Name: nerve-sparing mid-urethral obstruction (nemo) in female small rodents
Uploaded: 2017-04-25T13:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents at JoVE.com

The work was funded by a CHIR operating grant (DJB).

以下の実験プロトコルは、金融機関の動物管理委員会によって承認されました。 雌ラットの1 NEMO <ol><li> 準備 <ol><li> 、3％イソフルラン麻酔下で約200グラム体重の雌のSprague-Dawleyラットを配置し動物を秤量、およびヨウ素の外科用スクラブを用いて恥骨前領域を3回剃ると消毒。 </li><li>ダウン加熱パッド、テープ後肢および尾部に、また穏やかに摺動防止するダウンテープで20ゲージの血管カテーテルと膀胱カテーテルを挿入。 </li></ol></li><li> 解剖 <ol><li>尿道口への触知恥骨結合から8ミリメートル縦皮膚切開を行うためにメスを使用してください。後の創傷閉鎖を容易にするために、いずれかの側に皮膚をオフに持ち上げます。 </li><li>長手細かいハサミで拡散によりステント尿道を識別するために鈍切開。露出を高めるために、細かい自己保持、皮膚のフックを配置尿道の。 </li><li>いくつかの穏やかな長手方向が尿道と膣との間の面を開発するために尿道に湾曲したはさみの背部を使用して拡散行います。その後、ぶっきらぼうに尿道の背後に4-0絹縫合糸を渡し、外科結びの双投を準備します。 </li></ol></li><li> 部分的尿道閉塞 <ol><li>後者は依然として容易その後アウトスライドするように、血管カテーテルを除去尿道0.9ミリメートルの金属棒と平行に配置し、その後尿道と金属棒の周りに結び目を締めます。 </li><li> 3以上で結び目を固定し、ロッドを取り外す前に、穏やかテンションを使用してスローします。閉塞の後の放出は、実験の一部である場合、縫合糸は、長約3mm、または4ミリメートルを終了切断。 </li></ol></li><li> 創傷閉鎖 <ol><li>吸収性編組縫合糸4-0の埋め込み単一のステッチを使用して尿道傍腺を近似します。 </li><li>同じ吸収SUTを使用して埋め水平マットレスステッチで皮膚を閉じますURE。注意：それは皮膚浸透ステッチのための吸収性縫合糸を使用するように型にはまらないように思えるかもしれませんが、我々は、創傷感染症などの合併症が発生していなかった、とその縫合糸の除去を回避することができます。 </li></ol></li><li> アフターケア <ol><li>注入ブプレノルフィンを0.1mg / kgの皮下に回復ケージにラットを配置する前に。 </li><li>単一収容されたラットへの最初の24時間のためにいくつかのソフトリカバリの食事を提供します。この期間の後、手続き前と同じペアで再び家の動物は、彼らはよくやっと同じ処置グループに属して提供されます。 </li><li>膀胱のサイズと一般的な幸福のために毎日ラットをチェックしてください。 1週間に1回以上、体重を記録します。 </li></ol></li></ol>雌マウスで2 NEMO <ol><li> 準備 <ol><li>約18グラム体重の雌性C57BL / 6マウスを置き、ステップ1.1.1で詳述するように進みます。 </li><li> 24ゲージのANGを使用する以外、ステップ1.1.2で詳述するように進んでくださいiocatheter。 </li></ol></li><li> 解剖 <ol><li>触診可能な恥骨結合から尿道口に向けて6mmの長手方向の皮膚切開を行うためにメスを使用する。 </li><li>細かいはさみで縦に広げてステント留置尿道を確認するために鈍的に解剖する。類似の鈍的解剖によって尿道の腹側に走っている神経管束を持ち上げる。 </li><li>尿道と膣の間に平面を展開するために、尿道の後ろに湾曲したはさみを使っていくらか穏やかに縦方向に広げる。次に、尿道の後ろに5-0の非吸収性編組縫合糸をしっかりと通し、外科医の結び目の二重の投げを準備する。 </li></ol></li><li> 部分的な尿道閉塞 <ol><li>血管カテーテルを1 mLシリンジで接続し、生理食塩水0.1 mLを膀胱に注入します。 </li><li> 26ゲージのカニューレを尿道に平行に置きます。尿道とカニューレの周りの結び目を静かに締め、アンギオを戻します目。 </li><li> 3以上で結び目を固定したカニューレを取り外す前に、穏やかテンションを使用してスローします。 </li><li>動物の膀胱にやさしい圧力に尿道口に現れる尿のためのテスト。尿を発現することができない場合はネクタイを緩めます。それ以外の場合は、縫合糸は、長さ約3ミリメートルを終了カット。 </li></ol></li><li> 創傷閉鎖 <ol><li> 5-0編組吸収性縫合糸を使用して埋め水平マットレスステッチで皮膚を閉じます。 </li></ol></li><li> アフターケア <ol><li>回復ケージにマウスを置く前に0.1ミリグラム/ kgのブプレノルフィン皮下に注入します。 </li><li>単一収容され、マウスへの最初の48時間のためにいくつかのソフトリカバリの食事を提供します。この期間の後、手続き前と同じグループで、再び家の動物は、彼らはよくやっと同じ処置グループに属して提供されます。 注：過遮られ取り扱う際に無効にしていないとnの中に死んでしまうの手順の後に触知可能な膨張した膀胱24時間を有するマウスEXT 1 - 妨害結紮の除去のために麻酔下でバック取らない限り、2日間。 </li><li>膀胱のサイズと一般的な幸福のために毎日マウスを確認してください。 1週間に1回以上、体重を記録します。 </li></ol></li></ol>

<ol>
	<li>Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+and+voiding+symptoms:+pathophysiologic+aspects.">Storage and voiding symptoms: pathophysiologic aspects.</a> Urology. 62 (5), 3-10 (2003).</li><li>Boyle, P., 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=The+prevalence+of+lower+urinary+tract+symptoms+in+men+and+women+in+four+centres.+The+UrEpik+study.">The prevalence of lower urinary tract symptoms in men and women in four centres. The UrEpik study.</a> BJU Int. 92 (4), 409-414 (2003).</li><li>Coyne, K. S., Wein, A., Nicholson, S., Kvasz, M., Chen, C. I., Milsom, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+burden+of+urgency+urinary+incontinence+in+the+United+States:+a+systematic+review.">Economic burden of urgency urinary incontinence in the United States: a systematic review.</a> J Manag Care Pharm. 20 (2), 130-140 (2014).</li><li>Buttyan, R., Chen, M. W., Levin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+bladder+outlet+obstruction+and+molecular+insights+into+the+basis+for+the+development+of+bladder+dysfunction.">Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction.</a> Eur. Urol. 32 (Suppl 1), 32-39 (1997).</li><li>Mattiasson, A., Uvelius, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+contractile+properties+in+hypertrophic+rat+urinary+bladder.">Changes in contractile properties in hypertrophic rat urinary bladder.</a> J Urol. 128 (6), 1340-1342 (1982).</li><li>Chang, 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=Alteration+of+the+PKC-mediated+signaling+pathway+for+smooth+muscle+contraction+in+obstruction-induced+hypertrophy+of+the+urinary+bladder.">Alteration of the PKC-mediated signaling pathway for smooth muscle contraction in obstruction-induced hypertrophy of the urinary bladder.</a> Lab Invest. 89 (7), 823-832 (2009).</li><li>Moore, J. A., Brading, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+of+bladder+outlet+obstruction+incorporating+radio-telemetered+cystometry.">A porcine model of bladder outlet obstruction incorporating radio-telemetered cystometry.</a> BJU Int. 100 (5), 1192-1193 (2007).</li><li>Beamon, C. R., Mazar, C., Salkini, M. W., Phull, H. S., Comiter, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sildenafil+citrate+on+bladder+outlet+obstruction:+a+mouse+model.">The effect of sildenafil citrate on bladder outlet obstruction: a mouse model.</a> BJU Inte. 104 (2), 252-256 (2009).</li><li>Pandita, R. K., Fujiwara, M., Alm, P., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+evaluation+of+bladder+function+in+non-anesthetized+mice+with+and+without+bladder+outlet+obstruction.">Cystometric evaluation of bladder function in non-anesthetized mice with and without bladder outlet obstruction.</a> J Urol. 164 (4), 1385-1389 (2000).</li><li>Kang, Y. J., 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=Early+Sequential+Changes+in+Bladder+Function+after+Partial+Bladder+Outlet+Obstruction+in+Awake+Sprague-Dawley+Rats:+Focus+on+the+Decompensated+Bladder.">Early Sequential Changes in Bladder Function after Partial Bladder Outlet Obstruction in Awake Sprague-Dawley Rats: Focus on the Decompensated Bladder.</a> Korean J Urol. 52 (12), 835-837 (2011).</li><li>Schroeder, A., 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=Protective+effect+of+an+oral+endothelin+converting+enzyme+inhibitor+on+rat+detrusor+function+after+outlet+obstruction.">Protective effect of an oral endothelin converting enzyme inhibitor on rat detrusor function after outlet obstruction.</a> J Urol. 172 (3), 1171-1174 (2004).</li><li>Sidler, M., Aitken, K., Jiang, J., Bijos, D., B&#228;gli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="sometimes+a+sham+is+just+a+sham"+-+why+the+rodent+model+of+partial+bladder+outlet+obstruction+needs+adjustment.">"sometimes a sham is just a sham" - why the rodent model of partial bladder outlet obstruction needs adjustment.</a> J Urol. 195 (4), e379 (2016).</li><li>Jin, L. H., 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=Persistent+detrusor+overactivity+in+rats+after+relief+of+partial+urethral+obstruction.">Persistent detrusor overactivity in rats after relief of partial urethral obstruction.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 301 (4), R896-R904 (2011).</li></ol>

The authors have nothing to disclose.

動物の死亡率は、多くの報告10、11、13、15％以上で、近位尿道におけるpBOOの伝統的なモデルの主要な関心事の一つです。ニモは、雌ラットに適用した場合に、最小限の死亡率を持っているように見えます。マウスでは、近位尿道閉塞は、技術的に困難なラットに比べので、さらに発生しやすい合併症です。私...

部分膀胱出口閉塞（pBOO）は、高い有病率を有し、重度の膀胱機能不全1をもたらすことができます。スペクトルは、良性前立腺肥大、取得した尿道狭窄の上、そのような後部尿道弁又は尿道下裂などの先天性奇形の範囲です。男性60歳、2年上の30％以上に影響を与え、後者。グレート患者負担とpBOOに関連付けられている巨大な医療費が増加した流出抵抗3に対応して勉強膀胱改造に入れ、かなりの研究努力を正当化。 2006年から2015年まで、220以上のPubMedのインデックス付けの記事は、膀胱にpBOOの影響に関する発表されました。 pBOOのための動物モデルは、ラット5、ウサギ6、豚7、および覚書などのいくつかの種4に考案されてきたがE 8、9、間違いなく最も一般的に使用される動物モデルは、しかし、近位尿道の部分閉塞を受けた雌ラットです。動物の腹部へのアクセス、膀胱のexteriorizing、および膀胱頸部の周りの解剖は、この伝統的な近位尿道閉塞技術と避けられません。閉塞および動物の死亡率の程度の変動は、この手順10、11に関連した問題のいくつかです。私たちは、偽手術動物では、近位尿道の周りの解剖が膀胱頸部12で神経線維の損失と相関の生理的変化をもたらすことを示しました。この知見は、女性のげっ歯類で近位尿道にアクセスすることを含む、最も一般的に使用されるpBOO動物モデルは、影響を与える、膀胱の関連する構造的および機能的変化で除神経損傷につながることを示し、シャムと閉塞動物。したがって、除神経損傷を避け、代替的なアプローチが必要でした。偽手術動物は非操作コントロール動物と区別できなかった一方で我々の研究室では、そのような器官質量の増加および残留尿などの膀胱の予想される障害物に関連する変化を誘導するのに有効な、神経温存ミッド尿道閉塞（NEMO）アプローチを開発し、評価しました。それは解剖のレベルに近位に位置しても、横紋筋尿道括約筋はそのまま残りました。さらに、膀胱質量の閉塞により誘導される増加の変動は、死亡率はゼロであった伝統的な近位尿道閉塞および動物よりも有意に低かったです。 これまでに説明したマウスのためのすべてのpBOOモデルは50％の周りの死亡率と関連していた間、私達はまた成功し、閉塞した動物において10％未満の死亡率と雌マウスにニモを適用しました。マウスでのpBOOのコンテキストで膀胱の再構築を検討することはapplicabilの恩恵を受けるトランスジェニック改変の全体スペクトルのITY。 女性のげっ歯類で半ば尿道の周りに解剖することは、伝統的な近位閉塞モデルにおいて観察された膀胱における望ましくないと交絡構造的または機能的変化を誘導しません。それにもかかわらず、半ば尿道レベルでの部分的な閉塞を誘発することは、まだ膀胱肥大を誘導し、pBOOのための動物モデルから予想されるように、残尿を増加させました。重要なことに、マウスにおけるNEMOを行うことも大きいげっ歯類において実質使用できないトランスジェニック法にpBOOにおける膀胱リモデリングの調査を開きます。

<table><tbody><tr><td>Female rat&amp;nbsp;</td><td>Charles-River Canada</td><td>Sprague Dawley Rat (Crl:SD); Strain Code: 400</td><td>about 200 g</td></tr><tr><td>Isoflorane vaporizer</td><td>Benson medical industries inc.</td><td>TEC III Isoflurane; product code: 53-T3ISO&amp;nbsp;</td><td></td></tr><tr><td>Isoflorane</td><td>Fresenius Kabi Animal Health</td><td>Isoflorane; product code CP0406V2</td><td>&amp;nbsp;250 mL bottle</td></tr><tr><td>Disposable scalpel</td><td>Ted Pella, Inc.</td><td>#15 Sterile, Stainless Steel Scalpels; catalogue number 549-9-15S</td><td></td></tr><tr><td>Microscopy forceps</td><td>Ted Pella, Inc.</td><td>Aesculap Microscopy Forceps, Finepoint, 115mm; product number 5443</td><td></td></tr><tr><td>Adson forceps</td><td>Ted Pella, Inc.</td><td>Aesculap Adson Dissection Forceps, Curved, 120mm; product number 5002-9</td><td></td></tr><tr><td>Curved surgical scissors</td><td>Ted Pella, Inc.</td><td>Surgical Scissors, Curved, S/S, 105mm; catalogue number 1376</td><td></td></tr><tr><td>0.9 mm inox metal rod</td><td>goodfellow.com</td><td>Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td><td></td></tr><tr><td>Skin hooks - self made from inox steel</td><td>goodfellow.com</td><td>Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td><td></td></tr><tr><td>4-0 Silk suture</td><td>esutures.com</td><td>4-0 Sofsilk black 98&quot; strand; product number LS640</td><td></td></tr><tr><td>5-0 Vicry suture</td><td>esutures.com</td><td>5-0 Vicryl undyed 27&quot; RB-1 taper; product number J213H</td><td></td></tr><tr><td>Needle holder</td><td>Medsupplier.com</td><td>Integra Miltex Carb-N-Sert Baumgartner Needle Holder - 5 1/2&quot;; product code SKU NEE4484-8-40TC&lt;br/&gt; SKU NEE4484-8-40TC&lt;br/&gt; SKU NEE4484-8-40TC&amp;nbsp;</td><td></td></tr><tr><td>Temgesic (Buprenorphine)</td><td>first veterinary supply</td><td>Buprenex 0.3MG/ml inj amps; item #075701</td><td></td></tr><tr><td>1 mL syringe with 27 G needle</td><td>Fisher Scientific</td><td>BD&amp;nbsp;Tuberculin Syringe, BD 309623</td><td></td></tr><tr><td>20 G Angiocath</td><td>Fisher Scientific</td><td>Andwin Scientific&amp;nbsp;ANGIOCATH 20 GA; Catalog No. NC9561906</td><td></td></tr><tr><td>Recovery diet</td><td>clearh2o</td><td>DietGel 31M; 72-08-5022</td><td></td></tr><tr><td>C57Bl/6 female mice</td><td>Charles-River Canada</td><td>C57BL/6 Mouse (C57BL/6NCrI); Strain Code: 027</td><td>about 18 g</td></tr><tr><td>24 G angiocath</td><td>Fisher Scientific</td><td>Hanna Pharmaceutical Supply Co., Inc.&amp;nbsp;IV CATH ANGIOCATH 24GX3/4IN; Catalog No. NC9814340</td><td></td></tr><tr><td>5-0 Ethibond</td><td>esutures.com</td><td>5-0 Ethibond green 30&quot; RB-1 taper; product number X550H</td><td></td></tr><tr><td>26 G syringe needle</td><td>Sigma-Aldrich</td><td>BD Precisionglide syringe needle, Gauge 26; z192392</td><td></td></tr><tr><td>Normal Saline</td><td>Baxter</td><td>0.9% Sodium Chloride Injection, USP, 100 mL MINI-BAG; product code 2B0043</td><td></td></tr></tbody></table>

nerve-sparing mid-urethral obstruction (nemo) in female small rodents

部分膀胱出口閉塞（pBOO）、高い有病率を持っている重要な患者の負担、そして巨大な医療費が発生します。 pBOOで膀胱改造を調査するための最も一般的な動物モデルは、近位尿道の部分閉塞を受けた女性の齧歯類です。障害物や動物の死亡率の程度の変動は、近位閉塞との主要な関心事です。さらに、近位尿道や膀胱の首に解剖することは膀胱神経支配を脅かします。 私たちは、従来のモデルの欠点を回避pBOOための神経温存半ば尿道閉塞（NEMO）モデルを開発しました。私たちは、この分野での解剖のためだけでなく、開腹手術の必要性を回避恥骨結合、のすぐ下尿道に近づきました。また、横紋筋尿道括約筋はそのまま残りました。私たちは、20の閉塞（メスSprague-Dawleyラット（12の障害物、6匹の偽動物）ならびにメスC57 / BL6マウスでニモを行いましたS、18匹の偽動物）。 2週間後、私たちは、膀胱機能、膀胱質量、および体重を評価しました。 私たちは、obstructed-または偽手術雌ラットの間には死亡しませんでした。伝統的な近位pBOO法について記載したように、我々は、近位尿道の周りに縫合糸を結び、一時的に0.9ミリメートルの金属棒を入れました。 NEMOは、2週間後に膀胱質量の85％の増加を誘導した偽動物においてのみ0.03 mLのながら、平均残尿量は、部分的に閉塞ラットにおいて0.4 mLでした。マウスでは、我々は、縫合糸を結ぶときに我々が尿道に沿って配置されたカニューレの3つのサイズを試験しました。私たちは、27ゲージのカニューレを使用すると、50％の動物の死亡率の上にもたらしたことがわかりました。 25ゲージのカニューレを配置する膀胱質量を増加させることで所望の応答を生じませんでした。 26ゲージのカニューレを利用すると、最小限の動物の死亡率（1/8）まだ膀胱質量の有意な2倍の増加と良好な結果が得られました。

雌ラットの神経温存ミッド尿道閉塞（NEMO） 死亡 我々はこれまでに40以上の雌ラットに半ば尿道閉塞を行い、何の死亡がなかったです。 膀胱質量 閉塞ラットは85...

Watch this Scientific Journal Video about Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents at JoVE.com

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

げっ歯類の部分膀胱出口閉塞の伝統的なモデリングは、動物の死亡率をはらんでいます。近位尿道や膀胱頸部の周りの解剖から除神経損傷が主要な関心事でもあります。私たちは、従来のモデルの欠点を回避し、安全で信頼性の高いミッド尿道閉塞モデルを開発し、評価しました。

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 ...

nerve-sparing-mid-urethral-obstruction-nemo-in-female-small-rodents

Research

JoVE Journal

Medicine

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

9.6K ビュー.  The Hospital for Sick Children. げっ歯類の部分膀胱出口閉塞の伝統的なモデリングは、動物の死亡率をはらんでいます。近位尿道や膀胱頸部の周りの解剖から除神経損傷が主要な関心事でもあります。私たちは、従来のモデルの欠点を回避し、安全で信頼性の高いミッド尿道閉塞モデルを開発し、評価しました。 

ビデオ: Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

マウス尿路感染症の経尿道的誘導

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

免疫・感染学

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

小型げっ歯類における膀胱内圧測定の使用：膀胱Chemosensationの研究

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

医学

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小動物モデルにおける尿路感染症: 雌雄マウスの尿道カテーテル

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

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.

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 ...

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

インビボ げっ歯類における膨満誘発性尿路上皮ATP放出の内腔測定

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

シアノアクリレート皮膚バリアを用いた覚醒マウスの精密尿流動態測定

Optimizing Mouse Urodynamic Techniques for Improved Accuracy

Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions like neurogenic bladder post-traumatic spinal cord injury (SCI). However, conducting cystometry recordings in mice presents notable challenges. When mice are in a prone and restricted position during recording sessions, urine tends to be absorbed by the fur and skin, leading to an underestimation of voided volume (VV). The goal of this study was to enhance the accuracy of cystometry and external urethral sphincter electromyography (EUS-EMG) recordings in awake mice. We developed a unique method utilizing cyanoacrylate adhesive to create a waterproof skin barrier around the urethral meatus and abdomen, preventing urine absorption and ensuring precise measurements. Results show that after applying the cyanoacrylate, the sum of VV and RV remained consistent with the infused saline volume, and there were no wet areas observed post-experiment, indicating successful prevention of urine absorption. Additionally, the method simultaneously stabilized the electrodes connected with the external urethral sphincter (EUS), ensured stable electromyography (EMG) signals, and minimized artifacts caused by the movement of the awakened mouse and manipulation of the experimenter. Methodological details, results, and implications are discussed, highlighting the importance of improving urodynamic techniques in preclinical research.

著者スポットライト:神経因性膀胱を有する覚醒マウスにおける正確な尿測定のための強化された尿力学法

This protocol provides a guide for waterproofing the skin with cyanoacrylate to prevent urine absorption by fur and skin. It includes instructions for applying the glue to the skin, implanting a bladder catheter, and electrodes for cystometry and external urethral sphincter electromyography recordings in awake mice.

精度向上のためのマウス尿流動態技術の最適化 

Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions ...

神経科学

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

ラットモデルにおける尿道狭窄誘導とそれに続く頬粘膜移植尿道形成術

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...

Efficient Rat Model for Urethral Stricture: Electric Knife Method

Iatrogenic Injury Recapitulated: Electroexcision Technique for Urethral Stricture Modeling in Rats

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for US, such as urethral dilatation and internal urethrotomy, fail to fully resolve the condition and are associated with high rates of recurrence and complications. 
Additionally, the pathogenesis of US is not well understood. To explore the pathogenesis of US and develop new therapeutic strategies, it is crucial to establish a standardized rat model that accurately reflects the clinical manifestations. This study outlines a straightforward and repeatable method for inducing US in rats using a high-frequency electric knife. The method involves making a longitudinal incision with the electric knife set to a unipolar mixed cutting mode at 4 W, which inflicts significant urethral damage. Histopathological analysis shows thickening of the urothelium, inflammatory infiltration, and disorganized collagen fibers. This model effectively replicates iatrogenic injury through electroexcision in the rat urethra. In summary, this study successfully establishes a new, efficient, and stable rat model of US that closely mimics the clinical scenario, providing a valuable tool for further research into the mechanisms and novel treatments for US.

In this study, we present a novel, efficient, and stable rat model of urethral stricture created through electroexcision of the rat urethra, which effectively simulates iatrogenic injury observed in clinical settings.

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for ...

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）

要約

さらに動画を探す

この動画の章

マウス尿路感染症の経尿道的誘導

小型げっ歯類における膀胱内圧測定の使用：膀胱Chemosensationの研究

小動物モデルにおける尿路感染症: 雌雄マウスの尿道カテーテル

不可逆とリバーシブル片側尿管閉塞のマウスモデル

インビボげっ歯類における膨満誘発性尿路上皮ATP放出の内腔測定

精度向上のためのマウス尿流動態技術の最適化

精度向上のためのマウス尿流動態技術の最適化

精度向上のためのマウス尿流動態技術の最適化

ラットモデルにおける尿道狭窄誘導とそれに続く頬粘膜移植尿道形成術

Iatrogenic Injury Recapitulated: Electroexcision Technique for Urethral Stricture Modeling in Rats

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）

要約

さらに動画を探す

この動画の章

マウス尿路感染症の経尿道的誘導

小型げっ歯類における膀胱内圧測定の使用：膀胱Chemosensationの研究

小動物モデルにおける尿路感染症: 雌雄マウスの尿道カテーテル

不可逆とリバーシブル片側尿管閉塞のマウスモデル

インビボ げっ歯類における膨満誘発性尿路上皮ATP放出の内腔測定

精度向上のためのマウス尿流動態技術の最適化

精度向上のためのマウス尿流動態技術の最適化

精度向上のためのマウス尿流動態技術の最適化

ラットモデルにおける尿道狭窄誘導とそれに続く頬粘膜移植尿道形成術

Iatrogenic Injury Recapitulated: Electroexcision Technique for Urethral Stricture Modeling in Rats

インビボげっ歯類における膨満誘発性尿路上皮ATP放出の内腔測定