Gli studi attuali sono stati sostenuti da United States National Institutes of Health-HL092188 concessione R01, R01-DK083385 e R01HL098294, e la Fondazione per l&#39;educazione e la ricerca Anestesia (Faer) concede HKE e una (Deutsche Forschungsgemeinschaft) DFG Research Fellowship (GR2121/1- 1) e dell&#39;American Heart Association Grant per AG.

Osservazioni generali. Tutte le operazioni devono essere eseguite con un microscopio da dissezione montante (Leica) e utilizzando un coagulatore chirurgico. I topi nei gruppi sperimentali e sham deve essere trovato al dell&#39;età e del peso per garantire la comparabilità dei risultati. Amministrazione temperatura, pressione arteriosa, anestesia e fluido deve essere stabile e monitorato durante l&#39;intero esperimento. 1. Anestesia e Chirurgia <ol><li> Topi C57BL / 6 dovrebbe essere all&#39;interno di un range di età da 10 a 16 settimane e dovrebbero essere in genere abbinato nei rispettivi gruppi. </li><li> Anestetizzare topi con sodio pentobarbital (70 mg / kg ip peso corporeo) e mantenere l&#39;anestesia con ca. 10 mg / kg / h di sodio pentobarbital. Sovradosaggio può ridurre significativamente la pressione sanguigna e quindi in grado di alterare i risultati. </li><li> Topi, posizionarli su una temperatura controllata tabella riscaldata (RT, Effenberg, Monaco di Baviera, Germania) con una sonda del termometro rettale collegato a un feedback termicoregolatore per mantenere la temperatura corporea a 37 ° C. </li><li> Topi posto in posizione supina sul tavolo operatorio, con le estremità superiori e inferiori attaccate al tavolo con del nastro adesivo rimovibile. </li><li> In caso di esperimenti di sopravvivenza corrette tecniche asettiche tra cui ritaglio di capelli, la disinfezione della pelle con iodofori (es. Betadine), gestione sterile degli strumenti autoclavati, e utilizzando le maschere a guanti sterili per il chirurgo sono obbligatori. </li></ol> 2. Portal Triad occlusione <ol><li> Coprire l&#39;area incisione con olio minerale per evitare l&#39;inalazione di capelli mouse tagliare la pelle e quindi prevenire il rischio di allergie capelli del mouse. Eseguire laparotomia linea mediana e l&#39;incisione della linea alba fino alla fine superiore in modo che si può facilmente esporre il fegato. Se possibile eseguire l&#39;incisione con un elettrodo coagulazione (Erbe, ICC50, Tuebingen, Germania) per prevenire le emorragie. </li><li> Posizionare il tavolo di riscaldamento con il mouse in una posizione che la coda è direttaTed al chirurgo. Tale posizione vi offre la migliore impostazione per localizzare la triade e di collocare la sutura. </li><li> Spostare l&#39;intestino sul lato destro utilizzando un tampone umido punta di cotone per esporre la terna portale. </li><li> Spostare leggermente il lobo mediano e sinistro verso il diaframma e il lobo destra verso l&#39;intestino. Questo vi darà una visione chiara della triade portale sopra la biforcazione di destra, mediana / sinistra lobi. </li><li> Ispezionare con cura la triade portale e tenere premuto il lobo destro dolcemente con una pinza blande. Questo vi darà una bella vista sulla triade portale di sinistra. </li><li> Una volta identificato visivamente posto l&#39;ago con la sutura (nylon 7/0 sutura, Ethicon, Norderstedt, Germania) tramite un supporto dell&#39;ago sotto la sinistra portale triade, compreso l&#39;arteria epatica, vena epatica, e dotto biliare da sinistra a destra del mouse. </li><li> Il punto deve essere a stretto contatto con la triade. Un punto più profondo possono ferire l&#39;vena vaca inferiore che si trova sottola triade. Pertanto tenere i lobi del fegato destro verso il basso con le pinze per identificare la vena cava prima di mettere il punto. </li><li> Prendere la punta dell&#39;ago sul lato destro con una pinza e tirare delicatamente fuori della zona del fegato senza danneggiare il fegato. </li><li> Attaccare a ciascuna estremità della sutura un tubo Eppendorf (circa 3 g, riempito con acqua) e posizionare sia sutura estremità su supporti di sutura. Posizionare il titolare sutura distale del punto in modo che la sutura non interferisce con la terna destra. </li><li> Applicando i pesi della triade sarà immediatamente occlusa, causando l&#39;interruzione di fornitura di sangue ai lobi sinistra e mediana del fegato. Occlusione di successo può essere facilmente confermata mediante ispezione visiva delle sbiancamento pallida dei lobi ischemiche. Al contrario, il cambiamento di colore scompare immediatamente quando i pesi sospesi vengono rilasciati dai poli e il fegato è riperfuso. Questo modello di parziale ischemia mesenterica evitare la congestione epatica, preservando Blooil flusso d per i lobi del fegato giuste. </li><li> Sostituire l&#39;intestino nella cavità addominale. Tenere il fegato e intestino caldo con un tampone umido imbevuto di acqua a 37,0 ° C. Oltre a mantenere la temperatura corporea stabile coprire i topi con l&#39;involucro di cibo disponibile in commercio dopo aver terminato l&#39;intervento chirurgico. </li><li> Eseguire ischemia come previsto da protocollo sperimentale. Dopo un comunicato ischemia determinato periodo di tempo i pesi e la riperfusione dei lobi epatici mediani e di sinistra inizierà immediatamente. Questa tecnica permette di produrre ischemia reversibile nei topi. Se non i tempi di ischemia verranno condotte ulteriori rimuovere con attenzione la sutura. </li><li> Aggiungere circa 400 microlitri di cloruro di sodio nella cavità addominale 15 minuti prima e direttamente dopo la riperfusione. Continuare a sostituire fluido ogni 30 minuti in una quantità di 100 pl per compensare la perdita di fluido durante il tempo riperfusione. </li><li> Per gli esperimenti di sopravvivenza della ferita chirurgica viene chiusa mediante sutura continua della parete muscolare e la pelle wi° a 4/0 sutura. </li></ol> 3. Variazioni perfusione epatica I parametri seguenti risultati si consiglia di determinare danni al fegato ischemia epatica seguente: Determinare enzimi epatici (ad esempio ALT, AST) e istologia epatica (colorazione H &amp; E) ischemia e riperfusione del fegato seguente 3,9. <img alt="Figura 1" src="/files/ftp_upload/2550/2550fig1.jpg" /> Figura 1 Modello di un pensile peso del sistema per ischemia del fegato:. Un sistema di pesi sospensione è stato istituito per indurre ischemia del fegato del lobo sinistro e mediano 3. (A) Pertanto, il lobo caudato destro e sono stati leggermente separato dal lobo sinistro. Il lobo destro è stato poi leggermente spostata per visualizzare chiaramente la triade portale sopra la biforcazione di destra, lobi mediani e sinistra. Un 8,0 sutura in nylon è stata posta sotto la triade epatico sinistro. (B) Alla fine di ciascuna sutura un piccolo peso (3 g Eppendorf tubes pieni d&#39;acqua) è stato attaccato e applicata. Ischemia epatica è visibile da un cambiamento di colore del lobo sinistro e mediano dal rosso pallido. Questo disegno sperimentale consente occlusione precisa e riperfusione della triade epatico sinistro applicando e rilasciando il carico peso. <img alt="Figura 2" src="/files/ftp_upload/2550/2550fig2.jpg" /> Figura 2. Effetto di diversi tempi di ischemia epatica in alanina (ALT) e aspartato (AST) aminotransferasi nei topi. Parziale portale ischemia triade è stata indotta come indicato (0 - 60 ischemia min). Dopo 2 ore di riperfusione, ALT (A) e AST (B) sono stati misurati. I risultati sono espressi come media ± SE di 6 - 7 topi per gruppo. * P &lt;0.05 rispetto allo 0 min ischemia (sham di controllo).

<ol>
	<li>Rook, M., Rand, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+long-term+outcome+after+liver+transplant.">Predictors of long-term outcome after liver transplant.</a> Curr. Opin. Organ. Transplant. 16, 499-504 (2011).</li><li>Henrion, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia/reperfusion+injury+of+the+liver:+pathophysiologic+hypotheses+and+potential+relevance+to+human+hypoxic+hepatitis.">Ischemia/reperfusion injury of the liver: pathophysiologic hypotheses and potential relevance to human hypoxic hepatitis.</a> Acta. Gastroenterol. Belg. 63, 336-347 (2000).</li><li>Hart, M. L., Much, C., Kohler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+hanging-weight+system+for+liver+ischemic+preconditioning+in+mice.">Use of a hanging-weight system for liver ischemic preconditioning in mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 294, 1431-1440 (2008).</li><li>Schneider, M., Geyte, K., Fraisl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+or+silencing+of+the+PHD1+prolyl+hydroxylase+protects+livers+of+mice+against+ischemia/reperfusion+injury.">Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.</a> Gastroenterology. 138, 1143-1154 (2010).</li><li>Coriat, R., Leconte, M., Kavian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mangafodipir+Protects+against+Hepatic+Ischemia-Reperfusion+Injury+in+Mice.">Mangafodipir Protects against Hepatic Ischemia-Reperfusion Injury in Mice.</a> PLoS One. 6, e27005 (2011).</li><li>Sakai, N., Van Sweringen, H. L., Schuster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+activator+of+NF-kappaB+ligand+(RANKL)+protects+against+hepatic+ischemia/reperfusion+injury+in+mice.">Receptor activator of NF-kappaB ligand (RANKL) protects against hepatic ischemia/reperfusion injury in mice.</a> Hepatology. , (2011).</li><li>Zhang, J., Hu, W., Xing, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Protective+Role+of+CD59+and+Pathogenic+Role+of+Complement+in+Hepatic+Ischemia+and+Reperfusion+Injury.">The Protective Role of CD59 and Pathogenic Role of Complement in Hepatic Ischemia and Reperfusion Injury.</a> Am. J. Pathol. 179, 2876-2884 (2011).</li><li>Beldi, G., Banz, Y., Kroemer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+CD39+on+natural+killer+cells+attenuates+hepatic+ischemia/reperfusion+injury+in+mice.">Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice.</a> Hepatology. 51, 1702-1711 (2010).</li><li>Hart, M. L., Much, C., Gorzolla, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+adenosine+production+by+ecto-5'-nucleotidase+protects+during+murine+hepatic+ischemic+preconditioning.">Extracellular adenosine production by ecto-5'-nucleotidase protects during murine hepatic ischemic preconditioning.</a> Gastroenterology. 135, 1739-1750 (2008).</li><li>Beraza, N., Ludde, T., Assmus, U., Roskams, T., Vander Borght, S., Trautwein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte-specific+IKK+gamma/NEMO+expression+determines+the+degree+of+liver+injury.">Hepatocyte-specific IKK gamma/NEMO expression determines the degree of liver injury.</a> Gastroenterology. 132, 2504-2517 (2007).</li><li>Lentsch, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+function+of+hepatocyte+NF-kappaB+in+postischemic+liver+injury.">Activation and function of hepatocyte NF-kappaB in postischemic liver injury.</a> Hepatology. 42, 216-218 (2005).</li><li>Luedde, T., Assmus, U., Wustefeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+IKK2+in+hepatocytes+does+not+sensitize+these+cells+to+TNF-induced+apoptosis+but+protects+from+ischemia/reperfusion+injury.">Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury.</a> J. Clin. Invest. 115, 849-859 (2005).</li><li>Kapitsinou, P. P., Liu, Q., Unger, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+HIF-2+regulates+erythropoietic+responses+to+hypoxia+in+renal+anemia.">Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia.</a> Blood. , (2010).</li></ol>

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Il presente studio descrive una tecnica di esecuzione ischemia in un modello murino utilizzando un pensile peso sistema di occlusione della triade sinistra epatica portale. Anche se è un modo semplice per imparare le insidie ​​seguenti dovrebbero essere presi in considerazione. Un errore molto comune all&#39;inizio è un punto profondo che comprende il portale triade e la vena cava. La frequenza respiratoria e cardiaca rallenterà entro 15 minuti a causa della maggiore resistenza e la diminuzione ritorno del sangue al cuore con conseguente morte prematura dei topi. Il modo migliore per prevenire l&#39;compreso della vena cava è quello di localizzare la vena cava prima che il punto per tenere i lobi destro verso il basso con una pinza. Un altro aspetto importante è che i pesi non sono pesanti che possono provocare troppo stress sul processo portale sinistro e destro che si ferma anche il flusso di sangue ai lobi giuste. Pertanto la quantità di acqua nel flaconcini Eppendorf deve essere regolata al peso del mouse e la stitolari FUTURO deve essere posizionato distalmente dalla posizione del punto. Al contrario se i pesi sono a bassa pressione sanguigna non è completamente fermo. Entrambi gli errori sono facilmente riconoscibili da un cambiamento di colore aggiuntivo pallido del lobo destro o cambiamento di colore insufficiente dei lobi mediani e di sinistra, rispettivamente. Pertanto, il cambiamento di colore epatica dopo l&#39;applicazione dei pesi dovrebbe essere controllato ogni 10 minuti. Un&#39;altra complicazione comune chirurgica è il ferimento del fegato con l&#39;ago. Pertanto i lobi diritto deve essere premuto con una pinza blande in modo che la punta dell&#39;ago può essere facilmente visto e preso con un porta-aghi. Se queste trappole in principio sono prese in considerazione il pensile peso sistema di ischemia epatica fornisce una lesione altamente riproducibile a causa di ischemia riducendo al minimo la variabilità del danno epatico associato serraggio della triade portale. Utilizzando un sistema pensile peso, la terna portale viene manipolato sola volta durante l&#39;interoprocedura chirurgica, causando danni molto meno ai lobi epatici. Inoltre, nessuna congestione epatica o intestinale si verifica con questa tecnica. Trauma dei tessuti più avviene rimuovendo e sostituendo i morsetti delle navi, in particolare con riapplicazione della pinza durante i cicli di ischemia più come durante precondizionamento ischemico. L&#39;uso di appendere pesi che sono in una posizione remota dal tessuto epatico, secondo le nostre osservazioni, fornisce il vantaggio di occlusione affidabile evitando trauma tessuto dovuto alla manipolazione dei lobi epatica di reapplication di un morsetto. Considerati nel loro insieme, il presente studio fornisce fattibilità del pensile peso del sistema per il portale triade occlusione durante l&#39;ischemia, riducendo al minimo la variabilità ei limiti associati a bloccaggio. Quindi questa tecnica potrebbe essere di interesse per altri ricercatori che considerano lo studio ischemia epatica.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Pentobarbital di sodio (Plus Fatal) </td><td> Vortech Pharmaceutical Ls, Ltd </td><td> VPL 9372 </td><td> 4 mg / ml in soluzione salina </td></tr><tr><td> Suture, Prolene 8,0 </td><td> Ethicon, USA </td><td> M8739 </td><td> riutilizzabile </td></tr><tr><td> dissezione microscopio </td><td> Leica </td><td> n / a </td><td> considerare generosa distanza di lavoro </td></tr><tr><td> Riscaldamento Table </td><td> Rt, Effenberger, Germania </td><td> n / a </td><td> unico fornitore e un&#39;unica </td></tr><tr><td> Strumenti chirurgici </td><td> WPI, Dumont </td><td></td><td> Pinze, porta aghi </td></tr><tr><td> Coagulatore </td><td> Erbe, ICC50, Tuebingen, Germania </td><td> n / a </td><td> Diversi fornitori di coagulatori </td></tr></tbody></table> Tabella 1. Reagenti e attrezzature specifiche.

use of a hanging-weight system for liver ischemia in mice

Danno epatico acuto dovuto a ischemia può verificarsi durante una serie di procedure cliniche di trapianto ad esempio il fegato, tumore resezione epatica o la riparazione del trauma e può portare a insufficienza epatica che ha un alto tasso di mortalità 1-2. Pertanto gli studi murini di ischemia epatica sono diventati un importante campo di ricerca, fornendo l&#39;opportunità di utilizzare studi farmacologici e genetici 3-9. In particolare, i topi condizionali con delezione tessuto-specifica di un gene (CRE, flox sistema) fornire indicazioni sul ruolo delle proteine ​​nei tessuti particolari 10-13. A causa delle difficoltà tecniche associate manualmente serraggio della triade portale in topi, abbiamo eseguito una valutazione sistematica utilizzando un pensile peso sistema di occlusione triade portale che è stato precedentemente descritto 3. Utilizzando un pensile peso del sistema mettiamo una sutura intorno al ramo sinistro della triade portale senza causare danni ai lobi epatici, dal momento che anche ilmigliori disponibili morsetti può causare danni ai tessuti epatica a causa della vicinanza del tessuto epatico ai vasi. Inoltre, il ramo di destra della triade epatica è ancora perfuso congestione intestinale quindi non si verifica con questa tecnica, come il flusso di sangue ai lobi epatici destro è conservato. Inoltre, la terna portale viene manipolato sola volta durante tutta la procedura chirurgica. Come risultato, come procedure di pre-condizionamento, con brevi tempi di ischemia e riperfusione, può essere eseguita facilmente. Valutazione sistematica di questo modello eseguendo diversi e tempi di ischemia riperfusione ha rivelato una stretta correlazione di tempo ischemia epatica con danni al fegato come misurato da alanina (ALT) e aspartato (AST), i livelli sierici di transaminasi 3,9. Presi insieme, questi studi confermano danno epatico altamente riproducibili quando si utilizza il pensile peso sistema di ischemia e di riperfusione epatica intermittente. Così, questa tecnica può essere utile per altre interesse investigatorinegli studi ed ischemia del fegato nei topi. Pertanto il video clip fornisce una descrizione dettagliata della descrizione dettagliata di questa tecnica.

Watch this Scientific Journal Video about Use of a Hanging-weight System for Liver Ischemia in Mice at JoVE.com

Use of a Hanging-weight System for Liver Ischemia in Mice

Abbiamo stabilito un nuovo modello murino di un sistema di peso appeso per il portale triade occlusione. Questa tecnica può essere utile per future di ischemia in modelli murini epatiche.

L&#39;uso di una Volante peso Sistema di Ischemia fegato nei topi

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

use-of-a-hanging-weight-system-for-liver-ischemia-in-mice

Research

JoVE Journal

Medicine

15.9K Views.  University of Colorado, Denver. Abbiamo stabilito un nuovo modello murino di un sistema di peso appeso per il portale triade occlusione. Questa tecnica può essere utile per future di ischemia in modelli murini epatiche.

Video: Use of a Hanging-weight System for Liver Ischemia in Mice

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

L&#39;uso di un sistema di peso per appendere l&#39;occlusione dell&#39;arteria coronarica nei topi

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Ricerca

Medicina

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

L&#39;uso di un Hanging-peso di sistema per isolati di occlusione dell&#39;arteria renale

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Uso di un modello animale di sepsi per valutare nuove terapie a base di erbe

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Rilevazione intraoperatoria di endometriosi sottile: un nuovo paradigma per la rilevazione e il trattamento del dolore pelvico associato con la perdita di integrità peritoneale

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

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

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

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

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

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

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis-indotta ischemia focale come un modello di lesioni del midollo spinale nei topi

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

L&#39;utilizzo di un basso flusso di sistema per anestesia digitale per topi e ratti

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

La conferma di ischemia miocardica e riperfusione nei topi mediante il rilievo della superficie elettrocardiografia

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Ultrasuono-guida di iniezione intracardiaca di cellule staminali mesenchimali umane per aumentare Homing all'intestino per l'utilizzo in modelli murini di malattie intestinali infiammatorie sperimentali

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Murine Precision-Cut Liver Slices come un modello Ex Vivo di biologia del fegato

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...