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 border="0" cellpadding="0" cellspacing="0" width="1064">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Female rat&#160;</td>
			<td style="width:123px;">Charles-River Canada</td>
			<td style="width:559px;">Sprague Dawley Rat (Crl:SD); Strain Code: 400</td>
			<td style="width:167px;">about 200g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Isoflorane vaporizer</td>
			<td style="width:123px;">Benson medical industries inc.</td>
			<td style="width:559px;">TEC III Isoflurane; product code: 53-T3ISO&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Isoflorane</td>
			<td style="width:123px;">Fresenius Kabi Animal Health</td>
			<td style="width:559px;">Isoflorane; product code CP0406V2</td>
			<td style="width:167px;">&#160;250ml bottle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Disposable scalpell</td>
			<td style="width:123px;">Ted Pella, Inc.</td>
			<td style="width:559px;">#15 Sterile, Stainless Steel Scalpels; catalogue number 549-9-15S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microscopy forceps</td>
			<td style="width:123px;">Ted Pella, Inc.</td>
			<td style="width:559px;">Aesculap Microscopy Forceps, Finepoint, 115mm; product number 5443</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Adson forceps</td>
			<td style="width:123px;">Ted Pella, Inc.</td>
			<td style="width:559px;">Aesculap Adson Dissection Forceps, Curved, 120mm; product number 5002-9</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Curved surgical scissors</td>
			<td style="width:123px;">Ted Pella, Inc.</td>
			<td style="width:559px;">Surgical Scissors, Curved, S/S, 105mm; catalogue number 1376</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">0.9mm inox metal rod</td>
			<td style="width:123px;">goodfellow.com</td>
			<td style="width:559px;">Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Skin hooks - self made from inox steel</td>
			<td style="width:123px;">goodfellow.com</td>
			<td style="width:559px;">Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">4-0 Silk suture</td>
			<td style="width:123px;">esutures.com</td>
			<td style="width:559px;">4-0 Sofsilk black 98&#34; strand; product number LS640</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;width:216px;">5-0 Vicry suture</td>
			<td style="width:123px;">esutures.com</td>
			<td style="width:559px;">5-0 Vicryl undyed 27&#34; RB-1 taper; product number J213H</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Needle holder</td>
			<td style="width:123px;">Medsupplier.com</td>
			<td style="width:559px;">Integra Miltex Carb-N-Sert Baumgartner Needle Holder - 5 1/2&#34;; product code SKU NEE4484-8-40TC 
			SKU NEE4484-8-40TC 
			SKU NEE4484-8-40TC&#160;</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;width:216px;">Temgesic (Buprenorphine)</td>
			<td style="width:123px;">first veterinary supply</td>
			<td style="width:559px;">Buprenex 0.3MG/ml inj amps; item #075701</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="40" style="height:40px;width:216px;">1ml syringe with 27G needle</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:559px;">BD&#160;Tuberculin Syringe, BD 309623</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">20G Angiocath</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:559px;">Andwin Scientific&#160;ANGIOCATH 20 GA; Catalog No. NC9561906</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recovery diet</td>
			<td style="width:123px;">clearh2o</td>
			<td>DietGel 31M; 72-08-5022</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">C57Bl/6 female mice</td>
			<td style="width:123px;">Charles-River Canada</td>
			<td style="width:559px;">C57BL/6 Mouse (C57BL/6NCrI); Strain Code: 027</td>
			<td style="width:167px;">about 18g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">24G angiocath</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:559px;">Hanna Pharmaceutical Supply Co., Inc.&#160;IV CATH ANGIOCATH 24GX3/4IN; Catalog No. NC9814340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">5-0 Ethibond</td>
			<td style="width:123px;">esutures.com</td>
			<td style="width:559px;">5-0 Ethibond green 30&#34; RB-1 taper; product number X550H</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">26G syringe needle</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:559px;">BD Precisionglide syringe needle, Gauge 26; z192392</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Normal Saline</td>
			<td style="width:123px;">Baxter</td>
			<td style="width:559px;">0.9% Sodium Chloride Injection, USP, 100 mL MINI-BAG; product code 2B0043</td>
			<td style="width:167px;"></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

9.5K 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

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

マウスの長い持続時間の全身温熱療法のためのプロトコル

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

医学

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 ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

ラット同所性小腸移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

ヒアルロン酸ナトリウムの網膜下注入によるげっ歯類における網膜剥離モデル

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

リモート四肢虚血プレコンディショニング：げっ歯類における神経保護技術

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

懸濁液中の網膜色素上皮細胞を送達するげっ歯類における網膜下注射の実行

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

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 ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

コンシャスげっ歯類における腎機能の経皮評価

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e&#39;&#160;and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.

Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.

げっ歯類における大動脈デベンディングによる左心室逆改造の研究

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, ...