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.

Allgemeine Hinweise: Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften durch die Tiere verhängt (Scientific Procedures) Act 1986 Dieses Protokoll und das dazugehörige Video-Protokoll sind für einen Standard- UUO und einem R-UUO, die auf vielen durchgeführt werden durchgeführt Mausstämme. In der dazugehörige Video sind beide Verfahren auf die männliche C57BL / 6 Mäusen im Alter von 8 Wochen durchgeführt. Die in der repräsentative Ergebnisse Abschnitt präsentierten Daten wurden von männlichen FVB / n Mäusen erhalten. Hinweis: Dieses Protokoll und die Video-Informationen begleiten, wie eine Standard UUO zuführen und R-UUO Verwendung der linken Ureter, jedoch können die gleichen Techniken leicht auf der rechten Harnleiter angewendet werden. 1. Vorbereitung der Tiere und Laparotomie <ol><li> Führen Sie alle mit sterilem (autoklaviert) Instrumente und Verbrauchsmaterialien. </li><li> Injizieren Ketaminhydrochlorid (70 mg / kg) und Medetomidin Hydrochlorid (1 mg / kg) intraperitoneal anästhesiertdie Maus. Anmerkung: Die Dauer des resultierenden Anästhesieebene 4 h und keine zusätzliche Anästhesie erforderlich. </li><li> Bestätigen Sie die Narkosetiefe durch Verlust von Reflexen zB Zeh Prise. </li><li> Entfernen Sie alle Haare rund um den Schnittbereich und bereiten die Bauchhaut für die Operation durch die Auftragung einer verdünnten Lösung Chlorhexidin. </li><li> Platzieren Sie die Maus auf einem beheizten Operations Pad in Rückenlage und mit Low-Tack Klebeband fixieren die Gliedmaßen auf das Pad. </li><li> Während der Prozedur überwacht die Maus auf Anzeichen von Verbrennungen aufgrund des erwärmten chirurgischen Pad. Wenn möglich, verwenden Sie eine nicht-elektrische Wärmequelle. </li><li> Verabreichen analgetische durch eine subkutane Injektion von Buprenorphin-Hydrochlorid (0,06 mg / kg) und wenden Augensalbe korneale Trocknen zu verhindern. </li><li> Machen Sie eine Mittellinie Bauchschnitt und einen Einschnitt der avaskulären Linea alba mit Trenngewebe Schere, um den Zugriff auf die Bauchhöhle zu gewinnen. </li><li> Hängen Sie die Maus und legen Sie eine colibriRetraktors in die Inzision. </li></ol> 2. Einseitige Ureterobstruktion <ol><li> Mit sterilisierten Wattestäbchen Setzen Sie das linke Harnleiter durch Verschieben der Darm auf die rechte Seite der Bauchhöhle und sie mit feuchten Tücher. </li><li> Mit abgewinkelten Pinzette isolieren und heben Sie den linken Ureter. </li><li> Um eine Ureterobstruktion erstellen, zu ligieren die linke Harnleiter zweimal mit 6 / A schwarz geflochtenen Seidennaht irgendwo zwischen der Blase und Nierenbecken. Für Langzeitversuche, verwenden resorbierbares Nahtmaterial für alle Bauchoperationen. Alternativ kann eine Ligaturklammer zum Harnleiter angewendet werden. </li><li> Um die Blase aus dem Harnleiter zu isolieren, teilen Sie die Harnleiter zwischen den beiden Nähten. </li><li> Die Eingeweide vorsichtig wieder in die Bauchhöhle. </li><li> Befolgen Sie die in Abschnitt 4 aufgeführten Schritte - postoperative Wiederherstellung und Pflege der Schnitt zu schließen und umgekehrt die Anästhesie. </li></ol> 3. Vorbereitung ReversiblE Einseitige Ureterobstruktion <ol><li> Bereiten Sie eine Maus für die Chirurgie und zu isolieren, die linke Harnleiter wie in den Schritten 1,1-2,2 oben beschrieben. </li><li> Um eine Ureterobstruktion, die rückgängig gemacht werden kann erstellen, zu ligieren die linke Harnleiter zweimal mit 6 / A schwarz geflochtenen Seidenfaden in der Nähe der Blase. Verlassen das eine Ende des oberen Fadens solange dies wird verwendet, um den weichen Kunststoffschlauch walled der richtigen Stelle um den Harnleiter zu verankern. </li><li> Einen Längsschlitz in einem 5 mm Länge der weichen Kunststoffschlauch walled so dass er offen, damit es auf den Harnleiter eingesetzt werden gespreizt werden. Jede weiche walled Silikonkunststoffschlauch mit einem Innendurchmesser von 1 mm und Außendurchmesser von 2 mm, können verwendet werden. </li><li> Setzen Sie den weichen ummauerten Kunststoffschlauch sanft um den Harnleiter. Sicherzustellen, daß die langen Naht taucht von der Mitte des Schlitzes in dem Rohr, sobald es um den Harnleiter geschlossen. </li><li> Legen Sie eine Länge von 6 / A-schwarzen geflochtenen Seidenfaden um die weichen, isolierter Rohr Einschließen des Harnleiters und Krawatteeinmal. Nun das lange Ende des Nahtmaterials, die sich aus der Mitte des Schlitzes in der Röhre, in Längsrichtung über den Schlauch, so dass sie auf der Oberseite des zuvor gebundenen Naht liegt. </li><li> Binden Sie die Naht um den Schlauch befindet zweimal, um den Schlauch und den langen Nahtende in Position um den Harnleiter zu verankern. Hinweis: Um die Bildung von Adhäsionen an der Harnleiter und der weichen Wandkunststoffschlauch zu verhindern, kann ein Haftreduktionslösung auf den Bereich um den Schlauch aufgetragen werden. </li><li> Die Eingeweide vorsichtig wieder in die Bauchhöhle. </li></ol> 4. Postoperative Erholung und Pflege <ol><li> Schließen Sie das Bauchfell mit Zierstich mit 5 / A-schwarzen geflochtenen Seidennaht und annähernd die Haut mit metallischen Hautklammern. </li><li> Um das Risiko einer postoperativen Infektion zu minimieren, setzen ein Antiseptikum, wie Iod / Alkohol-Lösung auf die Bauchhaut. </li><li> Teilweise umgekehrten Anästhesie mit Atipamezol Hydrochlorid (2 mg / kg) subkutan verabreicht. </li&gt; <li> Verwalten Flüssigkeiten durch eine subkutane Injektion von 1 ml Kochsalzlösung erwärmt. </li><li> Überwachen Sie die Maus, bis er das Bewusstsein wieder. </li><li> Lassen Sie die Maus, um in einem beheizten Box bei 29 ° C für 24 Stunden gehalten erholen. Angefeuchtet Essen kann auch vorgesehen sein, um Flüssigkeit und Nahrungsaufnahme fördern. </li><li> Lassen Sie die Maus, um zu erholen, um einen gewünschten Grad der Behinderung, in der Regel 7 Tage zu induzieren. </li><li> Für langfristige Erholung Experimente liefern laufenden Analgetika. Wenn die Maus für mehr als 7 Tagen wiederhergestellt werden, entfernen Sie die Hautklammern 7 Tage nach der Operation. </li><li> Sobald der gewünschte Grad der Verstopfung induziert worden ist, entweder die Rückwärts UUO, wie unten beschrieben, oder euthanize die Maus durch zervikale Dislokation und sammeln die Nieren für histopathologische Analyse. </li></ol> 5. Reversible Einseitige Ureterobstruktion <ol><li> So führen Sie eine R-UUO bereiten eine Maus, die Vorbereitung auf die R-UUO unterzogen wurde, für eine Operation wie in den Schritten 10,1-1,7. </li><li> Falls vorhanden, entfernen Sie die Hautklammern und teilen oder entfernen Sie die Fäden in das Bauchfell, um den Zugang zur Bauchhöhle zu gewinnen. Hinweis: Wenn die Harnleiter wurde für ein Langzeitversuch Hautklammern behindert worden war, sollte entfernt 7 Tage nach der Anwendung haben. </li><li> Bereiten Sie die Maus und isolieren Sie die linke Harnleiter wie in den Schritten 1,8 bis 2,2 beschrieben. </li><li> Mit abgewinkelten Pinzette, müssen Sie den weichen ummauerten Kunststoffschlauch von jedem Granulationsgewebe, die sich gebildet haben können. </li><li> Schneiden Sie den Faden Halten der Kunststoffschlauch um den verstopften Ureter mit einem Skalpell und den Schlauch zu entfernen. </li><li> Bestätigen erfolgreiche UUO durch die Beurteilung für das Vorhandensein von Hydronephrose in der linken Niere, sollte die Nieren auch erscheinen blass. </li><li> Teilen Sie den Harnleiter zwischen den Nähten. </li><li> Legen Sie die Restlänge der Harnleiter in die Niere angeschlossen ist, auf einem kleinen Stück steriler Gaze. Dies wird verwendet, um den Urinsediment und toten Zellen, die aus dem Harnleiter ein Ablass sammeln werdennd Nierenbecken, sobald das Nahtmaterial entfernt wird. </li><li> Teilen Sie den Harnleiter oben, aber in der Nähe, die Naht und lassen Sie die Niere auf die Gaze zu entleeren. Verlassen Sie die untere Naht, am nächsten an der Blase, intakt. Dies ist, um sicherzustellen, daß kein Urin aus der Blase und in die Bauchhöhle austreten. </li><li> Sobald die Harnleiter und Nierenbecken wurden abgelassen, gelten ein lang 6 / A-schwarzen geflochtenen Seidennaht bis zum Ende der Restlänge des Harnleiters. Dies wird verwendet, um den Harnleiter zu Blasen Anastomose später durchgeführt unterstützen. </li><li> Gehen Sie folgendermaßen vor, um die verbleibende Länge der Harnleiter in die Blase anastomosieren: <ol><li> Drehen des Harnleiters, daß er liegt vor in ihre ursprüngliche Position und liegt über die Niere. </li><li> Legen Sie eine einzelne 9 / A-Polyamid-Monofilament Heftnaht 2 mm vom Ende des Harnleiters, dass er in Richtung der Blase entsteht. Achten Sie darauf, dass der Faden bleibt in der Muskelschicht und nicht in die Harnröhre Lumen. Die Heftnaht will verwendet werden, um den Harnleiter in die Blase zu verankern. </li><li> Um einen Kanal durch die Blase zu erstellen, übergeben Sie eine 21G Nadel diagonal durch die Blase, so dass er in Richtung der vorderen (ventralen) Wand der Blase verlässt. </li><li> Seien Sie ein Augen Nadel in der Schräge der 21G Nadel. Verwenden Sie die 21G Nadel, um die Augen Nadel durch die Blase zu führen. Die gemusterte Nadel verwendet, um den Harnleiter durch die Blase zu tragen. </li><li> Passieren die 9 / O Heftnaht aufgebracht, um den Harnleiter in Schritt 5.11.2, durch den ersten Einschnitt in der Harnblase und durch die Blasenwand angrenzend an die Eintrittsstelle. Sobald der Harnleiter durch die Blase geleitet wird dies gebunden werden, um die Harnröhre in die Blase zu verankern. </li><li> Legen Sie die lange 6 / A-schwarzen geflochtenen Seidenfaden durch die Augen Nadel in Schritt 5.10 bis zum Ende des Harnleiters angewendet. </li><li> Die Augen Nadel vorsichtig herausziehen aus der Blase gleichzeitig sicherzustellen, dass der Harnleiter wird auch durch die Blase gezogen. </li><li> Sobald die Harnleiter geht aus the Blase entfernen gemustert Nadel und Anwendung einer Klammer an der langen Naht am Ende des Harnleiters Zurückziehen wieder in die Blase zu verhindern. </li><li> Um den Harnleiter im Ort in der Blase zu verankern, das Ja-9 / A-Polyamid-Monofilament Heftnaht im Schritt 5.11.5 angewendet. </li><li> Bewerben zusätzlichen Einzel 9 / A-Polyamid-Monofilament Krawatte Nähten wie in den Schritten 5.11.2 und 5.11.5 an zwei Standorten rund um den Einstieg beschrieben fest zu verankern den Harnleiter in die Blase. </li><li> Mit der Rückseite geöffnet Schere, drücken Sie die Blasenwand ein wenig zurück, um mehr des austretenden Harnleiter aus. </li><li> Unterteilen den Harnleiter über dem lang Naht am Ende des Harnleiters angeordnet. Der Harnleiter ist in den Körper der Blase zurückzuziehen. Es ist üblich, um Urin aus der Öffnung in der Blase fließt, zu beobachten, was bestätigt, das freie Lumen des Harnleiters. </li><li> Schließen Sie die Ausgabe in der Blase aufgewickelt mit einem 9 / A-Polyamid-Monofilament Naht. </li></ol></li><li> Die Eingeweide vorsichtig wieder indie Bauchhöhle. </li><li> Geben post-operative Betreuung wie in Schritten von 4,1 bis 4,8 beschrieben. </li><li> Lassen Sie die Maus, um zu erholen, bis die Nieren dekomprimiert, in der Regel 7 Tage. </li><li> Bei der experimentellen Endpunkt, einschläfern die Maus durch Genickbruch und sammeln die Nieren zur histopathologischen Analyse. </li></ol>

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Die Autoren haben keine Konkurrenz oder widerstreitende Interessen offen zu legen.

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.

Das Aussehen der Nierenveränderungen deutlich folgende Ureterobstruktion und es wird heller und angespannt Palpation mit der Zeit (Abbildung 1). Es gibt zunehmende Ausdehnung des Ureters proximal der Obstruktion und des Nierenbeckens. Die Niere wird zunehmend atrophische wie die Dauer obstruktiver Nephropathie steigt mit längerer Obstruktion was zu einer deutlichen Verdünnung der Nierenrinde und Medulla. Nach der Auflösung des Ureterobstruktion, wird die Farbe der Niere dunkler Blutfluss erhöht und...

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.

Ein Mausmodell der irreversiblen und reversiblen Einseitige Ureterobstruktion

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

30.4K Aufrufe.  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.

Serie: 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.

Erstellung von Reversible Cholestatische Rattenmodell

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

Forschung

Medizin

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.

Ein Modell von gequetscht Einseitige Cervical Spinal Cord Injury Mit dem 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 ...

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.

Quantitative Echtzeit-PCR-Bewertung von microRNA-Expressions in der Mausniere mit einseitiger ureteraler Obstruktion

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

Genetik

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 sparende Mittelharnröhrenobstruktion (NEMO) in Female Kleine Nagetiere

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.

Detrusor-Unteraktivitätsmodell bei Ratten durch Conus medullaris-Durchtrennung

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

Neurowissenschaften

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.

Ein effektives Mausmodell der einseitigen renalen Ischämie-Reperfusionsverletzung

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.

Mit der 2-Photonen-Mikroskopie die Auswirkungen einer chronischen einseitigen Harnleiterobstruktion auf glomeruläre Prozesse quantifizieren

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.

Ein unreifes Mausmodell einer reversiblen einseitigen Harnleiterobstruktion

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.

Einseitiges Harnleiterobstruktionsmodell zur Untersuchung der interstitiellen Nierenfibrose

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.

Einseitige Harnleiterobstruktion Mausmodell: Ein chirurgischer Eingriff zur Behinderung des Harnflusses aus einer Niere in Mausmodellen

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