Laboratorio del Dr. de CAESTECKER è supportato dal NIH 1RO1 HL093057-01 e 1RC4DK090770-01. Laboratorio Dr. Harris &#39;è supportata da DK38226, DK51265, DK62794 e finanziamenti del Veterans Administration. Il supporto per il mouse rene ambulatori lesioni, creatinina sierica, e saggi fibrosi anche forniti dalla Vanderbilt O&#39;Brien Kidney Injury Centro concessione 1P30 DK079341.

<ol><li> Autoclave tutti gli strumenti chirurgici prima di un intervento chirurgico. Si noti che se si sta eseguendo interventi chirurgici multipli su diversi topi, sciacquare gli strumenti dopo l&#39;uso e poi sterilizzare utilizzando uno sterilizzatore tallone caldo. Non è sufficiente a bagno in 70% di etanolo. </li><li> Dare 0,5 ml S / C soluzione fisiologica sterile prima dell&#39;intervento e subito dopo l&#39;intervento per compensare la perdita di liquidi durante l&#39;intervento chirurgico. </li><li> Pesare i topi. </li><li> Anestetizzare il mouse utilizzando IP Xilazina / miscela ketamina. Di solito ci vogliono 3-5 minuti per i topi di raggiungere aereo chirurgico di anestesia. </li><li> Shave il sito chirurgico con zona di confine abbastanza per tenere i capelli di contaminare il sito di incisione. Questo deve essere eseguita in una zona di preparazione, non dove viene eseguito l&#39;intervento chirurgico. </li><li> Applicare pomata oftalmica lubrificante per gli occhi contro l&#39;essiccamento durante la procedura. </li><li> Posizionare il mouse prono sulla superficie riscaldata coperta con tampone assorbente panchina. Gambe nastro alla superficie chirurgico. </li><li> Se si utilizzail sistema bagnomaria, impostare termico a 38 ° C 1 h prima dell&#39;intervento. </li><li> Preparazione asettica usando Betadine soluzione tampone bastone: strofinare dal centro del sito verso la periferia 3x, ogni volta seguito da lavaggio con clorexidina per rimuovere il Betadine. Tutto Betadine deve essere rimosso dal campo operatorio prima dell&#39;incisione mediante risciacquo con clorexidina. Prima di iniziare la procedura di guanti asettici dovrebbe essere modificato per essere sterili. </li><li> Coprire campo chirurgico con un telo chirurgico sterile. Ciò impedirà capelli randagi di entrare nel campo chirurgico e fornire una superficie su cui posare strumenti sterili durante la chirurgia. </li><li> Palpare posizione del rene attraverso la pelle. </li><li> Tagliare la pelle dorsale lungo la linea mediana del topo (circa 1,5 cm) con forbici e pinze. </li><li> Pelle separata e strati sottocutanei sopra i lati dorsale sinistro e destro attraverso questa incisione per via smussa con forbici e pinze. </li><li> Fai una piccola incisione attraverso la fl dirittoank muscolo e fascia sopra il rene ed esteriorizzare il rene destro. </li><li> Sezionare attentamente i poli superiori e inferiori del rene liberi dal tessuto circostante. Si noti che il tessuto adiposo intorno al polo superiore contiene la ghiandola surrenale portando la propria fornitura di sangue che può essere spinta fuori dal rene ma ovviamente non deve essere rimosso dal topo (Figura 1). Dopo reni liberatorio dal tessuto circostante, legare la sutura di seta 4-0 intorno l&#39;ilo del rene destro con un doppio nodo chirurgico. Lascia una estremità della sutura ~ 2 a lungo per reggere il rene come si sta tagliando distale al nodo in modo da poter visualizzare il sito da tagliare. Irrigare il moncone con soluzione salina sterile. </li><li> Chiudere lo strato muscolare mediante sutura assorbibile. </li><li> Fare una piccola incisione attraverso il muscolo fianco sinistro e fascia sopra del rene e esteriorizzarsi rene sinistro (Figura 2A). </li><li> Tenere accuratamente rene con pinze smussato mentre rilasciando renalepeduncolo dal circostante tessuto adiposo con pinze (Figure 2B-2F). Troppa pressione sul rene usando la pinza smussato può causare danno renale così uno ha bisogno di ricordare questo durante la progettazione di controlli &quot;farsa&quot; per gli studi. Se si scopre che i controlli sham stanno sviluppando danno renale (come evidenziato da un aumento della creatinina sierica), un metodo alternativo si può prendere in considerazione per rilasciare il tessuto adiposo circostante dal peduncolo renale è di rotolare delicatamente i tessuti con soluzione salina sterile imbevuto di cotone tamponi con punta. Nella nostra esperienza il grasso è abbastanza saldamente attaccato al peduncolo renale e non può essere facilmente rimosso con tamponi di cotone, quindi questo non può essere il primo approccio che si vorrebbe utilizzare. Tuttavia, come altri aspetti di questo modello, questo è qualcosa che si dovrà stabilire per se stessi con la pratica e l&#39;esperienza. </li><li> Bloccare lasciato peduncolo renale utilizzando pinza vascolare non traumatico con Pinza (per facilitare il posizionamento pinza), utilizzare il timer per diverso clamp volte a seconda del ceppo di topi e di gravità degli infortuni prescritta (vedi Tabella 1). </li><li> Coprire il rene sinistro con la pelle (Figura 2H). </li><li> Coprire incisione cutanea con soluzione fisiologica garza imbevuta. </li><li> Esteriorizzare rene, confermare aspetto fosco uniforme e rilasciare il morsetto dopo gli orari indicati, e confermare che l&#39;aspetto fosco inverte in modo uniforme in tutto il rene (Figura 2I / G). Se i reni non rapidamente e in modo uniforme ri-profumato (fino rosa), il mouse deve essere esclusa da ulteriori analisi. Spingere delicatamente rene indietro nello spazio retroperitoneale. </li><li> Chiudere lo strato muscolare mediante sutura assorbibile. </li><li> Chiudere lo strato di pelle utilizzando monofilo di nylon sutura non assorbibile. </li><li> Somministrare buprenorfina analgesia. </li><li> Trasferire i topi lontano dal campo chirurgico su un rilievo di riscaldamento fino a quando si svegliano. </li><li> Riportare i topi per camera animale. </li><li> Monitorare attentamente i topi e dare ulteriori dosidella buprenorfina come indicato sopra ogni 8-12 ore per il dolore o disagio. I segni clinici con conseguente somministrazione di analgesici includono depressione o altre alterazioni del comportamento, aspetto anomalo, o la postura come pilo-erezione, la postura ingobbita, mancanza di governare e di mangiare, o immobilità. </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure.+II.+Experimental+models+of+acute+renal+failure:+Imperfect+but+indispensable.">Acute renal failure. II. Experimental models of acute renal failure: Imperfect but indispensable.</a> Am. J. Physiol. Renal Physiol. 278, F1-F12 (2000).</li><li>Dunn, S. R., Qi, Z., Bottinger, E. P., Breyer, M. D., Sharma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+endogenous+creatinine+clearance+as+a+measure+of+renal+function+in+mice.">Utility of endogenous creatinine clearance as a measure of renal function in mice.</a> Kidney Int. 65, 1959-1967 (2004).</li><li>Yuen, P. S., Dunn, S. R., Miyaji, T., Yasuda, H., Sharma, K., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+HPLC+determination+of+creatinine+in+mouse+serum.">A simplified method for HPLC determination of creatinine in mouse serum.</a> Am. J. Physiol. Renal Physiol. 286, F1116-F1119 (2004).</li><li>Hetu, P. O., Gingras, M. E., Vinet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+rapid+liquid+chromatography+isotope+dilution+tandem+mass+spectrometry+(LC-IDMS/MS)+method+for+serum+creatinine.">Development and validation of a rapid liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for serum creatinine.</a> Clin. Biochem. 43, 1158-1162 (2010).</li><li>Keppler, A., Gretz, N., Schmidt, R., Kloetzer, H. M., Groene, H. J., Lelongt, B., Meyer, M., Sadick, M., Pill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+creatinine+determination+in+mice+and+rats:+An+enzymatic+method+compares+favorably+with+a+high-performance+liquid+chromatography+assay.">Plasma creatinine determination in mice and rats: An enzymatic method compares favorably with a high-performance liquid chromatography assay.</a> Kidney Int. 71, 74-78 (2007).</li><li>Yang, L., Besschetnova, T. Y., Brooks, C. R., Shah, J. V., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+cycle+arrest+in+G2/M+mediates+kidney+fibrosis+after+injury.">Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.</a> Nat. Med. 16, 535-543 (2010).</li><li>Shanley, P. F., Rosen, M. D., Brezis, M., Silva, P., Epstein, F. H., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+focal+proximal+tubular+necrosis+after+ischemia+with+reflow+in+the+rat+kidney.">Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.</a> Am. J. Pathol. 122, 462-468 (1986).</li><li>Chawla, L. S., Amdur, R. L., Amodeo, S., Kimmel, P. L., Palant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+severity+of+acute+kidney+injury+predicts+progression+to+chronic+kidney+disease.">The severity of acute kidney injury predicts progression to chronic kidney disease.</a> Kidney Int. 79, 1361-1369 (2012).</li><li>Lo, L. J., Go, A. S., Chertow, G. M., McCulloch, C. E., Fan, D., Ordonez, J. D., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialysis-requiring+acute+renal+failure+increases+the+risk+of+progressive+chronic+kidney+disease.">Dialysis-requiring acute renal failure increases the risk of progressive chronic kidney disease.</a> Kidney Int. 76, 893-899 (2009).</li><li>Burne, M. J., Haq, M., Matsuse, H., Mohapatra, S., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+to+renal+ischemia+reperfusion+injury+revealed+in+a+murine+model.">Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model.</a> Transplantation. 69, 1023-1025 (2000).</li><li>Muller, V., Losonczy, G., Heemann, U., Vannay, A., Fekete, A., Reusz, G., Tulassay, T., Szabo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+renal+ischemia-reperfusion+injury+in+rats:+Possible+role+of+endothelin.">Sexual dimorphism in renal ischemia-reperfusion injury in rats: Possible role of endothelin.</a> Kidney Int. 62, 1364-1371 (2002).</li><li>Schmitt, R., Marlier, A., Cantley, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zag+expression+during+aging+suppresses+proliferation+after+kidney+injury.">Zag expression during aging suppresses proliferation after kidney injury.</a> J. Am. Soc. Nephrol. 19, 2375-2383 (2008).</li><li>Oxburgh, L., de Caestecker, M. P. <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+mouse+kidney.">Ischemia-reperfusion injury of the mouse kidney.</a> Methods Mol. Biol. 886, 363-379 (2012).</li></ol>

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Descriviamo due modelli di IR-AKI per studiare gli effetti di danno renale moderata e grave. Questi modelli ci permettono di indurre lesioni coerente e prevedibile con una bassa mortalità. Il nostro protocollo delinea molte delle difficoltà e le insidie ​​tradizionalmente associate a questo modello. Inoltre, abbiamo dimostrato che in base alla lunghezza del peduncolo renale bloccaggio, il modello induce una gran parte reversibile lieve e moderata AKI, o più grave AKI con recupero incompleto e fibrosi renale persi...

Una varietà di modelli sperimentali di danno renale acuto (AKI) sono stati sviluppati per soddisfare la diversità e la complessità della condizione umana (cfr. domanda di recente, revisione completa 1). Ognuno di questi modelli ha i propri punti di forza e di debolezza, e mentre ogni imita le corrispondenti condizioni umane con diverse efficienze, nessuno modellare precisamente la fisiopatologia delle loro controparti umane. Ischemia riperfusione (IR) indotta AKI è stato sviluppato come un modello di danno renale indotta da ischemia acuta nei roditori. Mentre la gravità del danno tubulare renale visto in questo modello si riscontra raramente in pazienti con lesioni ipoperfusione renale 2, nonostante i suoi limiti e in gran parte a causa della natura relativamente riproducibile di questo modello, il suo largo uso ha fornito e, si prevede, continuerà fornire importanti intuizioni molti dei meccanismi comuni di base di AKI, la riparazione e la terapia 3. Chirurgia IR richiede familiarity con l&#39;anatomia rene di topo, che abbiamo illustrato in forma semplificata in Figura 1. Chirurgia danno da ischemia-riperfusione renale (IR) può essere effettuata tramite (retroperitoneale) approcci ventrali (laparotomia) o dorsale. Usiamo un approccio dorsale dal momento che è meno traumatico, consentendo tempi di recupero più rapidi e una migliore sopravvivenza (in particolare quando prima imparare la procedura). Lesioni IR renale può essere eseguita unilateralmente o bilateralmente. Tuttavia, le differenze tra le effettive pressioni clamp (che possono derivare da interposizione di grasso peri-ilare fra le ganasce della pinza) e / o differenze nelle risposte renali ai ischemia tra le parti porta a risultati più variabili. Mentre questo non è un problema insormontabile, si può aumentare la variabilità tra esperimenti, che è una questione importante per questo modello. IR unilaterale può essere effettuata con nefrectomia controlaterale. Questo è il nostro metodo di scelta poiché riduce variabilità nella chiusura tra peduncoli, e allo stesso tempo unllows uno per valutare la funzione renale, che non è influenzato solo con IR unilaterale. C&#39;è stata discussione su quello che è il metodo più pratico vs ottimale per valutare la funzione renale nei topi. Azoto ureico ematico (BUN) fornisce una misura della funzione renale ed è un utile &quot;primo sguardo&quot; marcatore in alcuni modelli di AKI, tra cui lesioni IR. Tuttavia, i livelli di BUN possono essere influenzati dallo stato del volume dei topi che possono essere colpiti, in particolare a seguito di approccio ventrale infortunio IR, quando ritardato recupero riduce l&#39;assunzione orale di liquidi per un numero di giorni post-operatorie. La creatinina sierica è meno influenzato da stato di idratazione, ma è chiaramente influenzata dalla massa muscolare. Una delle difficoltà con i livelli di creatinina sierica è stato problemi con il rilevamento di cromogeni non-creatinina nel siero del mouse usando le tecniche base di acido picrico. In alternativa, un certo numero di centri hanno sviluppato un metodo HPLC-based per quantificare creatinina topo che non è influenzata da questo artefatto 4. Tuttavia, UNLike BUN e dell&#39;acido picrico test della creatinina, che richiedono solo il 5-10 ml di siero, HPLC-saggi richiedono ~ 25 microlitri di siero per dosaggio, che se eseguita in duplice copia richiederà ~ 100 ml di sangue intero per ogni dosaggio. Questo può essere limitante per gli studi del mouse. Alcuni centri hanno sviluppato metodi HPLC e spettrometria di massa basata più sensibili che permettono l&#39;analisi di piccoli volumi di campione 5, 6. Tuttavia, queste tecnologie non sono ampiamente disponibili. Un&#39;alternativa, saggio cascata enzimatica (che richiede solo 5-10 ml di siero) è stato valutato nel topo e nel ratto campioni di siero e mostrato a parallelo strettamente misure mediante HPLC della creatinina sierica, mentre dosaggi dell&#39;acido picrico sempre i valori di creatinina sovrastimare 7. Mentre questo dosaggio non è ampiamente utilizzato in letteratura AKI, il saggio è disponibile in commercio, semplice da utilizzare, e troviamo dà risultati affidabili con questo modello di IR indotta AKI nei topi.

<table border="1">
	<tbody>
		<tr>
			<td>Drug or solution</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ketamine (anesthetic)</td>
			<td>Webster Veterinary</td>
			<td>07-881-9413 (100 mg/ml)</td>
			<td>Final concentration 30 mg/ml Ketamine with 3 mg/ml Xylazine administered at 3-4 ml/kg (100 mg/kg Ketamine and 10 mg/kg Xylazine)</td>
		</tr>
		<tr>
			<td>Xylazine (analgesic)</td>
			<td>Webster Veterinary</td>
			<td>07-808-1939 (100 mg/ml)</td>
			<td>As above</td>
		</tr>
		<tr>
			<td>Antisedan (anti-Xylazine)</td>
			<td>Webster Veterinary</td>
			<td>07-867-7097 (5 mg/ml)</td>
			<td>Optional: to reverse effects of Xylazine in order to increase the rate of recovery after surgery Use 0.5-0.7 mg/kg</td>
		</tr>
		<tr>
			<td>Buprenorphine (analgesic)</td>
			<td>Bedford Laboratories</td>
			<td>NDC 55390-100-10 (0.3 mg/ml)</td>
			<td>Dose 0.05-0.1 &mu;g/g every 8-12 hr. Make up a 1:5 dilution in sterile normal saline preoperatively and give 2.5-5 &mu;l/g of the diluted solution.</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>NDC 17033-211-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine swab sticks</td>
			<td>Purdue Products L.P.</td>
			<td>NDC 67618-153-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nolvasan Surgical Scrub</td>
			<td>Pfizer Animal Care</td>
			<td>300253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Drugs and solutions for I/R surgery.</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Integra Miltex</td>
			<td>VWR 21909-404</td>
			<td>For skin and muscle dissection</td>
		</tr>
		<tr>
			<td>Curved iris forceps</td>
			<td>FST</td>
			<td>11065-07</td>
			<td>To hold skin and muscle, to remove peri-nephric fat</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>ROBOZ</td>
			<td>RS-5228</td>
			<td>To manipulate kidneys</td>
		</tr>
		<tr>
			<td>Vascular clamp (795 g pressure)</td>
			<td>ROBOZ</td>
			<td>RS-5459</td>
			<td>Good for C57BL/6 and BALB/c mice strains</td>
		</tr>
		<tr>
			<td>Schwartz clip applying forceps</td>
			<td>ROBOZ</td>
			<td>RS-5450</td>
			<td>For RS-5459 clamps</td>
		</tr>
		<tr>
			<td>Vascular clamp (75-85 g pressure)</td>
			<td>FST</td>
			<td>18055-02</td>
			<td>Optimal for CD1 mice strain, but also can be used for C57BL/6 and BALB/c</td>
		</tr>
		<tr>
			<td>Micro-Serrefine Clamp Applicator-14 cm with Lock</td>
			<td>FST</td>
			<td>18056-14</td>
			<td>For FST 18055-02 clamps</td>
		</tr>
		<tr>
			<td>Gayman T/Pump (water-bath heated platform)</td>
			<td>Braintree Scientific</td>
			<td>TP-650</td>
			<td>The water heater is set at 38 &deg;C 1 hr prior surgery</td>
		</tr>
		<tr>
			<td>15" W x 24" Heated platform</td>
			<td>Braintree Scientific</td>
			<td>HHP-2</td>
			<td>Connect to Gayman heated water pump</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree Scientific</td>
			<td>Model 39DP</td>
			<td>Uses microwave for heating. This is useful for warming during recovery</td>
		</tr>
		<tr>
			<td>Absorbable suture (Vicryl 5-0)</td>
			<td>ETHICON</td>
			<td>VCP834G</td>
			<td>For fascia and muscle</td>
		</tr>
		<tr>
			<td>Non-absorbable suture: monofilament nylon MONOMID 6/0 660B)</td>
			<td>CP Medical</td>
			<td>CP-B660B-03</td>
			<td>For skin</td>
		</tr>
		<tr>
			<td>Halsey needle holder: Tungsten Carbide/13 cm</td>
			<td>FST</td>
			<td>12501-13</td>
			<td>To hold suture needles</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>FST</td>
			<td>06-662-3</td>
			<td>To quantify ischemia time</td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>FST</td>
			<td>18000-45</td>
			<td>To sterilize instruments between mice if you are performing multiple surgeries (it is not sufficient to soak instruments in ethanol)</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Braintree Scientific</td>
			<td>CLP-22965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Durapore</td>
			<td>1538-0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>EXELint</td>
			<td>26044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicators</td>
			<td>Kendall</td>
			<td>8884541300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent BenchPad</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical drapes</td>
			<td>VWR</td>
			<td>21902-985</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr>
			<td>2'' x 2'' Gauze Pads</td>
			<td>Medi-First</td>
			<td>60673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Cardinal Health</td>
			<td>2D7251</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Equipment and instruments for I/R surgery.</td>
		</tr>
	</tbody>
</table>

ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice

Ischemia-riperfusione danno renale acuto indotto (IR-AKI) è ampiamente usato come modello di AKI nei topi, ma i risultati sono spesso molto variabile, con alti tassi di mortalità, spesso non dichiarata che potrebbero confondere le analisi. Bilaterale renale peduncolo di bloccaggio è comunemente usato per indurre IR-AKI, ma le differenze tra le pressioni efficaci clamp e / o risposte renali a ischemia tra reni spesso portano a risultati più variabili. Inoltre, i tempi di serraggio più brevi sono noti per indurre più variabile danno tubulare, e mentre i topi sottoposti a lesione bilaterale con tempi più lunghi clamp sviluppano più consistente danno tubulare, spesso muoiono entro i primi 3 giorni dopo la lesione dovuta a grave insufficienza renale. Per migliorare la sopravvivenza post-infortunio e ottenere risultati più coerenti e prevedibili, abbiamo sviluppato due modelli di unilaterale ischemia-riperfusione seguita da nefrectomia controlaterale. Entrambi gli interventi chirurgici vengono eseguiti utilizzando un approccio dorsale, riducendo lo stress chirurgico conseguente from laparotomia ventrale, comunemente utilizzata per il mouse IR-AKI ambulatori. Per l&#39;induzione di lesioni lievi topi BALB / c subiscono unilaterale di serraggio del peduncolo renale per 26 min e anche sottoposti a nefrectomia simultanea controlaterale. Usando questo approccio, il 50-60% dei topi sviluppano moderata AKI 24 ore dopo la lesione, ma 90-100% dei topi sopravvivere. Per indurre più grave AKI, topi BALB / c sono sottoposti peduncolo renale bloccaggio per 30 min seguita da nefrectomia controlaterale 8 giorni dopo la lesione. Questo permette la valutazione funzionale del recupero renale dopo l&#39;infortunio con il 90-100% di sopravvivenza. All&#39;inizio danno tubulare post-infortunio, così come la fibrosi messaggio lesioni sono altamente coerenti con questo modello.

Moderato IR indotta AKI. IR unilaterale con simultanea nefrectomia controlaterale riduce sostanzialmente variabilità nei risultati, ma con tempi più brevi di serraggio necessarie per i topi per sopravvivere a questa procedura, abbiamo ancora trovato che solo il 50-60% dei topi ha sviluppato l&#39;atteso insufficienza renale 24 ore dopo la lesione (Figura 3A). In termini pratici, questo crea difficoltà di valutazione dei dati a meno che gli studi riguardano i regimi di trattamento che...

Watch this Scientific Journal Video about Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice at JoVE.com

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Descriviamo modelli di danno renale da ischemia-riperfusione indotta moderata e grave in cui i topi sottoposti unilaterale peduncolo renale serraggio seguita da nefrectomia controlaterale simultaneo o ritardato, rispettivamente. Questi modelli sempre danno luogo a disfunzione renale e fibrosi post-infortunio, ma la gravità delle lesioni e la sopravvivenza dipendono da sfondo, età e attrezzature chirurgiche mouse.

Ischemia-riperfusione Modello di danno renale acuta e post Infortunio fibrosi nei topi

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

ischemia-reperfusion-model-acute-kidney-injury-post-injury-fibrosis

Research

JoVE Journal

Medicine

39.4K Views.  Vanderbilt University Medical Center. Descriviamo modelli di danno renale da ischemia-riperfusione indotta moderata e grave in cui i topi sottoposti unilaterale peduncolo renale serraggio seguita da nefrectomia controlaterale simultaneo o ritardato, rispettivamente. Questi modelli sempre danno luogo a disfunzione renale e fibrosi post-infortunio, ma la gravità delle lesioni e la sopravvivenza dipendono da sfondo, età e attrezzature chirurgiche mouse.

Video: Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Normotermico arresto cardiaco e rianimazione cardiopolmonare: un modello murino di ischemia-riperfusione

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Ricerca

Medicina

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Induzione e valutazione di ischemia-riperfusione Injury in Langendorff-perfusi Hearts Rat

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Modello murino di ischemia-riperfusione intestinale Injury

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

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Valutazione della riserva del flusso coronario dopo la miaocardia Ischemia Reperfusione nei ratti

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Un modello di ratto preclinico per lo studio delle lesioni ischemia-reperfusione in microchirurgia ricostruttiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Consegna intramiocardica ritardata di cellule staminali dopo l'infortunio di ricottamento ischemia in un modello Murine

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Modello di lesione renale acuta indotto dal cisplatino nel pesce zebra adulto

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lesione da ischemia-riperfusione dell'organo simulando cambiamenti emodinamici nel modello di trapianto di fegato di ratto

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Un modello di trapianto di polmone di ratto di ischemia calda / danno da riperfusione: ottimizzazioni per migliorare i risultati

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

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 ...