Name: a murine model of irreversible and reversible unilateral ureteric obstruction
Uploaded: 2014-12-20T15:00:00.000+00:00
Duration: 14 min 5 s
Description: Watch this Scientific Journal Video about A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction at JoVE.com

The present study was supported by grants from Kidney Research UK (ST4/2011), the Cunningham Trust (CT11/14), the Mrs AE Hogg Charitable Trust for Kidney Research and the Renal Endowment Fund of the Royal Infirmary of Edinburgh.

Guida Generale: Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e regolamenti imposti dagli Animali (Procedure Scientifiche) Act 1986. Questo protocollo e il protocollo video che accompagna sono sia un UUO standard e un R-UUO, che può essere eseguita su molti ceppi di topi. Nel video che accompagna, entrambe le procedure sono effettuate su maschi C57BL / 6 topi di età compresa tra 8 settimane. I dati presentati nella sezione risultati rappresentativi sono stati ottenuti da maschi FVB / n topi. NOTA: Questo protocollo e accompagnamento dettagli video come eseguire un UUO standard R-UUO utilizzando dell&#39;uretere sinistro, ma le stesse tecniche possono essere facilmente applicati al dell&#39;uretere destra. 1. Preparazione animali e laparotomia <ol><li> Eseguire tutte le procedure con (autoclave) gli strumenti e materiali di consumo sterili. </li><li> Iniettare ketamina cloridrato (70 mg / kg) e cloridrato medetomidina (1mg / kg) per via intraperitoneale per anestetizzareil mouse. Nota: La durata del piano anestetico risultante è 4 ore e non necessita di anestesia supplementare. </li><li> Confermare la profondità dell&#39;anestesia dalla perdita di riflessi, per esempio pizzico punta. </li><li> Rimuovere tutti i capelli che circonda l&#39;area di incisione e preparare la pelle per la chirurgia addominale mediante applicazione di una soluzione diluita di clorexidina. </li><li> Posizionare il mouse su un tappetino riscaldato chirurgica in posizione supina e fissare gli arti al pad con bassa aderenza del nastro adesivo. </li><li> Durante la procedura, monitorare il mouse per segni di ustioni a causa del pad chirurgica riscaldato. Se possibile utilizzare una fonte di calore non elettriche. </li><li> Amministrare analgesico da una iniezione sottocutanea di buprenorfina cloridrato (0.06mg / kg) e applicare il lubrificante occhio per evitare l&#39;essiccazione della cornea. </li><li> Effettuare una laparotomia mediana e una incisione delle alba avascolare Linea usando forbici tessuto separazione per ottenere l&#39;accesso alla cavità peritoneale. </li><li> Proietta il mouse e inserire un colibririavvolgitore nella incisione. </li></ol> 2. unilaterale Ureteric Ostruzione <ol><li> Usando bastoncini di cotone sterilizzati esporre dell&#39;uretere sinistro spostando l&#39;intestino verso il lato destro della cavità addominale e coprirli con teli inumiditi. </li><li> Utilizzando pinze ad angolo isolano e sollevare dell&#39;uretere sinistro. </li><li> Per creare una ostruzione ureterale, legare dell&#39;uretere sinistro due volte con 6 / O nera intrecciata sutura di seta ovunque tra la vescica e pelvi renale. Per gli esperimenti a lungo termine, di utilizzare suture assorbibili per tutti gli interventi chirurgici addominali. In alternativa, una clip di legatura può essere applicato dell&#39;uretere. </li><li> Per isolare la vescica dal dell&#39;uretere, dividere l&#39;uretere tra i due punti di sutura. </li><li> Ricollocare accuratamente l&#39;intestino nella cavità peritoneale. </li><li> Seguire le istruzioni di cui alla sezione 4 - recupero post-operatorio e cura per chiudere l&#39;incisione e invertire la anestesia. </li></ol> 3. Preparazione Reversible unilaterale Ureteric Ostruzione <ol><li> Preparare un mouse per la chirurgia e isolare dell&#39;uretere sinistro come descritto nei passaggi 1,1-2,2 sopra. </li><li> Per creare una ostruzione ureterale che può essere invertito, legare l&#39;uretere sinistro due volte con 6 / O nero intrecciato sutura di seta vicino alla vescica. Lasciare una estremità della sutura superiore finché questo sarà utilizzato per ancorare il tubo di plastica morbida murato in luogo intorno dell&#39;uretere. </li><li> Effettuare una feritoia longitudinale in una lunghezza di 5 mm di tubazione di plastica morbida murata tale che possa essere strombato aperto per permettere di essere applicato al dell&#39;uretere. Qualsiasi tubazione di plastica morbido silicone murata, con un diametro interno di 1 mm e diametro esterno di 2 mm, può essere utilizzato. </li><li> Posizionare il tubo di plastica murato morbida dolcemente intorno l&#39;uretere. Assicurarsi che il lungo sutura emerge dal centro della fessura nel tubo una volta chiuso attorno dell&#39;uretere. </li><li> Mettere una lunghezza di 6 / O nera intrecciata sutura di seta attorno al tubo murato morbido che racchiude l&#39;uretere e cravattauna volta. Ora posizionare l&#39;estremità lunga della sutura, emergente dal centro della fessura nel tubo, longitudinalmente attraverso il tubo in modo da giacere sopra della sutura precedentemente legato. </li><li> Legare la sutura si trova attorno al tubo per due volte per ancorare il tubo e la fine lunga sutura sul posto intorno al uretere. Nota: Per evitare la formazione di adesione su dell&#39;uretere e il tubo di plastica morbida murata, una soluzione di riduzione adesione può essere applicato alla zona attorno al tubo. </li><li> Ricollocare accuratamente l&#39;intestino nella cavità peritoneale. </li></ol> 4. Post-Operative recupero e cura <ol><li> Chiudere il peritoneo con punto coperta con 5 / O nera intrecciata sutura di seta e approssimare la pelle con clip pelle metallica. </li><li> Per minimizzare il rischio di infezioni post-operatorie, applicare tale antisettico come soluzione di iodio / alcool alla pelle addominale. </li><li> Parzialmente anestesia indietro con cloridrato atipamezolo (2 mg / kg) per via sottocutanea. </li&gt; <li> Somministrare fluidi da una iniezione sottocutanea di 1 ml di soluzione salina riscaldato. </li><li> Monitorare il mouse fino a che non ha recuperato la coscienza. </li><li> Lasciare il mouse per recuperare in una scatola riscaldata mantenuto a 29 ° C per 24 ore. Cibo umido può essere fornita anche per incoraggiare l&#39;assunzione di liquidi e la nutrizione. </li><li> Lasciare il mouse per recuperare per indurre un livello desiderato di ostruzione, tipicamente 7 giorni. </li><li> Per gli esperimenti di recupero a lungo termine, fornire analgesici in corso. Se il mouse è da recuperare per più di 7 giorni, rimuovere le clip di pelle 7 giorni dopo l&#39;intervento chirurgico. </li><li> Una volta che il livello desiderato di ostruzione è stata indotta, sia invertire la UUO, come descritto di seguito, o eutanasia il mouse per dislocazione cervicale e raccogliere i reni per l&#39;analisi istopatologica. </li></ol> 5. reversibile unilaterale Ureteric Ostruzione <ol><li> Per eseguire un R-UUO preparare un topo, che ha subito la preparazione per la R-UUO, per la chirurgia come descritto ai punti 10,1-1,7. </li><li> Se presente, rimuovere i fermagli pelle e dividere o rimuovere le suture nel peritoneo per accedere alla cavità addominale. Nota: Se l&#39;uretere è stato ostacolato per un lungo periodo clip pelle esperimento avrebbe dovuto essere rimosso 7 giorni dopo l&#39;applicazione. </li><li> Preparare il mouse e isolare dell&#39;uretere sinistro come descritto nei punti 1,8-2,2. </li><li> Utilizzando pinze ad angolo, liberare il tubo di plastica morbida murato da qualsiasi tessuto granulomatoso che si possono formare. </li><li> Tagliare la sutura tenendo il tubo di plastica intorno l&#39;uretere ostruito con un bisturi e rimuovere il tubo. </li><li> Conferma successo UUO valutando la presenza di idronefrosi nel rene sinistro, il rene dovrebbe apparire pallido. </li><li> Dividete l&#39;uretere tra le suture. </li><li> Posizionare la lunghezza rimanente dell&#39;uretere, attaccato al rene, su un piccolo pezzo di garza sterile. Questo verrà utilizzato per raccogliere il sedimento urinario e cellule morte che si scarica dal dell&#39;uretere unnd pelvi renale volta la sutura viene rimosso. </li><li> Dividere l&#39;uretere sopra, ma vicino, la sutura e consentire il rene di drenare sulla garza. Lasciare la sutura inferiore, più vicino alla vescica, intatta. Questo per garantire che nessun urina fuoriuscire dalla vescica e nella cavità peritoneale. </li><li> Una volta che l&#39;uretere e pelvi renale sono state drenato applicare una lunga 6 / O nero intrecciato sutura di seta alla fine della lunghezza rimanente dell&#39;uretere. Questo sarà usato per aiutare l&#39;uretere per anastomosi vescica eseguita successivamente. </li><li> Seguire i passi di seguito per anastomosi la durata residua del uretere nella vescica: <ol><li> Ruotare l&#39;uretere tale che si trova anteriormente alla sua posizione originale e si trova sopra il rene. </li><li> Inserire un singolo 9 / O poliammide monofilamento incollamento sutura 2 mm dal terminale dell&#39;uretere tale che emerge nella direzione della vescica. Fare attenzione al fine di garantire che la sutura rimane nella tunica muscolare e non entra il lume uretere. Il wil virata sutural essere utilizzato per ancorare l&#39;uretere nella vescica. </li><li> Per creare un canale attraverso la vescica, passare un ago 21G diagonalmente attraverso la vescica tale che esce verso il frontale (ventrale) parete della vescica. </li><li> Resto un ago eyed nella smussatura dell&#39;ago 21G. Utilizzare l&#39;ago 21G per guidare l&#39;ago occhi attraverso la vescica. L&#39;ago occhi sarà usato per prendere l&#39;uretere attraverso la vescica. </li><li> Passare il 9 / O incollamento sutura, applicato al dell&#39;uretere nel passaggio 5.11.2, attraverso la prima incisione nella vescica e fuori attraverso la parete della vescica adiacente al punto di ingresso. Una volta che l&#39;uretere viene fatto passare attraverso la vescica sarà legato per ancorare l&#39;uretere alla vescica. </li><li> Posizionare la lunga 6 / O intrecciato nero sutura di seta applicata all&#39;estremità dell&#39;uretere nel passaggio attraverso l&#39;ago 5.10 occhi. </li><li> Sfilare delicatamente l&#39;ago occhi fuori della vescica assicurando al contempo che l&#39;uretere è anche tirato attraverso la vescica. </li><li> Una volta che l&#39;uretere emerge da the vescica rimuovere l&#39;ago occhi e applicare un morsetto alla lunga sutura alla fine dell&#39;uretere per impedire retrazione nella vescica. </li><li> Per ancorare l&#39;uretere all&#39;interno posto nella vescica, legare il 9 / O poliammide monofilamento tack sutura, applicato in fase 5.11.5. </li><li> Applicare unici aggiuntivi 9 / O poliammide suture monofilamento cravatta come descritto nei punti 5.11.2 e 5.11.5 a due posizioni intorno al punto di ingresso per ancorare saldamente l&#39;uretere all&#39;interno della vescica. </li><li> Utilizzando la parte posteriore di forbici aperte, spingere la parete della vescica leggermente indietro per esporre più dell&#39;uretere emergente. </li><li> Dividere dell&#39;uretere sopra lungo sutura situato all&#39;estremità dell&#39;uretere. L&#39;uretere deve rientrare nel corpo della vescica. Si è soliti osservare urine scorre fuori dall&#39;apertura nella vescica, confermando il lume aperte dell&#39;uretere. </li><li> Chiudere l&#39;uscita avvolto nella vescica con una poliammide sutura monofilamento 9 / O. </li></ol></li><li> Ricollocare accuratamente l&#39;intestino inla cavità peritoneale. </li><li> Fornire assistenza post-operatoria, come descritto nei passaggi 4,1-4,8. </li><li> Lasciare che il mouse per recuperare fino a quando il rene viene decompresso, tipicamente 7 giorni. </li><li> Al punto finale sperimentale, eutanasia il mouse dislocazione cervicale e raccogliere i reni per l&#39;analisi istopatologica. </li></ol>

<ol>
	<li>Chevalier, R. L., Forbes, M. S., Thornhill, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureteral+obstruction+as+a+model+of+renal+interstitial+fibrosis+and+obstructive+nephropathy.">Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.</a> Kidney Int. 75, 1145-1152 (2009).</li><li>Kipari, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+is+an+important+mediator+of+renal+tubular+epithelial+cell+death+in+vitro+and+in+murine+experimental+hydronephrosis.">Nitric oxide is an important mediator of renal tubular epithelial cell death in vitro and in murine experimental hydronephrosis.</a> Am J Pathol. 169, 388-399 (2006).</li><li>Henderson, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-3+expression+and+secretion+links+macrophages+to+the+promotion+of+renal+fibrosis.">Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis.</a> Am J Pathol. 172, 288-298 (2008).</li><li>Kitamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+liposome+clodronate+on+renal+leukocyte+populations+and+renal+fibrosis+in+murine+obstructive+nephropathy.">Effects of liposome clodronate on renal leukocyte populations and renal fibrosis in murine obstructive nephropathy.</a> J Pharmacol Sci. 111, 285-292 (2009).</li><li>Tapmeier, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reimplantation+of+the+ureter+after+unilateral+ureteral+obstruction+provides+a+model+that+allows+functional+evaluation.">Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation.</a> Kidney Int. 73, 885-889 (2008).</li><li>Cochrane, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+structural+and+functional+repair+in+a+mouse+model+of+reversal+of+ureteral+obstruction.">Renal structural and functional repair in a mouse model of reversal of ureteral obstruction.</a> J Am Soc Nephrol. 16, 3623-3630 (2005).</li><li>Puri, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+kidney+disease+induced+in+mice+by+reversible+unilateral+ureteral+obstruction+is+dependent+on+genetic+background.">Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background.</a> Am J Physiol Renal Physiol. 298, F1024-F1032 (2010).</li><li>Heikkila, J., Holmberg, C., Kyllonen, L., Rintala, R., Taskinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+risk+of+end+stage+renal+disease+in+patients+with+posterior+urethral+valves.">Long-term risk of end stage renal disease in patients with posterior urethral valves.</a> J Urol. 186, 2392-2396 (2011).</li><li>Ravanan, R., Tomson, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+postobstructive+nephropathy:+a+single-center+retrospective+study.">Natural history of postobstructive nephropathy: a single-center retrospective study.</a> Nephron Clin Pract. 105, c165-c170 (2007).</li><li>Bascands, J. L., Schanstra, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obstructive+nephropathy:+insights+from+genetically+engineered+animals.">Obstructive nephropathy: insights from genetically engineered animals.</a> Kidney Int. 68, 925-937 (2005).</li><li>Park, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postobstructive+regeneration+of+kidney+is+derailed+when+surge+in+renal+stem+cells+during+course+of+unilateral+ureteral+obstruction+is+halted.">Postobstructive regeneration of kidney is derailed when surge in renal stem cells during course of unilateral ureteral obstruction is halted.</a> Am J Physiol Renal Physiol. 298, F357-F364 (2010).</li><li>Manson, S. R., Niederhoff, R. A., Hruska, K. A., Austin, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+BMP-7+is+a+critical+molecular+determinant+of+the+reversibility+of+obstruction-induced+renal+injuries.">Endogenous BMP-7 is a critical molecular determinant of the reversibility of obstruction-induced renal injuries.</a> Am J Physiol Renal Physiol. 301, F1293-F1302 (2011).</li><li>Manson, S. R., Niederhoff, R. A., Hruska, K. A., Austin, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP-7-Smad1/5/8+pathway+promotes+kidney+repair+after+obstruction+induced+renal+injury.">The BMP-7-Smad1/5/8 pathway promotes kidney repair after obstruction induced renal injury.</a> J Urol. 185, 2523-2530 (2011).</li><li>Chaves, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+effects+of+systemic+monocyte/macrophage+and+CD4++T+cell+depletion+in+a+reversible+ureteral+obstruction+mouse+model+of+chronic+kidney+disease.">Contrasting effects of systemic monocyte/macrophage and CD4+ T cell depletion in a reversible ureteral obstruction mouse model of chronic kidney disease.</a> Clin Dev Immunol. 2013, 836-989 (2013).</li><li>Haque, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+changes+in+MRI+markers+in+a+reversible+unilateral+ureteral+obstruction+mouse+model:+preliminary+experience.">Longitudinal changes in MRI markers in a reversible unilateral ureteral obstruction mouse model: preliminary experience.</a> J Magn Reson Imaging. 39, 835-841 (2014).</li><li>Bai, Z. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterially+transplanted+mesenchymal+stem+cells+in+a+mouse+reversible+unilateral+ureteral+obstruction+model:+in+vivo+bioluminescence+imaging+and+effects+on+renal+fibrosis.">Arterially transplanted mesenchymal stem cells in a mouse reversible unilateral ureteral obstruction model: in vivo bioluminescence imaging and effects on renal fibrosis.</a> Chinese Med J. 126, 1890-1894 (2013).</li><li>Chaabane, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+functional+decline+and+glomerulotubular+injury+are+arrested+but+not+restored+by+release+of+unilateral+ureteral+obstruction+(UUO).">Renal functional decline and glomerulotubular injury are arrested but not restored by release of unilateral ureteral obstruction (UUO).</a> Am J Physiol Renal Physiol. 304, F432-F439 (2013).</li><li>Thornhill, B. A., Burt, L. E., Chen, C., Forbes, M. S., Chevalier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+chronic+partial+ureteral+obstruction+in+the+neonatal+rat:+a+new+model+of+ureteropelvic+junction+obstruction.">Variable chronic partial ureteral obstruction in the neonatal rat: a new model of ureteropelvic junction obstruction.</a> Kidney Int. 67, 42-52 (2005).</li><li>Thornhill, B. A., Chevalier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+partial+unilateral+ureteral+obstruction+and+its+release+in+the+neonatal+and+adult+mouse.">Variable partial unilateral ureteral obstruction and its release in the neonatal and adult mouse.</a> Methods Mol Biol. 886, 381-392 (2012).</li></ol>

Gli autori non hanno concorrenti o conflittuali interessi da dichiarare.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. The R-UUO model is of relevance to human disease as patients often present with obstruction of the renal tract secondary to prostatic hypertrophy, posterior urethral valves etc. that requires decompression with patients commonly left with significant residual kidney impairment that has no current treatment options and may eventually lead to end stage kidney disease.8,9 The models detailed here enable ...

The overall aim of the experimental model described here is to induce obstructive nephropathy by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). A simple irreversible model of UUO is presented in which the left ureter is permanently obstructed by ligation with a suture or by the application of a ligating clip. This results in marked dilatation of the ureter together with reduced renal blood flow and glomerular filtration. Renal histology demonstrates tubular dilatation and increasingly severe interstitial renal inflammation and fibrosis. Irreversible UUO is a useful model and has been adopted by many researchers in the study of both renal inflammation and fibrosis.1-4 Although the irreversible model of UUO requires some surgical expertise it is relatively straightforward and is often used to seek insights into the pathogenesis of interstitial renal injury and the ensuing fibrosis. Also presented is a less frequently used R-UUO model using a modification of the method originally described by Tapmeier et al.5 The R-UUO model has much future potential for the study of inflammatory and immune processes, cellular and tissue regeneration as well as the subsequent tissue remodeling following the removal of an injurious stimulus. 
The more challenging R-UUO model has been used by a limited number of investigators with some groups employing a significantly different surgical technique to that described here6,7 though with interesting results. In the R-UUO model presented, the ureter is ligated to induce complete ureteric obstruction for a period of time sufficient to induce the level of injury and fibrosis desired: 7 days of UUO was chosen in the method described here. The ureteric obstruction is reversed and the kidney is allowed to decompress for a period of time determined by the investigator before the mice are culled and the kidney removed for analysis: 7 days of decompression was chosen in the method described here though a longer period would be chosen if the resolution of inflammation and fibrosis was being studied. Although the method described here requires significant surgical expertise, it offers several advantages over other R-UUO models. The application of soft walled plastic tubing to the obstructed ureter prevents excessive ureteric dilation and this facilitates the subsequent manipulation and anastomosis of the ureter. Furthermore, in the described R-UUO model the ureter is divided thereby allowing the removal of any residual urinary sediment and debris. This confirms that the remaining ureter lumen is de-obstructed and patent prior to anastomosis to the bladder. 
Experiments incorporating both the irreversible and reversible UUO models can provide researchers with a powerful insight into the molecular and cellular mechanisms of both injury and subsequent resolution and regeneration. Thus, the model of R-UUO described here would be highly relevant to researchers interested in post-inflammatory tissue remodeling and how this can be modulated.

<table><tbody><tr><td>Blunt Dissecting Scissors</td><td>Fine Science Tools</td><td>14072-10</td><td></td></tr><tr><td>Spring Scissors &amp;ndash; straight</td><td>Fine Science Tools</td><td>15000-10</td><td></td></tr><tr><td>Toothed forceps</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>Angled forceps x 2</td><td>Fine Science Tools</td><td>00649-11</td><td></td></tr><tr><td>Straight forceps</td><td>Fine Science Tools</td><td>00632-11</td><td></td></tr><tr><td>Colibri 3 cm wire retractor</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Castroviejo needle holder with lock</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Wound clip applicator</td><td>Fine Science Tools</td><td>12031-07</td><td></td></tr><tr><td>7 mm wound clips</td><td>Fine Science Tools</td><td>12032-07</td><td></td></tr><tr><td>Castroviejo micro needle holder with lock</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>OPMI pico microscope</td><td>Carl Zeiss</td><td>S100</td><td></td></tr><tr><td>Heat electronic pad</td><td>Cozee Comfort</td><td>n/a</td><td></td></tr><tr><td>6/O silk braided suture</td><td>Harvard Apparatus</td><td>72-3287</td><td></td></tr><tr><td>9/O Dafilon (polyamide) suture</td><td>B-Braun</td><td>G1111434</td><td></td></tr><tr><td>5/O braided silk suture</td><td>Harvard Apparatus</td><td>51-7680</td><td></td></tr><tr><td>Regular bevel needle, 1 inch, 21G</td><td>Becton, Dickinson and Company</td><td>305175</td><td></td></tr><tr><td>1 ml syringe slip tip</td><td>Becton, Dickinson and Company</td><td>300184</td><td></td></tr><tr><td>Wypall paper swabs</td><td>Kimberley-Clark</td><td>L40</td><td>Sterilised (Autoclave)</td></tr><tr><td>Cotton wool buds</td><td>Johnson and Johnson</td><td>n/a</td><td>Sterilised (Autoclave)</td></tr><tr><td>Plain drapes</td><td>Guardian</td><td>CB03</td><td>Sterilised (Autoclave)</td></tr><tr><td>Soft wall silicone rubber tubing</td><td></td><td></td><td>Silicone tubing - internal diameter 1.0 mm; external diameter 2.0 mm; wall thickness 0.6 mm</td></tr><tr><td>(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2%</td><td>Allergan Ltd</td><td>21956GB10X</td><td></td></tr><tr><td>(Videne) Povidone-iodine 10%</td><td>Ecolab Ltd</td><td>PL 04509/0041</td><td></td></tr><tr><td>(Vetalar V) Ketamine hydrochloride</td><td>Pfizer Animal Health</td><td>Vm 42058/4165</td><td>100mg/ml solution</td></tr><tr><td>(Domitor) Medetomidine hydrochloride</td><td>Orion Pharma</td><td>Vm 06043/4003</td><td>1mg/ml</td></tr><tr><td>(Vetergesic) Bupernorphine hydrochloride</td><td>Alsto Animal Health</td><td>Vm 00063/4002</td><td>0.3mg/ml</td></tr><tr><td>(Antisedan) Atipamezole hydrochoride</td><td>Orion Pharma</td><td>Vm 06043/4004</td><td>5mg/ml</td></tr><tr><td>(Adept) 4% Icodextrin</td><td>Baxter</td><td></td><td>Adhesion reduction solution</td></tr><tr><td>NaCl 0.9%</td><td>Baxter</td><td>FKE1323</td><td></td></tr></tbody></table>

a murine model of irreversible and reversible unilateral ureteric obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

L&#39;aspetto delle modifiche renali marcatamente seguente ostruzione degli ureteri e diventa pallido e teso alla palpazione con il tempo (Figura 1). Vi è una crescente dilatazione dell&#39;uretere prossimale all&#39;ostruzione e pelvi renale. Il rene diventa sempre atrofica come la durata della nefropatia ostruttiva aumenta con ostruzione prolungata con conseguente notevole assottigliamento della corteccia renale e midollare. Dopo l&#39;inversione di ostruzione ureterale, il colore del rene diventa pi...

Watch this Scientific Journal Video about A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction at JoVE.com

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Un modello murino di irreversibile e reversibile unilaterale Ureteric Ostruzione

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

a-murine-model-irreversible-reversible-unilateral-ureteric

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

30.3K Views.  University of Edinburgh. The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Video: A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Creation of Reversible Cholestatic Rat Model

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and affect the nutritional status and surgical outcomes. Study of the pathophysiological changes occurring in the liver and other organs is of importance. Various studies have been done in cholestatic rat models.
We used a reversible cholestatic rat model in our recent study looking at the role of methylprednisolone in the ischemia reperfusion injury. Various techniques for creation of a reversible cholestatic model have been described. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.
This model can be used in various studies, such as looking at the changes in nutritional, physiological, pathological, histological and immunological changes in the gastrointestinal tract. This model can also be used to see the effects of cholestasis and various therapeutic interventions on major hepatic surgeries.

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.

Creazione del Modello reversibile Rat colestatico

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and ...

Ricerca

Medicina

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modello contusivo unilaterale di lesioni del midollo spinale cervicale Uso della Impactor Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

Valutazione PCR in tempo reale quantitativa delle espressioni di microRNA nel rene del topo con ostruzione ureterale unilaterale

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Genetica

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Nerve-sparing Mid-ostruzione uretrale (Nemo) nel femminile piccoli roditori

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was performed in a total of 40 female Wistar rats (control group: 10 rats; test group: 30 rats) weighing 200&#8211;220 g, and the conus medullaris was transected at the L4&#8210;L5 level in the test group. All the rats were housed and fed under the same environmental conditions for six weeks. In the test group, urine voiding was performed twice daily for six weeks, and mean residual urine volume was recorded. A cystometrogram was performed in both groups. Maximum cystometric capacity (MCC), detrusor opening pressure (DOP), and compliance of the bladder were recorded and calculated. The test group showed significant urinary retention after the surgery, both during and after the spinal shock. However, no abnormality was observed in the control group. When compared to the control group, the MCC and compliance of bladder in the test group was significantly higher than that of the test group (3.24 &#177; 2.261 mL versus 1.04 &#177; 0.571 mL; 0.43 &#177; 0.578 mL/cmH2O versus 0.032 &#177; 0.016 mL/cmH2O), whereas DOP in the test group was lower than control (20.28 &#177; 14.022 cmH2O versus 35 &#177; 13.258 cmH2O). This method of establishing an animal model of DU by the conus medullaris transection offers an excellent opportunity to understand DU&#8217;s pathophysiology in a better manner.

We present a method for establishing a detrusor underactivity model by conus medullaris transection in rats. Detrusor underactivity was successfully stimulated in these animals. The model can be used for studying urinary tract function.

Modello di sottoattività del detrusore nei ratti mediante transezione di Conus medullaris

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

Neuroscienze

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 ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Utilizzo della microscopia a 2 fotoni per quantificare gli effetti dell'ostruzione ureterale unilaterale cronica sui processi glomerulari

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Un modello murino immaturo di ostruzione ureterale unilaterale reversibile

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely used to elucidate the molecular and cellular mechanisms underlying kidney interstitial fibrosis and to identify potential therapeutic targets. This model is established in mice through surgical ligation of a unilateral ureter, a procedure that is relatively straightforward and easy to perform. However, the UUO mouse model is known to exhibit significant variability and inconsistency, influenced by factors such as mouse strain, age, sex, anesthesia type, duration of surgery, body temperature during the procedure, the operator's surgical skills, feeding conditions, and the overall health status of the mice. Variations in surgical techniques, suture placement, and the duration of obstruction contribute to the variability in outcomes. Additionally, inconsistent sampling of obstructed kidneys further increases variability in the assessment of kidney fibrosis. This study outlines the process of developing the UUO mouse model and evaluating interstitial fibrosis, discusses the technical challenges contributing to the model's unpredictability, and proposes potential solutions. These insights aim to establish a more standardized and universally applicable approach for investigating kidney fibrosis.

This protocol describes a method for inducing unilateral ureteral obstruction (UUO) in mice to study the progression of tissue fibrosis in obstructive nephropathy. It includes surgical procedures, post-operative care, and methods for fibrosis assessment.

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely ...

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

Source: Yanai, K. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/61383" target="_blank">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Modello murino di ostruzione ureterale unilaterale: una procedura chirurgica per ostruire il flusso urinario da un rene in modelli murini

Source: Yanai, K. et al. Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction. J. Vis. Exp. ...