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.

一般的なガイダンス：動物実験は、動物によって課せられたガイドラインおよび規則に従って（科学的処置）法1986このプロトコルと多くの上で実行することができます付属のビデオプロトコル標準UUOとR-UUO両方のためのもの、で行ったマウス系統。添付のビデオでは、両方の手順は、8週齢のオスのC57BL / 6マウスで実行されます。代表的な結果のセクションで提示されたデータは、オスのFVB / Nマウスから得た。 注：このプロトコルは、左尿管を利用して、標準的なUUO及びR-UUOを実行する方法ビデオの詳細を伴うが、しかし、同じ技術を容易に右尿管にも適用することができる。 1.動物の準備および開腹<ol><li>無菌（オートクレーブ処理）楽器や消耗品を持つすべての手順を実行します。 </li><li>麻酔腹腔内に塩酸ケタミン（70mgの/ kg）及びメデトミジン塩酸塩（1mgの/ kg）を注入マウス。注意：結果の麻酔薬平面の持続時間は4時間で、全く補足麻酔は必要ありません。 </li><li>反射神経例えば、つま先のピンチの損失によって麻酔の深さを確認してください。 </li><li>切開領域周辺の毛を削除し、希薄クロルヘキシジン溶液の適用を介して手術のために腹部の皮膚を準備します。 </li><li>仰臥位で加熱された外科パッドの上にマウスを置き、低粘着性接着テープを使用してパッドに手足を固定する。 </li><li>手順の間に、加熱された手術用パッドの結果として、熱傷の兆候がないマウスを監視します。可能であれば、非電気的熱源を使用しています。 </li><li>塩酸ブプレノルフィン（0.06mg / kg）を皮下注射によって鎮痛を投与し、角膜の乾燥を防ぐために、眼の潤滑剤を適用する。 </li><li>正中開腹と腹腔へのアクセスを得るために組織分離ハサミを使って無血管白線の切開を行います。 </li><li>マウスをドレープコリブリを挿入切開に開創。 </li></ol> 2.片側尿管閉塞<ol><li>滅菌綿棒を使用すると、腹腔の右側に向かって腸を移動させることによって、左尿管を露出し、湿らせたドレープでそれらをカバーしています。 </li><li>角度付き鉗子を使用すると、隔離し、左尿管を持ち上げます。 </li><li>尿管閉塞を作成するには、どこにでも膀胱と腎盂の間に6 / O黒編組絹縫合糸で二回、左尿管を結紮。長期実験のために、すべての腹部の手術のために吸収性縫合糸を使用しています。あるいは、結紮クリップは、尿管にも適用することができる。 </li><li>尿管から膀胱を単離するために、2本の縫合糸の間に尿管を分割。 </li><li>慎重に腹腔内に腸を交換してください。 </li><li>第4節に記載されている手順に従ってください - 術後の回復とケア切開を閉じ、麻酔を逆にする。 </li></ol> Reversibl 3.準備E片側尿管閉塞<ol><li>手術のためにマウスを準備し、上記の手順1.1から2.2で詳述するように、左尿管を隔離する。 </li><li>逆にすることができる尿管閉塞を作成するには、膀胱に近い6 / O黒編組絹縫合糸で二回、左尿管を結紮。これは尿管の周りの場所に柔らかい壁のプラスチックチューブを固定するために使用されるように、長いアッパー縫合糸の一端を残します。 </li><li>それは尿管に適用されるように開放スプレイすることができるように柔らかい肉プ​​ラスチック管5mmの長さの縦方向のスリットを作る。 1ミリメートルと2ミリメートルの外径の内径を有する任意の柔らかい壁シリコーンプラスチック管を使用することができる。 </li><li>尿管の周りに優しく柔らかい壁のプラスチックチューブを置きます。それは尿管の周りに閉鎖された後、長い縫合糸は、チューブのスリットの中心から出ていることを確認してください。 </li><li>尿管を囲む柔らかい肉の管の周りに6 / O黒編み絹縫合糸の長さを置き、ネクタイ一回。今、縦方向にそれが以前に結ばれた縫合糸の上に位置するように、管全体に、チューブのスリットの中央から出てくる、縫合糸の長い方の端を置く。 </li><li>チューブおよび尿管の周りの場所で長い縫合糸端を固定する二回のチューブの周囲に位置する縫合糸を結ぶ。 注：尿管およびソフト薄肉プラスチックチューブ上の癒着形成を防止するために、接着還元溶液は、チューブの周りの領域に適用することができる。 </li><li>慎重に腹腔内に腸を交換してください。 </li></ol> 4.術後の回復とケア<ol><li> 5 / O黒編み絹縫合糸を使用したブランケットステッチで腹膜を閉じて、金属製の皮膚クリップを使用して皮膚に近似している。 </li><li>術後感染のリスクを最小限にするために、そのような腹部の皮膚にヨウ素/アルコール溶液のような防腐剤を加える。 </li><li>部分的に皮下アチパメゾール塩酸塩（2mgの/ kg）で麻酔を逆転。 </lI&gt; <li> 1mlを皮下注射することにより、流体を投与は、生理食塩水を温めた。 </li><li>それが意識を回復するまでマウスを監視します。 </li><li>マウスは24時間29℃に保たれ、加熱ボックスに回復することを許可します。湿らせた食品は、流体および栄養摂取を促進するために提供することができる。 </li><li>通常は7日、障害物の所望のレベルを誘導するために回復するマウスのままにしておきます。 </li><li>長期的な回復実験のために、継続的な鎮痛を提供する。マウスよりも長い7日間回復される場合、皮膚クリップを手術後7日を削除します。 </li><li>閉塞の所望のレベルが誘発された後、いずれかの以下に説明するように、UUOを逆転、または頸椎脱臼によってマウスを安楽死させると組織病理学分析のために腎臓を収集する。 </li></ol> 5.リバーシブル片側尿管閉塞<ol><li> R-UUOを実行するには、手順で説明するように手術のために、R-UUOのための準備が行われたマウスを、準備10.1から1.7。 </li><li>存在する場合、皮膚クリップを削除して、腹腔へのアクセスを得るために腹膜に縫合糸を分割または削除。注：尿管は、長期実験皮膚クリップの妨げされている場合は、アプリケーション以下の7日間を除去されている必要があります。 </li><li>マウスを準備し、ステップ1.8から2.2で説明したように、左尿管を隔離する。 </li><li>角度付き鉗子を使用して、形成された可能性の肉芽腫組織から柔らかい壁のプラスチックチューブを解放する。 </li><li>メスで閉塞尿管の周りにプラスチックチューブを保持している縫合糸を切り、チューブを外す。 </li><li>左の腎臓における水腎症の存在を評価することによって成功したUUOを確認し、腎臓にも淡い表示されます。 </li><li>縫合糸の間に尿管を分割します。 </li><li>滅菌ガーゼの小片に、腎臓に付着し、尿管の残りの長さを置きます。これは尿管aから排出されます尿沈渣と死細胞を収集するために使用されますND腎盂縫合糸が除去されると。 </li><li>上記の尿管を分割したが、近くに、縫合糸と腎臓は、ガーゼの上に排出させる。膀胱に最も近い、そのまま、下縫合のままにしておきます。これは、尿が膀胱から腹腔に漏れないことを確実にすることである。 </li><li>尿管と腎盂が排出されると、尿管の残りの長さの最後に長い6 / O黒編組絹縫合糸を適用する。これは、後の膀胱尿管吻合部を補助するために使用される。 </li><li>膀胱に尿管の残りの長さを吻合するには、以下の手順に従ってください。 <ol><li>それは元の位置の前方に位置し、腎臓の上に位置するように尿管を回します。 </li><li>それは膀胱の方向に出てくるような尿管の端から縫合糸2ミリメートルをタッキングシングル9 / Oポリアミドモノフィラメントを置きます。縫合糸が筋層に残り、尿管の内腔に入らないように注意してください。タッキング縫合糸ウィルlは、膀胱内の尿管を固定するために使用すること。 </li><li>膀胱を通してチャネルを作成するには、それが膀胱の正面（腹側）壁の方に出るように、膀胱を通して斜めに21G針を渡す。 </li><li> 21G針のベベルで目の針を休ま。膀胱を通して目の針を導くために21G針を使用してください。目の針は、膀胱を通って尿管を取るために使用される。 </li><li> 9 / Oタッキング縫合糸を渡し、膀胱内と外のエントリポイントに隣接した膀胱壁を介して第切開部を介して、ステップ5.11.2で尿管に適用される。尿管、膀胱を通過したらこれは、膀胱に尿管を固定するために結ばれます。 </li><li>目の針を介してステップ5.10で尿管の一端に印加長い6 / O黒編組絹縫合糸を配置します。 </li><li>慎重に尿管が膀胱を通って引っ張られることを確実にしながら、膀胱の外に目を針を撤回。 </li><li>尿管が目から出たらEの膀胱は、目の針を削除し、バック膀胱へ後退を防止するために、尿管の終わりに長い縫合糸にクランプを適用する。 </li><li>膀胱内に場所内の尿管を固定するには、手順5.11.5で適用9 / Oポリアミドモノフィラメントタック縫合糸を、ネクタイ。 </li><li>しっかりと膀胱内の尿管を固定するエントリポイントの周りの2箇所に段階5.11.2と5.11.5で説明したように、追加のシングル9 / Oポリアミドモノフィラメントタイ縫合糸を適用します。 </li><li>開いたハサミの裏を使用して、緊急尿管のより多くを露出するために戻って少し膀胱壁を押してください。 </li><li>尿管の端部に位置する長い縫合上記の尿管を分割します。尿管、膀胱の体内に後退する必要があります。これは、尿管の開放管腔を確認し、膀胱内の開口部から流れ出る尿を観察することが通常である。 </li><li> 9 / Oポリアミドモノフィラメント縫合糸で膀胱の出口傷を閉じます。 </li></ol></li><li>慎重に腸を交換腹腔。 </li><li>ステップ4.1から4.8に詳述術後ケアを提供。 </li><li>腎臓は通常、7日、圧縮解除されるまで、マウスが回復することを許可します。 </li><li>実験の終了時点で、頸椎脱臼によりマウスを安楽死させると組織病理学的分析のために腎臓を収集します。 </li></ol>

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著者らは、開示する、競合または競合する利害を有していない。

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.

腎臓の変化の外観が著しく尿管閉塞に続く、それはより薄いと時間との触診に緊張した（ 図1）となる。障害物に尿管の近位および腎盂の増加拡張があります。腎臓は腎皮質と髄質の著しい間伐結果として長期の閉塞閉塞性腎症の増加期間としてますます萎縮になる。尿管閉塞の逆転後、腎臓の色が血流が増加し、組織浮腫が解消するように暗くなる。彼らは長期の膨満の期間（...

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.

不可逆とリバーシブル片側尿管閉塞のマウスモデル

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

Research

JoVE Journal

Medicine

30.3K ビュー.  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.

ビデオ: 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.

リバーシブル胆汁うっ滞ラットモデルの作成

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

医学

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.

無限の地平線インパクターを用いた片側頚髄損傷の打撲のモデル

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.

一方的な尿管閉塞を有するマウス腎臓におけるマイクロRNA表現の定量リアルタイムPCR評価

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

遺伝学

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.

女性の小型げっ歯類における神経温存ミッド尿道閉塞（NEMO）

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.

円錐髄質断面によるラットの排尿筋活動不足モデル

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

神経科学

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.

片側性腎虚血再灌流傷害の有効マウスモデル

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.

2光子顕微鏡を使用して、糸球体プロセスに対する慢性片側尿管閉塞の影響を定量化

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.

可逆的片側尿管閉塞の未熟マウスモデル

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://www.jove.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.

片側尿管閉塞マウスモデル:マウスモデルにおける腎臓からの尿の流れを遮断する外科的処置

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