano evacuazione dei gas filtro viene posizionato correttamente a garantire le luci di scarico sul fondo del contenitore non sono bloccati o occlusa in alcun modo. Pesare gas scavenging bomboletta prima procedura e il peso documento sul contenitore. Eliminare il contenitore quando il peso contenitore supera i 50 g di peso accumulato (~ 12 ore). </li></ol> 2. Anestesia <ol><li> Anestetizzare mouse con 3% isoflurano in una camera di induzione (1 L / minO 2). <ol><li> Valutare profondità dell&#39;anestesia da una incapacità di rimanere in posizione verticale, perdita di movimento intenzionale volontaria, la perdita del riflesso corneale, rilassamento muscolare, e la perdita di risposta alla stimolazione reflex (punta o la coda pizzico con pressione costante). </li><li> Valutare la frequenza respiratoria e il modello osservando parete toracica e addominale movimenti. In anestesia ottimale, la frequenza respiratoria dovrebbe essere ~ 55-65 respiri al minuto. </li><li> Rimuovere mouse dalla camera di induzione e radere rapidamente l&#39;area addome del mouse. </li></ol></li><li> Per prevenire l&#39;essiccamento della cornea, posizionare blanda pomata oftalmica negli occhi. </li><li> Posizionare il mouse sul tappetino riscaldato e collegarlo tramite cono di sistema di anestesia. Assicurarsi membrana cono lattice naso si inserisce saldamente sopra la testa del mouse e non vi è alcuna perdita di isoflurano. </li><li> Ridurre tasso isoflurano al 1,5%, e iniettare buprenorfina (0,1 mg / kg) e ketamina (10 mg / kg) per via sottocutanea per impedire avvolgimento del dolore in cascata. </li><li> pulire °e pelle della zona operazione con un tampone di cotone sterile imbevuto di soluzione chirurgica Betadine seguita da etanolo al 70%. </li></ol> 3. Chirurgia <ol><li> Fare un cm laparotomia linea mediana 3-5 con le forbici di funzionamento. zona di operazione di copertura con tampone non aderente sterili inumidito con soluzione fisiologica. Isolare cieco e ileo ed esporre l&#39;arteria mesenterica superiore utilizzando tamponi di cotone inumidito con soluzione salina. </li><li> Per facilitare l&#39;applicazione di clip, fare piccole incisioni nel mesentere circostante l&#39;arteria mesenterica superiore con iris belle scissors.To fare questo, alzare delicatamente l&#39;intestino con una pinza medicazione e tagliare mesentery su entrambi i lati dell&#39;arteria mesenterica superiore alla posizione clip desiderata (Figura 1A). Quindi, aggiungere alcune gocce di soluzione salina sterile per l&#39;area di posizione della clip desiderata prima di applicare clip. Nota: per eseguire l&#39;intervento chirurgico farsa, seguire la procedura chirurgica fino al punto 3.2. Non applicare clip. Invece, mantenere il tessuto umido con aggiunta s caldoaline come descritto nel 3,6 per 1 ora. In seguito, passare al punto 4.1, </li><li> Occlude i primi rami ordine dell&#39;arteria mesenterica superiore con clip microvascolari (70 g forza) utilizzando una clip applicatore per creare una regione 5-7 cm dell&#39;ileo ischemico adiacente cieco (Figura 1B). Anche se la posizione dei vasi è conservatore, ci potrebbero essere lievi differenze tra i topi (vedere esempi nella Figura 1). Pertanto, 2 o 3 clip sono solitamente richiesti (vedi la posizione dei clip su figura 1A, D, E, frecce nere). Nota: utilizzare clip del vaso di alta qualità. clip ad alta pressione può danneggiare i vasi e prevenire la rigenerazione mentre clip a bassa pressione (&lt;30 g) non possono bloccare completamente il flusso di sangue. </li><li> Bloccare il flusso di sangue attraverso l&#39;intestino collaterale utilizzando due clip microvascolari tutti navi (40 g forza), demarking regione dell&#39;intestino ischemico (Figura 1). L&#39;occlusione dei vasi collaterali è necessario perevitare afflusso di sangue dai vasi sanguigni adiacenti (vedi la posizione dei clip su figura 1A, D, E, frecce verdi). </li><li> Opzionale: Aggiungere la soluzione di eparina (6 USP unità / ml), per prevenire la coagulazione del sangue. Gocciolata aggiungere 0,5 ml di soluzione di eparina per l&#39;intestino isolato. </li><li> Wet pad non aderente salviettine delicate sterili con soluzione salina pre-riscaldato a 37 ° C e si applicano a zona chirurgica. Assicurarsi che salviettine rimane umido durante l&#39;intera procedura. </li><li> Mantenere ischemia per 60 min usando 1-1,5% anestesia isoflurano in tutto. Se la procedura ischemia è eseguita correttamente, la regione ischemica cambia in vino rosso in colore in circa 30 min. Si noti che i vasi sanguigni distali alla posizione della clip sono ingrandite durante l&#39;ischemia (figura 1, pannelli di destra) che indica l&#39;occlusione di successo. </li><li> Seguire da vicino il mouse durante la fase di ischemia. Continuare ad applicare soluzione salina per il pad non aderente che copre il sito chirurgico. </li><li> marchioi bordi della zona ischemica pipettando 20 ml di Gill`s 3 ematossilina sul tessuto per agevolare la raccolta del tessuto ischemico e tessuto sano adiacente dal medesimo mouse per il confronto (Figura 1E, pannello di destra). </li></ol> 4. Fase riperfusione <ol><li> Alla fine della ischemia aggiungere qualche goccia di soluzione salina sull&#39;area fermaglio e rimuovere delicatamente clip microvascolari con clip applicatore. Poi, spingere delicatamente l&#39;intestino torna alla cavità addominale con punte di cotone inumidito saline. Rimuovere pad non aderente e chiudere la parete addominale e la pelle con 9 mm in acciaio inox clip ferita. Se riperfusione viene eseguita più di 3 ore, utilizzare una sutura Vicryl assorbibile per chiudere la parete addominale prima di applicare clip ferita sulla pelle. </li><li> Mantenere topi in una gabbia pulita riscaldata per tempo desiderato (30 min, 60 min, 120 min, o 180 min) per la fase di riperfusione. </li><li> Controllare gli animali, almeno ogni 30 min per assicurare la stabilità. </li></ol><pclass = &quot;jove_title&quot;&gt; 5. Necroscopia e raccolta dell&#39;intestino tenue <ol><li> Euthanize topi da CO 2 overdose seguita da dislocazione cervicale al momento desiderato seguendo riperfusione. </li><li> Aprire cavità addominale e raccogliere il tessuto intestinale ischemico per ulteriori analisi. Harvest sano tessuto normale adiacente al tessuto danneggiato come controllo interno per tenere conto di qualsiasi reazione sistemica di un infortunio. Nota: Questo controllo è più appropriato rispetto alla farsa topi di controllo operato perché i topi sham operato non subiscono una reazione sistemica al danno IR-indotta. </li><li> Lavare contenuto intestinale con 30 ml siringa con l&#39;ago collegato sonda gastrica riempito di soluzione salina e poi tagliare l&#39;intestino in senso longitudinale. Se è richiesto un campione di intestino per l&#39;analisi di espressione genica, tagliare un frammento di 1,5 millimetri longitudinalmente, e utilizzare il pezzo rimanente per l&#39;analisi istologica. </li><li> Per l&#39;analisi istologica, preparare un rotolo svizzero con un paio di pinze a rotolare la intestine. </li><li> Per mantenere la forma arrotolata, posizionare i pezzi di intestino tra imbottiture biopsia in cassette di tessuto (Figura 2). Mettere le cassette in 10% formalina tamponata. </li><li> Fix tessuto in formalina per almeno 24 ore. Sostituire formalina con il 70% di etanolo per altre 24 ore. Conservare tessuto in 70% di etanolo indefinitamente a temperatura ambiente. </li><li> Incorporare in paraffina, taglio 5 sezioni micron e macchia con ematossilina ed eosina utilizzando un protocollo standard (Figura 3). </li></ol> 6. punteggio <ol><li> Punteggio ottenuto il murino danno da ischemia-riperfusione come riassunto nella tabella 2. Scegliere un metodo di punteggio appropriato. </li><li> Opzionale: Dividere il campo visivo in quattro sezioni poiché la gravità del danno varia in tutta la sezione. </li><li> Calcolare la media di ogni sezione da punteggi ottenuti alla cieca. </li><li> Confrontare il grado del tessuto danneggiato tra casi e di controllo nonché di the indenne tessuto utilizzando un test di Kruskal-Wallis, seguito dal test di confronto multiplo di un Dunn. </li></ol>

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<li>Tatum, P. M., Harmon, C. M., Lorenz, R. G., Dimmitt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-like+receptor+4+is+protective+against+neonatal+murine+ischemia-reperfusion+intestinal+injury%5BTitle%5D)+AND+%22J+Pediatr+Surg%22%5BJournal%5D)">Toll-like receptor 4 is protective against neonatal murine ischemia-reperfusion intestinal injury.</a> J Pediatr Surg. 45, 1246-1255 (2010).</li>
<li>Fleming, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-phospholipid+antibodies+restore+mesenteric+ischemia%2Freperfusion-induced+injury+in+complement+receptor+2%2Fcomplement+receptor+1-deficient+mice%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Anti-phospholipid antibodies restore mesenteric ischemia/reperfusion-induced injury in complement receptor 2/complement receptor 1-deficient mice.</a> J. Immunol. 173, 7055-7061 (2004).</li>
<li>Fleming, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mice+deficient+in+complement+receptors+1+and+2+lack+a+tissue+injury-inducing+subset+of+the+natural+antibody+repertoire%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire.</a> J. Immunol. 169, 2126-2133 (2002).</li>
<li>Lapchak, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelets+orchestrate+remote+tissue+damage+after+mesenteric+ischemia-reperfusion%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Platelets orchestrate remote tissue damage after mesenteric ischemia-reperfusion.</a> Am J Physiol Gastrointest Liver Physiol. 302, G888-G897 (2012).</li>
<li>Rehrig, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement+inhibitor%2C+complement+receptor+1-related+gene%2Fprotein+y-Ig+attenuates+intestinal+damage+after+the+onset+of+mesenteric+ischemia%2Freperfusion+injury+in+mice%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Complement inhibitor, complement receptor 1-related gene/protein y-Ig attenuates intestinal damage after the onset of mesenteric ischemia/reperfusion injury in mice.</a> J. Immunol. 167, 5921-5927 (2001).</li>
<li>Hoffman, S. M., Wang, H., Pope, M. R., Fleming, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+infection+alters+MyD88+and+Trif+signalling+in+response+to+intestinal+ischaemia-reperfusion%5BTitle%5D)+AND+%22Exp+Physiol%22%5BJournal%5D)">Helicobacter infection alters MyD88 and Trif signalling in response to intestinal ischaemia-reperfusion.</a> Exp Physiol. 96, 104-113 (2011).</li>
<li>Moses, T., Wagner, L., Fleming, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TLR4-mediated+Cox-2+expression+increases+intestinal+ischemia%2Freperfusion-induced+damage%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">TLR4-mediated Cox-2 expression increases intestinal ischemia/reperfusion-induced damage.</a> J Leukoc Biol. 86, 971-980 (2009).</li>
<li>Feinman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIF-1+mediates+pathogenic+inflammatory+responses+to+intestinal+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">HIF-1 mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury.</a> Am J Physiol Gastrointest Liver Physiol. 299, G833-G843 (2010).</li>
<li>Lapchak, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+platelet+factor+4+in+local+and+remote+tissue+damage+in+a+mouse+model+of+mesenteric+ischemia%2Freperfusion+injury%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">The role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury.</a> PloS one. 7, e39934(2012).</li>
<li>Wen, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemic+postconditioning+during+reperfusion+attenuates+intestinal+injury+and+mucosal+cell+apoptosis+by+inhibiting+JAK%2FSTAT+signaling+activation%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Ischemic postconditioning during reperfusion attenuates intestinal injury and mucosal cell apoptosis by inhibiting JAK/STAT signaling activation.</a> Shock. 38, 411-419 (2012).</li>
<li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+variations+of+the+ileal+microbiota+in+intestinal+ischemia+and+reperfusion%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Temporal variations of the ileal microbiota in intestinal ischemia and reperfusion.</a> Shock. 39, 96-103 (2013).</li>
<li>Zou, L., Attuwaybi, B., Kone, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+NF-kappa+B+inhibition+on+mesenteric+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Effects of NF-kappa B inhibition on mesenteric ischemia-reperfusion injury.</a> Am J Physiol Gastrointest Liver Physiol. 284, G713-G721 (2003).</li>
<li>Hassoun, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alpha-melanocyte-stimulating+hormone+protects+against+mesenteric+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Alpha-melanocyte-stimulating hormone protects against mesenteric ischemia-reperfusion injury.</a> Am J Physiol Gastrointest Liver Physiol. 282, G1059-G1068 (2002).</li>
<li>Stallion, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemia%2Freperfusion%3A+a+clinically+relevant+model+of+intestinal+injury+yielding+systemic+inflammation%5BTitle%5D)+AND+%22J+Pediatr+Surg%22%5BJournal%5D)">Ischemia/reperfusion: a clinically relevant model of intestinal injury yielding systemic inflammation.</a> J Pediatr Surg. 40, 470-477 (2005).</li>
<li>Blikslager, A. T., Roberts, M. C., Rhoads, J. M., Argenzio, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+reperfusion+injury+an+important+cause+of+mucosal+damage+after+porcine+intestinal+ischemia%3F%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">Is reperfusion injury an important cause of mucosal damage after porcine intestinal ischemia?</a> Surgery. 121, 526-534 (1997).</li>
<li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li>
<li>Victoni, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+and+remote+tissue+injury+upon+intestinal+ischemia+and+reperfusion+depends+on+the+TLR%2FMyD88+signaling+pathway%5BTitle%5D)+AND+%22Med+Microbiol+Immunol%22%5BJournal%5D)">Local and remote tissue injury upon intestinal ischemia and reperfusion depends on the TLR/MyD88 signaling pathway.</a> Med Microbiol Immunol. 199, 35-42 (2010).</li>
<li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-like+receptor+2+mediates+ischemia-reperfusion+injury+of+the+small+intestine+in+adult+mice%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Toll-like receptor 2 mediates ischemia-reperfusion injury of the small intestine in adult mice.</a> PloS one. 9, e110441(2014).</li>
<li>Pope, M. R., Fleming, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TLR2+modulates+antibodies+required+for+intestinal+ischemia%2Freperfusion-induced+damage+and+inflammation%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">TLR2 modulates antibodies required for intestinal ischemia/reperfusion-induced damage and inflammation.</a> J. Immunol. 194, 1190-1198 (2015).</li>
<li>Leung, F. W., Su, K. C., Passaro, E. Jr, Guth, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+differences+in+gut+blood+flow+and+mucosal+damage+in+response+to+ischemia+and+reperfusion%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Regional differences in gut blood flow and mucosal damage in response to ischemia and reperfusion.</a> Am J Physiol. 263, G301-G305 (1992).</li>
<li>Chiu, C. J., McArdle, A. H., Brown, R., Scott, H. J., Gurd, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+mucosal+lesion+in+low-flow+states.+I.+A+morphological%2C+hemodynamic%2C+and+metabolic+reappraisal%5BTitle%5D)+AND+%22Arch+Surg%22%5BJournal%5D)">Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal.</a> Arch Surg. 101, 478-483 (1970).</li>
<li>Quaedackers, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+evaluation+of+methods+for+grading+histologic+injury+following+ischemia%2Freperfusion+of+the+small+bowel%5BTitle%5D)+AND+%22Transplant+Proc%22%5BJournal%5D)">An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel.</a> Transplant Proc. 32, 1307-1310 (2000).</li>
<li>Bianciardi, P., Scorza, R., Ghilardi, G., Samaja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xanthine+oxido-reductase+activity+in+ischemic+human+and+rat+intestine%5BTitle%5D)+AND+%22Free+Radic+Res%22%5BJournal%5D)">Xanthine oxido-reductase activity in ischemic human and rat intestine.</a> Free Radic Res. 38, 919-925 (2004).</li>
<li>Yandza, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pig+as+a+preclinical+model+for+intestinal+ischemia-reperfusion+and+transplantation+studies%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">The pig as a preclinical model for intestinal ischemia-reperfusion and transplantation studies.</a> J Surg Res. 178, 807-819 (2012).</li>
</ol>

The authors declare no conflict of interest

Lo sviluppo di modelli murini di intestinale lesioni IR hanno notevolmente migliorato la comprensione dei meccanismi di danno tissutale e aiutato nello sviluppo di potenziali strategie terapeutiche per ridurre al minimo i danni ai tessuti 7,9,11,34. I passaggi critici di questo protocollo sono il corretto posizionamento delle clip microvascolari, corretta tempistica della valutazione ischemia e propria istologica di lesioni IR. La durata di ischemia è fondamentale per la successi...

L&#39;intestino è molto sensibile alla interruzione del flusso di sangue che provoca ischemia e epiteliale danni. Riperfusione dopo ischemia fornisce ri-ossigenazione dei tessuti, e può promuovere ulteriormente la patologia. Pertanto, intestinale danno da ischemia e riperfusione è associata ad una vasta gamma di patologie, tra cui enterocolite necrotizzante, il rigetto nel trapianto di intestino tenue, complicanze della chirurgia addominale aneurisma aortico, bypass cardiopolmonare, e infiammatoria cronica dell&#39;intestino 1,2. Lesioni intestinali IR, ischemia mesenterica particolarmente acuta, è una condizione di pericolo di vita con conseguente morbilità e mortalità 3. Anche se poco conosciuta, intestinale ischemia-riperfusione (IR) danno è pensato per essere associate a cambiamenti nella flora intestinale, nonché la produzione di specie reattive dell&#39;ossigeno e citochine infiammatorie e chemochine 1,4-6. Questo porta alla attivazione sia inNate e meccanismi immunitari adattivi che promuovono l&#39;infiammazione dei tessuti e lesioni 1,7,8. I modelli animali sono fondamentali per la comprensione dei meccanismi di danno IR, in quanto consentono un facile guadagno-e la perdita-di-funzione di esperimenti genetici. Diversi modelli animali di IR sono state sviluppate che includono occlusione vascolare completo, a basso flusso ischemia, e segmentato occlusione vascolare (riassunto in una recente revisione globale 9). Ischemia intestinale causata da occlusione vascolare completa di mesenterica superiore (SMA) è un modello semplice e comunemente usato di IR in grandi animali e roditori 9-11. Tuttavia, diverse aree del intestino hanno diversa suscettibilità al danno. Inoltre, la vasta gamma di anestetici, analgesici, tecniche di occlusione, così come incoerenza nella durata del danno ischemico e risultato del recupero in gradi variabili di danno confondendo la nostra comprensione della biologia di IR attraverso Studie multiplas. La tabella 1 mostra queste incongruenze negli studi IR murini. Il più grande svantaggio di utilizzare i tempi d&#39;ischemia più brevi (30-45 minuti) si rivolge la finestra di recupero su cui possono essere osservate differenze discernibili tra casi e controlli. lieve lesione all&#39;epitelio può essere risolto un&#39;ora dopo riperfusione, quindi specializzata metriche patologiche possono essere necessari per trovare le differenze di restituzione epiteliale. Al contrario, danni eccessivi, come visto da 100 min di lesione ischemica può comportare la denudement completa dell&#39;epitelio, qualora la restituzione non è più possibile, aumentare il tasso di mortalità, e tempo di recupero. Pertanto, qui si descrive la procedura dettagliata di IR intestinale nei topi che causa ferite riproducibili senza la mortalità per favorire la standardizzazione di questa tecnica in tutto il nostro campo. Questo modello di lesione intestinale IR può essere utilizzato per studiare i meccanismi cellulari e molecolari di lesioni e di rigenerazione.

<table><tbody><tr><td>Heated Pad</td><td>Sunbeam</td><td>E12107-819</td><td>Alternative: Braintree Scientific heated pad</td></tr><tr><td>Table top research anesthesia Machine</td><td>Vasco</td><td>UCAP 0001-0000171</td><td>Alternative: Parkland Scientific, V3000PS</td></tr><tr><td>Nose Cone</td><td>Parkland Scientific</td><td>ARES500</td><td></td></tr><tr><td>Scavenger canister and replacement cartridge</td><td>Parkland Scientific</td><td>80000, 80120</td><td></td></tr><tr><td>Induction Chamber</td><td>Surgivet</td><td>V711802</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare</td><td>NDC 66794-013-10</td><td>Controlled substance, contact IACUC</td></tr><tr><td>Animal clipper&amp;nbsp;</td><td>Oster&amp;nbsp;</td><td>Oster Golden A5 078005-050-003</td><td></td></tr><tr><td>Ophthalmic ointment</td><td>Webster</td><td>8804604</td><td></td></tr><tr><td>Buprenorphine</td><td>McKesson</td><td>562766</td><td>Controlled substance,contact IACUC</td></tr><tr><td>Ketaset (Ketamine HCl)</td><td>Pfizer</td><td>NADA 45-290</td><td>Controlled substance, contact IACUC</td></tr><tr><td>Cotton tips</td><td>Puritan medical products</td><td>806-WC</td><td>Autoclave before use</td></tr><tr><td>Betadine</td><td>Purdue Products</td><td>67618-150-17</td><td>10% Povidone-Iodine</td></tr><tr><td>Sterile saline solution</td><td>Aspen</td><td>46066-807-60</td><td>Adjust to room temperature before use</td></tr><tr><td>IR rodent thermometer</td><td>BIOSEB</td><td>BIO-IRB153</td><td></td></tr><tr><td>Micro vascular clips, 70 g</td><td>Roboz Surgical&amp;nbsp;</td><td>RS5424, RS5435</td><td>Alternative: WPI 14121, for SMA occlusion</td></tr><tr><td>Micro vascular clips, 40 g</td><td>Roboz Surgical&amp;nbsp;</td><td>RS6472</td><td>Alternative:WPI 14120, for collateral vessels occlusion</td></tr><tr><td>Clip applying forceps</td><td>World Precision Instruments</td><td>14189</td><td>Alternative: Roboz #RS-5410 or&amp;nbsp; #RS-5440</td></tr><tr><td>Gill&#039;s 3 hematoxylin</td><td>Thermo Scientific</td><td>14-390-17</td><td></td></tr><tr><td>Surgical staples, Reflex 9 mm</td><td>Cell Point Scientific</td><td>201-1000</td><td></td></tr><tr><td>Autoclip applier</td><td>Beckton Dickinson</td><td>427630</td><td></td></tr><tr><td>Byopsy foam pad</td><td>Simport</td><td>M476-1</td><td></td></tr><tr><td>Tissue cassette</td><td>Fisher Healthcare</td><td>15182701A</td><td>Histosette II combination lid and base</td></tr><tr><td>10% buffered formalin</td><td>Fisher Scientific</td><td>245-684</td><td></td></tr><tr><td>Surgical iris scissors</td><td>World Precision Instruments</td><td>501263-G SC</td><td>Alternative: Roboz RS6816</td></tr><tr><td>Operating scissors</td><td>World Precision Instruments</td><td>501219-G</td><td>Alternative: Roboz RS6814</td></tr><tr><td>Dressing forceps</td><td>Roboz Surgical&amp;nbsp;</td><td>RS-5228, RS-8122</td><td>Alternative: World Precision Instruments 1519-G</td></tr><tr><td>Heparin, endotoxin free, 300 USP units/vial, 50 mg</td><td>Sigma</td><td>2106</td><td></td></tr><tr><td>Reflex wound clip removing forceps</td><td>Roboz Surgical&amp;nbsp;</td><td>RS-9263</td><td>Alternative: World Precision Instruments: 500347</td></tr><tr><td>Mice C57BL/6J mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>Stock No 0664</td><td></td></tr><tr><td>Telfa non-adherent dressings, 3 x 4, sterile</td><td>Coviden</td><td>1050</td><td></td></tr><tr><td>Fisherbrand transfer pipets</td><td>Fischer Scientific</td><td>13-711-5AM</td><td>Use pipets to dropwise add saline</td></tr></tbody></table>

murine model of intestinal ischemia-reperfusion injury

ischemia intestinale è una condizione pericolosa per la vita associata con una vasta gamma di condizioni cliniche, tra cui l&#39;aterosclerosi, trombosi, ipotensione, enterocolite necrotizzante, il trapianto di intestino, traumi e l&#39;infiammazione cronica. Intestinal ischemia-riperfusione (IR) infortunio è una conseguenza di ischemia mesenterica acuta, causata dal flusso sanguigno insufficiente attraverso i vasi mesenterici, causando danni intestinali. Riperfusione dopo ischemia può esacerbare ulteriori danni dell&#39;intestino. I meccanismi di danno IR sono complessi e poco conosciuta. Pertanto, modelli sperimentali piccoli animali sono fondamentali per comprendere la fisiopatologia di lesioni IR e lo sviluppo di nuove terapie. Qui si descrive un modello murino di danno IR intestinale acuta che fornisce lesioni riproducibili del piccolo intestino, senza mortalità. Ciò si ottiene ischemia inducendo nella regione dell&#39;ileo distale temporalmente occludinag i rami collaterali periferici e terminali dell&#39;arteria mesenterica superiore per 60 minuti utilizzando clip microvascolari. Riperfusione per 1 ora, o 2 ore dopo i risultati di lesioni a danno riproducibile dell&#39;intestino esaminato da analisi istologica. La corretta posizione delle clip microvascolari è fondamentale per la procedura. Pertanto, il video clip fornisce una dettagliata descrizione visiva step-by-step di questa tecnica. Questo modello di lesione intestinale IR può essere utilizzato per studiare i meccanismi cellulari e molecolari di lesioni e di rigenerazione.

Abbiamo ottimizzato il protocollo sperimentale di chirurgia IR avere riproducibile lesioni IR indotta dell&#39;ileo nei topi. I risultati rappresentativi sono dimostrati in questa sezione. La Figura 1 mostra alcuni esempi di posizione clip microvascolari per indurre ischemia dell&#39;ileo. frecce nere indicano la posizione dei principali clip occlusione primi rami ordine di mesenterica superiore. Le frecce ver...

Watch this Scientific Journal Video about Murine Model of Intestinal Ischemia-reperfusion Injury at JoVE.com

Murine Model of Intestinal Ischemia-reperfusion Injury

Modello murino di ischemia-riperfusione intestinale Injury

Qui si descrive la procedura dettagliata di intestinale ischemia-riperfusione nei topi che causa ferite riproducibili senza la mortalità per favorire la standardizzazione di questa tecnica in tutto il campo. Questo modello di intestinale danno da ischemia-riperfusione può essere utilizzato per studiare i meccanismi cellulari e molecolari di lesioni e di rigenerazione.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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Medicine

19.5K Views.  Trudeau Institute.  Qui si descrive la procedura dettagliata di intestinale ischemia-riperfusione nei topi che causa ferite riproducibili senza la mortalità per favorire la standardizzazione di questa tecnica in tutto il campo. Questo modello di intestinale danno da ischemia-riperfusione può essere utilizzato per studiare i meccanismi cellulari e molecolari di lesioni e di rigenerazione. 

Video: Murine Model of Intestinal Ischemia-reperfusion Injury

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

A Murine chiuso del torace modello di ischemia miocardica e riperfusione

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

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Medicina

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Modello Murine dei tumori metastatici del fegato nell'impostazione della lesione alla riperfusione dell'ischemia

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Ricerca sul cancro

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

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

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

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

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

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Isolamento delle cellule staminali intestinale e cultura in un modello porcino di segmentale piccola Ischemia intestinale

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

Occlusione del vaso due Mouse modello di Ischemia-riperfusione cerebrale

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modello di infarto miocardico Murine utilizzando la legatura permanente dell'arteria coronaria discendente anteriore sinistra

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Un modello murino efficace di danno unilaterale da ischemia-riperfusione renale

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Induzione di infarto miocardico e danno da ischemia-riperfusione miocardica nei topi

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Modello di roditore di danno da ischemia-riperfusione intestinale attraverso l'occlusione dell'arteria mesenterica superiore

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...

Modello murino per lo studio del danno da ischemia-riperfusione polmonare

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows for&#160;detailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

Autore in primo piano: Approfondimenti sull'effetto della riperfusione ischemica sul trapianto di polmone

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse models.