すべてのラットは、首都医科大学北京友好病院の動物実験委員会によって承認されたプロトコルに従って使用されました。
1.外科的準備、麻酔、および外科的技術
注:本研究では、体重200〜220 gの合計40匹の雌のWistarラットが商業的に入手されました。40匹のラットのうち、10匹を対照群として無作為に選択し、残りを試験群として治療した。すべての動物は、首都医科大学北京友好病院の動物施設の無菌環境に収容されました。
<ol><li>ペントバルビタールナトリウムを腹腔内投与して全身麻酔を行います(40 mg / kg)。.あるいは、3%〜4%のイソフルランを使用して麻酔を誘発し、1%〜3%(吸入)に維持する。乾燥を防ぐために眼科用軟膏を目に塗ります。次に、ラットを手術プラットフォームに置き、熱サポートを提供します。 注:手順の開始時に、ブプレノルフィン、0.05 mg / kg、SC、0.1〜0.2mLなどの鎮痛薬を投与します。</li>
	<li>つま先のつまみに対する反応の欠如によって麻酔の深さを確認してください。かみそりで背中全体から毛皮を剃ります。</li>
	<li>クロルヘキシジンやポビドンヨードなどの2段階のスクラブとそれに続くイソプロピルアルコールで少なくとも3サイクルの手術部位を滅菌します。手足をサージカルテープで固定し、手術用ハサミで背中の中央値約3cmを切開します。</li>
	<li>外科用ハサミを使用して皮下組織を通して切開を深くし、脊椎に付着した筋肉を切り落とします。</li>
	<li>13番目の 肋骨を視覚的に識別して露出させます(その肋骨に接続されている椎間腔は間隔T13\u2012L1です)。縫合糸を使用して13番目の 肋骨に印を付けます。</li>
	<li>識別後、脊椎に付着した筋肉を注意深く切除し、脊柱を露出させます。脊柱を正確に識別するために、棘上靭帯と棘間靭帯を切除します。外科用ハサミと鉗子でL4〜L5のレベルを公開します。 注:棘上靭帯は、薄い皮下組織が存在するため、簡単に識別できます。棘上靭帯の切除後、棘突起間の靭帯は棘間靭帯である。</li>
	<li>ケリー鉗子を使用して、L4〜5椎骨棘突起と横突起の一部を慎重に解剖し、脊髄を露出させます(図1)。</li>
	<li>円錐髄をL4〜L5レベルで完全に露出させ、円錐髄錐を虹彩切除ハサミで完全に横断します。脊髄の回復をブロックするために、組織パッキングを挿入します。</li>
	<li>4-0非吸収性縫合糸を使用して、外側の皮膚層の上に横たわる筋肉と皮膚を閉じます。</li>
	<li>コントロールグループについては、手順1.1〜7を実行し、円錐髄をそのまま残します。ステップ1.9に従って切開部を閉じます。</li>
</ol>2.動物の回復
<ol><li>術後最初の1時間はラットを温度管理されたインキュベーター(37°C)に入れ、胸骨または活発に動くまで監視します。 注:完全に回復するには約30分かかります。</li>
	<li>動物を十分な食物と水の入った清潔なケージに移します。ラットを別々のケージに入れてください。 注:切断の成功は、テストグループのラットが前足の助けを借りてのみ動くのに対し、コントロールグループのラットは正常に歩くことができた場合に示されます。</li>
</ol>3. 運用後管理
<ol><li>抗生物質であるペニシリンG(動物あたり50,000 U / mL)を腹腔内に注射します。.術後48時間、ブプレノルフィン、0.05 mg / kg、SC、0.1〜0.2 mLなどの鎮痛薬を6〜12時間ごとに投与します。.</li>
	<li>排尿を助けるために下腹部で膀胱を圧迫します。これを1日2回、同時に(午前8時と午後8時)6週間実行します。 注:排尿筋の正常な狭窄の喪失は、DUの象徴です。</li>
	<li>すべてのラットを代謝ケージに入れ、それぞれに尿収集漏斗を事前に計量した吸収紙の上に置き、排尿と失禁を監視します。</li>
	<li>排尿量(VV)を示す吸収紙の重量変化と残留尿量を別々に収集してメモします。</li>
</ol>4.尿流動態検査
<ol><li>術後6週間で、以下のように尿流動態測定装置を用いて膀胱鏡検査を行う。<ol><li>10%抱水クロラールを腹腔(3 mL / kg)に注射してラットを麻酔します。.</li>
		<li>排尿のために膀胱を圧縮し、次にテープを使用してラットを手術プラットフォームに固定します。</li>
		<li>硬膜外カテーテル(3F)を膀胱に挿入し、尿流動態測定装置、硬膜外カテーテル、輸液ポンプを三肢チューブで接続します。</li>
		<li>尿流動態測定のために生理食塩水を0.2 mL / minの速度でポンプで送ります( 材料の表を参照)。MCCとDOP、および膀胱のコンプライアンス(膀胱容積δ排尿筋の圧力で割って計算δ記録します)。</li>
	</ol></li>
</ol>5.統計分析
<ol><li>市販のソフトウェアを使用して統計分析を実行します。</li>
	<li>コルモゴロフ-スミルノフ検定を使用して、データの正規性を検定します。</li>
	<li>正規分布変数を標準偏差の平均値で表します。両側対応のスチューデント のt検定を使用して、両方のグループの膀胱メトログラムのパラメーターを比較します。 注: p < 0.05は、差に統計的有意性があることを示します。</li>
</ol>

<ol>
	<li>van Koeveeringe, G. 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=Detrusor+underactivity:+Pathophysiological+considerations,+models+and+proposals+for+future+research.">Detrusor underactivity: Pathophysiological considerations, models and proposals for future research.</a> Neurourology and Urodynamics. 33 (5), 591-596 (2014).</li><li>Osman, N. I., Esperto, F., Chapple, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrusor+Underactivity+and+the+Underactive+Bladder:+A+Systematic+Review+of+Preclinical+and+Clinical+Studies.">Detrusor Underactivity and the Underactive Bladder: A Systematic Review of Preclinical and Clinical Studies.</a> European Urology. 74 (5), 633-643 (2018).</li><li>Osman, N. I., Chapple, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contemporary+concepts+in+the+aetiopathogenesis+of+detrusor+underactivity.">Contemporary concepts in the aetiopathogenesis of detrusor underactivity.</a> Nature Reviews. Urology. 11 (11), 639-648 (2014).</li><li>Panicker, J. N., Nagaraja, D., Kovoor, J. M. E., Nair, K. P. S., Subbakrishna, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+urinary+tract+dysfunction+in+acute+disseminated+encephalomyelitis.">Lower urinary tract dysfunction in acute disseminated encephalomyelitis.</a> Multiple Sclerosis. 15 (9), 1118-1122 (2009).</li><li>Lee, W. C., Wu, H. P., Tai, T. Y., Yu, H. J., Chiang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+urodynamic+characteristics+and+bladder+sensory+function+in+the+early+stages+of+diabetic+bladder+dysfunction+in+women+with+type+2+diabetes.">Investigation of urodynamic characteristics and bladder sensory function in the early stages of diabetic bladder dysfunction in women with type 2 diabetes.</a> The Journal of Urology. 181 (1), 198-203 (2009).</li><li>Araki, I., Kitachara, M., Oida, T., Kuno, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voiding+dysfunction+and+Parkinson’s+disease:+urodynamic+abnormalities+and+urinary+symptoms.">Voiding dysfunction and Parkinson’s disease: urodynamic abnormalities and urinary symptoms.</a> The Journal of Urology. 164 (5), 1640-1643 (2000).</li><li>Meng, N. 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=Incomplete+bladder+emptying+in+patients+with+stroke:+is+detrusor+external+sphincter+dyssynergia+a+potential+cause.">Incomplete bladder emptying in patients with stroke: is detrusor external sphincter dyssynergia a potential cause.</a> Archives of Physical Medicine and Rehabilitation. 91 (7), 1105-1109 (2010).</li><li>FitzGerald, M. P., Brubaker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+etiology+of+urinary+retention+after+surgery+for+genuine+stress+incontinence.">The etiology of urinary retention after surgery for genuine stress incontinence.</a> Neurourology and Urodynamics. 20 (1), 13-21 (2001).</li><li>Rahman, M., Siddik, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroanatomy,+Pontine+Micturition+Center.">Neuroanatomy, Pontine Micturition Center.</a> StatPearls. , (2020).</li><li>Wrobel, A., Lancut, M., Rechberger, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+detrusor+overactivity+in+conscious+rats+induced+by+retinyl+acetate+instillation.">A new model of detrusor overactivity in conscious rats induced by retinyl acetate instillation.</a> Journal of Pharmacological and Toxicological Methods. 74 (7), 16 (2015).</li><li>Rosenzweig, E. S., McDonald, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+treatment+of+spinal+cord+injury:+research+trends+and+progress+toward+useful+repair.">Rodent models for treatment of spinal cord injury: research trends and progress toward useful repair.</a> Current Opinion in Neurology. 17 (2), 121-131 (2004).</li><li>Yoo, K. H., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+animal+models+of+neurogenic+bladder+dysfunction.">Experimental animal models of neurogenic bladder dysfunction.</a> International Neurourology Journal. 14 (1), 1-6 (2010).</li><li>Kanai, 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=Sophisticated+models+and+methods+for+studying+neurogenic+bladder+dysfunction.">Sophisticated models and methods for studying neurogenic bladder dysfunction.</a> Neurourology and Urodynamics. 30 (5), 658-667 (2011).</li><li>Nomiya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+vascular+damage+may+lead+to+bladder+underactivity+in+rats.">Progressive vascular damage may lead to bladder underactivity in rats.</a> The Journal of Urology. 191 (5), 1462-1469 (2014).</li><li>Seki, T., Hida, K., Tada, M., Koyanagi, I., Iwasaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+contusion+model+of+the+mouse+spinal+cord+using+a+pneumatic+impact+device.">Graded contusion model of the mouse spinal cord using a pneumatic impact device.</a> Neurosurgery. 50 (5), 1075-1081 (2002).</li><li>Yeo, S. 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=Development+of+a+rat+model+of+graded+contusive+spinal+cord+injury+using+a+pneumatic+impact+device.">Development of a rat model of graded contusive spinal cord injury using a pneumatic impact device.</a> Journal of Korean Medical Science. 19 (4), 574-580 (2004).</li><li>Bergman, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+transplants+permit+the+growth+of+serotonergic+axons+across+the+site+of+neonatal+spinal+cord+transection.">Spinal cord transplants permit the growth of serotonergic axons across the site of neonatal spinal cord transection.</a> Brain Research. 431 (2), 265-279 (1987).</li><li>Chancellor, M. B., 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=Underactive+bladder;+Review+of+progress+and+impact+from+the+International+CURE-UAB+Initiative.">Underactive bladder; Review of progress and impact from the International CURE-UAB Initiative.</a> International Neurourology Journal. 24 (1), 3-11 (2020).</li><li>Pfisterer, M. H. D., Griffiths, D. J., Schaefer, W., Resnick, N. M. <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+age+on+lower+urinary+tract+function:+a+study+in+women.">The effect of age on lower urinary tract function: a study in women.</a> Journal of the American Geriatrics Society. 54 (3), 405-412 (2006).</li><li>Duchen, L. W., Anjorin, A., Watkins, P. J., Mackay, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+autonomic+neuropathy+in+diabetes+mellitus.">Pathology of autonomic neuropathy in diabetes mellitus.</a> Annals of Internal Medicine. 92 (2), 301-303 (1980).</li><li>Schneider, T., Hein, P., Bai, J., Michel, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+muscarinic+receptors+or+rho-kinase+in+hypertension+associated+rat+bladder+dysfunction.">A role for muscarinic receptors or rho-kinase in hypertension associated rat bladder dysfunction.</a> The Journal of Urologoy. 173 (6), 2178-2181 (2005).</li><li>Drake, M. J., Harvey, I. J., Gillespie, J. I., Van Duyl, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+contractions+in+the+normal+human+bladder+and+in+urinary+urgency.">Localized contractions in the normal human bladder and in urinary urgency.</a> BJU International. 95 (7), 1002-1005 (2005).</li><li>Suskind, A. M., Smith, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+detrusor+underactivity:+impaired+contractility+versus+afferent+dysfunction.">A new look at detrusor underactivity: impaired contractility versus afferent dysfunction.</a> Current Urology Reports. 10 (5), 347-351 (2009).</li><li>Osman, N. I., 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=Detrusor+underactivity+and+the+underactive+bladder:+A+new+clinical+entity?+A+review+of+current+terminology,+definitions,+epidemiology,+aetiology,+and+diagnosis.">Detrusor underactivity and the underactive bladder: A new clinical entity? A review of current terminology, definitions, epidemiology, aetiology, and diagnosis.</a> European Urology. 65 (2), 389-398 (2014).</li></ol>

著者は開示するものは何もありません。

DUは、男性と女性の両方の下部尿路症状の一般的な原因です。これは、影響を受けた人々の生活の質(Qol)を大幅に低下させる可能性のある治療オプションがほとんどない複雑な症状の星座です18。DUは多因子性であると考えられているが、その病因の理解は初歩的なままである。研究によると、DUの病因は筋原性および神経原性の要因に関連している可能性があります。
<p c...

排尿筋活動低下症(DU)は、研究が続けられている典型的な下部尿路機能障害である。DUは国際排泄学会(ICS)1によって定義されていますが、この病気を指すために、「排尿筋不全」、「収縮性膀胱」、「排尿筋性反射症」2など、さまざまな用語が使用されています。2002年に国際禁制学会(ICS)によって定義されたDUは、強度と持続時間の低下の収縮であり、その結果、膀胱が排出される時間が長時間増加し、それによって通常の期間内に完全な膀胱排出を達成できなくなります。
DUは、男性の48%、女性の12%(>70歳)3 に、尿路症状が下がる可能性がある。それは多因子性のようであり、効果的な治療法は存在しません。DUは、多発性硬化症4、糖尿病5、パーキンソン病6、脳脳卒中7などの神経因性膀胱機能障害の患者に遍在していることが報告されています。DUは、腹腔鏡下子宮摘出術、前立腺全摘除術、または小骨盤8の他の外科的介入などの医原性神経損傷によっても引き起こされる可能性があります。DUの病態生理学の変化と利用可能な治療法は、研究に適した動物モデルがないため、依然として混乱しています。
排尿反射は、橋排尿中枢、仙骨副交感神経核、およびより上級の皮質中枢を組み合わせた脊髄球脊髄経路によって制御されています9。排尿反射の活性化と維持は、主に膀胱からより上級の皮質センターへの感覚信号の定期的な輸送に依存します。感覚機能障害がDUに寄与すると仮定することができます。
下部尿路機能障害に関連するほとんどの実験動物研究は、過活動膀胱(OAB)モデルに焦点を当てています10。これらのモデルは、OABの病態生理学および予後の合理的な理解を提供する。しかし、報告されているDUモデルはごくわずかであり、例えば、脊髄上損傷(局所病変、衰弱、および中大脳動脈閉塞)、脊髄切断または挫傷損傷、全身性(例:.、シクロホスファミド)または膀胱内投与刺激性または炎症性薬剤(例:.、酸、アクロレイン、およびリポ多糖)11,12,13,14。.これらの方法のうち、DU13の動物モデルを確立する際に使用できるのは、脊髄離断法または挫傷損傷法のみである。橋排尿センターと高次皮質センターの損傷を伴う試みは、重度の外傷のために放棄されました。そのため、排尿反射中枢の正確な位置を見つけて、最小限の副作用でDUを誘発することにますます注意が払われています。
前述のように、DUを誘導するメカニズムの1つは、脊髄を傷つけて排尿反射のシグナル伝達経路を損傷することです。アレンの体重減少法は、脊髄損傷を有する実験動物を確立するために開発された15。ただし、この方法に関するこれ以上の実験データはありません。さらに、動物の一部はDUなしで脳卒中後に脊髄機能を回復したので、DU動物モデル16を生成するための完全な方法とは考えられない。
1987年、ブレグマンはDU動物モデルを生成するために脊髄を切断するプロセスを解明し、実験データ17を取得しました。それにもかかわらず、この方法はDU動物モデルを確立するために適用されなかった。当時、研究者たちはまだDUの病因について混乱していました。OABまたはDUの誘導に関連する脊髄内の位置は互いに隣接しているため、DU17を誘導するための脊髄の損傷部位を正確に見つけることができませんでした。OABとDUは、この方法で一緒にまたは別々に導入されました。そのため、この方法はDUを導入しましたが、不正確であり、DUの発生と処理の理解には使用できませんでした。
上記のように、DUの適切な動物モデルの欠如は、DUの研究の主な障害の1つです。 研究者は、DUの病理をシミュレートできる正確で管理しやすいモデルを継続的に探しています。DUの治療選択肢でさえ、過去20年間に大幅に改善されていません。まとめると、DUの動物モデルを確立するための標準プロトコルを記述することが大いに必要とされている。
そこで本稿では、円錐髄郭清によりDUのラットモデルを首尾よく確立する方法について述べる。円錐髄質を分離するためにL4〜5レベルで切断を行った。最大膀胱測定容量(MCC)、排尿筋開放圧(DOP)、および膀胱のコンプライアンスを記録および分析して、プロトコルを検証しました。以下に述べるプロトコルは、実現可能性と信頼性の両方を標準化された方法で組み合わせて、DU動物モデルを確立し、DUの発生と処理をシミュレートします。このプロトコルは、DUのさらなる研究のための技術として使用することができる。

<table><tbody><tr><td>0.9% saline</td><td>Wuhan Prosai Company</td><td>EY-C1178</td><td>pump for urodynamic measurement</td></tr><tr><td>10% chloral hydrate</td><td>Shandong Yulong Co., Ltd</td><td>H37022673</td><td>3mL/kg, administered intraperitoneally</td></tr><tr><td>Buprenorphine Hydrochloride Injection</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td><td>H12020275</td><td>0.05mg/kg subcutaneously 24h and 48h postoperation</td></tr><tr><td>Epidural Catheter</td><td>Shandong Xinghua Co, Ltd</td><td>VABR3L</td><td>for urodynamic measurement</td></tr><tr><td>Penicillin G</td><td>Alta Technology Co., Ltd</td><td>1ST5637</td><td>50,000 unit/ml per animal</td></tr><tr><td>pentobarbital</td><td>Beijing solabo Technology Co., Ltd</td><td>NK-WF0001</td><td>40 mg/kg, administered intraperitoneally</td></tr><tr><td>Suture line(4-0)</td><td>ETHICON</td><td>VCP422H</td><td>suture the injury</td></tr><tr><td>Three-limb tube</td><td>Shandong Xinghua Co, Ltd</td><td>VAB3T</td><td>for urodynamic measurement</td></tr><tr><td>Trace infusion pump</td><td>Zhejiang Smith Medical Instrument Co., Ltd</td><td>20162540335</td><td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td></tr><tr><td>Urodynamic measurement equipment</td><td>Medical Measurement SystemsB.V.</td><td>08-0467</td><td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td></tr><tr><td>Wistar Rats</td><td>HFK Biotechnology Co.Ltd,Beijing ,China</td><td>SCXK2012-0023</td><td>200-220g</td></tr></tbody></table>

detrusor underactivity model in rats by conus medullaris transection

提示されたプロトコルの目標は、円錐髄膜剥離を通じてラットの排尿筋活動低下(DU)モデルを確立することでした。体重200〜220 gの雌Wistarラット40匹(対照群:10匹、試験群:30匹)で椎弓切除術を行い、試験群では髄膜円錐をL4〜5レベルで切除しました。全てのラットを同じ環境条件下で6週間飼育し、給餌した。試験群では、排尿を1日2回6週間行い、平均残尿量を記録した。膀胱メトログラムは両方のグループで実施されました。最大膀胱測定容量(MCC)、排尿筋開口圧(DOP)、および膀胱のコンプライアンスが記録され、計算されました。試験群は、脊髄ショック中および手術後の両方で、手術後に有意な尿閉を示した。しかし、対照群に異常は認められなかった。対照群と比較した場合、試験群のMCCおよび膀胱のコンプライアンスは試験群よりも有意に高かった(3.24 ± 2.261 mL 対 1.04 ± 0.571 mL;0.43 ± 0.578 mL/cmH 2 O 対 0.032 ± 0.016 mL/cmH 2 O)、試験群のDOPは対照群よりも低かった(20.28 ± 14.022 cmH 2 O vs 35 ± 13.258 cmH2円錐髄膜解離によってDUの動物モデルを確立するこの方法は、DUの病態生理学をよりよく理解する絶好の機会を提供します。

円錐髄膜切開の全手順は、経験豊富な外科医によって45分以内に完了することができます。当研究室では、100例以上の円錐髄膜切開手術を行ってきました。成功率は、ラットの生存率とDUの誘導の成功によって定義されるように、95%以上です。尿流動態検査によりDUの誘導が確認された。
我々の経験に基づいて、DUの誘導は、残留尿量によって事前に評価することができま...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

ラットにおける円錐髄膜剥離による排尿筋活動低下モデルを確立する方法を提示する。排尿筋活動低下は、これらの動物において首尾よく刺激された。このモデルは、尿路機能の研究に使用できます。

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 ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

2.0K ビュー.  Capital Medical University. ラットにおける円錐髄膜剥離による排尿筋活動低下モデルを確立する方法を提示する。排尿筋活動低下は、これらの動物において首尾よく刺激された。このモデルは、尿路機能の研究に使用できます。

ビデオ: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

各種齧歯類モデルで内圧分析を用いた膀胱補強のためのバイオマテリアルの評価

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

生物工学

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 Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

マウスの膀胱痛に対するモデルとしての膀胱膨満誘発内臓運動応答

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

下部尿路薬理学を評価するための方法として膀胱平滑筋収縮ストリップ

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

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 ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

マウスモデルにおける覚醒時膀胱内圧測定の実施手順の評価

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

膀胱充填中にスブロテリウムに直接アクセスするために脱皮筋を除去した分散型(Ex Vivo)マウス膀胱モデル

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

注入カテーテル電極の繰り返し測定を可能にすると目がさめているラットの膀胱内圧測定と外部尿道括約筋の測定

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

生物学

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 ...