研究は、NHLBIリサーチサイエンティスト開発助成金(1K01HL135461-01)によってJAGに支援されました。David Carmona-BerrioとIsabel Adarve-Rengifoの技術支援に感謝します。

マウスは、米国国立衛生研究所(NIH)のガイドラインおよび米国保健福祉省の実験動物の世話と使用のためのガイドに従って、ヴァンダービルト大学医療センター(VUMC)動物ケア部門で飼育され、世話をされました。すべての動物手順は、実験を開始する前にVUMC機関動物ケアおよび使用委員会によって承認されました。
1. 動物の準備と解剖
<ol><li>手術を開始する前に、加熱パッドの発芽器とウォーターポンプを約30分オンにします。</li>
	<li>長さ0.5mmのポリウレタンチューブを鋭いメスで切断します。袖口を作るために縦に切って円周の0.2mmを外します。 注:これは、腎動脈狭窄手順の重要な部分であり、極端な精度、細部への注意、忍耐が必要です。一度に数本のポリウレタンチューブを切断してみてください。顕微鏡下でこの手順のすべてを実行します。</li>
	<li>先に進む前に、手術用滅菌手袋と手術用マスクを着用してください。</li>
	<li>6〜8週間のC57BL/6野生型(WT)マウスを使用する。性的な偏見を避けるために、同じ数の雄と雌のマウスを使用してください。</li>
	<li>手術を行う前にマウスの体重を記録する。ポリウレタンチューブで腎動脈狭窄を行うのに理想的な体重は18〜22gです。マウスを優しく扱い、麻酔を注入しながら攪拌しないでください。術前鎮痛薬(ケトフゲン、5mg / kgボディウィーヒト)を投与する。 注:マウスを収容室から手術室に移す際は、動揺を避けるために、非常に優しさと注意を払ってマウスを運んでください。トロリーの代わりにマウスケージを手に持ち歩くことを強くお勧めします。</li>
	<li>腹腔内(I.P.)注射により、ケタミン(100mg/kg BW)とキシラジン(10mg/kg BW)の混合物でマウスを麻酔する。</li>
	<li>完全に麻酔がかかるまでマウスをケージの中に戻します。マウスが完全に意識を失うまでに約4〜5分かかります。鉗子で尾やつま先をつまんで、マウスが完全に意識不明で手術の準備ができているかどうかを確認します。</li>
	<li>マウスをペーパータオルの上に置きます。育毛の反対方向に沿って電気バリカンを使用して側腹部の毛を除去します。手術部位を剃った後、ヨウ素(またはクロルヘキシジン)を含む外科用スクラブで領域を清掃し、続いて70%エタノール(または滅菌生理食塩水)ですすいでください。 これを3回繰り返します。 注:脱毛手順は、手術手順中の毛髪の干渉や毛髪汚染を避けるために、ある程度の距離で、または好ましくは手術手順ベンチとは異なるベンチで実行する必要があります。</li>
	<li>加熱パッドを滅菌シートで覆います。マウスを手術台に持ってきて、マウスの右側の側面を顕微鏡に向けて滅菌シートの上に置きます。循環水で37°Cの一定のパッド温度を維持します。</li>
	<li>すべての手術器具が入った滅菌袋を開けます。解剖顕微鏡と滅菌鋭利はさみを使用して、小さな側面切開(マウスの最後の肋骨である13番目の 胸部肋骨に近い)を椎骨から約0.5cm離します。腰椎に沿って進み、1インチの切開を行います。</li>
	<li>皮膚と筋肉を後ろに引いて腎臓を露出させます。</li>
	<li>腎動脈を隔離するために滅菌綿棒を使用して周囲の脂肪をきれいにし、除去します。湾曲した鉗子を使用して腎動脈から腎神経を単離する。</li>
	<li>偽の手術を行うときは、縫合糸を塗って皮膚を閉じ、閉じた創傷に抗生物質を塗布し、 術後ケアに進みます。 そうでない場合は、次のセクションに進んで動脈を圧迫します。 注:すべての実験は、外科的処置のコントロールとして偽の動物を持つべきです。偽の動物は、袖口を置かずに腎動脈を露出させる外科的処置を経たマウスからなる。</li>
</ol>2.右腎動脈狭窄症
<ol><li>2つのナイロン縫合糸を右腎動脈の下に置き、ゆるい結び目を作り、次に腎臓と大動脈の分岐の間にほぼ等距離にある主腎動脈の周りに袖口を置きます</li>
	<li>ナイロン縫合糸を使用して袖口を閉じます。手術後に縫合糸を失う可能性を避けるために、縫合糸ごとに4つの結び目を作ります。</li>
	<li>簡単な連続縫合糸を適用して筋肉の切開部を閉じる。</li>
	<li>皮膚を閉じるために簡単な中断縫合糸を作ります。</li>
	<li>IACUC承認鎮痛剤を投与する。 注:使用する前にオートクレーブ手術器具を保管してください。一度に複数の手術を行う場合は、使用済みのすべてのツールを滅菌アルコールゲージで拭き取り、すべての手術後15〜30秒間熱い発芽器に入れます。滅菌手袋もマウスごとに交換してください。</li>
</ol>3. 術後ケア
<ol><li>マウスをケージに戻し、ケージを半分オン、ハーフオフ、循環水加熱パッドで2〜3時間放置します。ケージの中にゲルダイエット回復食品を追加します。</li>
	<li>鎮痛剤(ケトプロフェン)を翌日腹腔内投与(投与量:5mg / kg BW)する。</li>
	<li>次の2日間マウスを計量する;一部のマウスが体重の20%以上を失った場合は、獣医師に相談し、適切なIACUC認定手順に従って動物を安楽死させる必要があるかどうかを判断してください。</li>
	<li>マウスを毎日監視して、発赤、腫れ、痛み、感染を評価します。</li>
	<li>IACUCの制度的方針に従って、術後の合併症について獣医の相談を受ける。</li>
</ol>4. 組織採取
<ol><li>外科的処置の3週間後に組織を採取する。各マウスの体重を記録します。</li>
	<li>IACUCが承認した手順でマウスを安楽死させる。</li>
	<li>マウスを仰臥位の滅菌プラットフォームに置き、解剖する。</li>
	<li>手足を固定して伸ばし、動きを制限します。</li>
	<li>マウスに70%エタノールを十分に噴霧する。</li>
	<li>鋭いはさみを使用して腹部と胸部を開くために正中線切開を行います。</li>
	<li>皮膚と腹膜壁を引き戻します。</li>
	<li>慎重に心臓を露出させ、右心室を穿刺し、マウスを放血する。</li>
	<li>鉗子を使用して両方の腎臓を削除します。腎臓はマウスの背中にあります。 注:両方の腎臓を混ぜないでください。狭窄、偽、および対側腎臓に注意してください。</li>
	<li>腎臓カプセルを取り出し、脂肪からそれらをきれいにし、各腎臓の重量を別々に記録します。</li>
	<li>両腎臓の縦切片を切り取り、パラフィン包埋のために後で処理する4°Cで一晩4%PFAで固定し、in situハイブリダイゼーション(ISH)および免疫組織化学(IHC)を行う。報告されたようにISHおよびIHCプロトコルに従ってください23,24.</li>
	<li>残りの腎臓の皮質を単離し、液体窒素中でフラッシュフリーズしてウェスタンブロットを行う。分析までサンプルを-80°Cで保存します。</li>
	<li>文献23、24に記載されているように、レニン、およびN-GAL発現をウェスタンブロットで定量化する。</li>
</ol>5. 統計
<ol><li>3つ以上の条件を持つ実験には一元配置分散分析または二元配置分散分析を使用し、その後、個々のグループ間の比較のためにボンフェローニ事後検定を使用します。p値が0.05以下であれば有意であると考えてください。ソフトウェア(グラフパッドプリズム8.2など)を使用して、すべての統計分析を実行します。</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+haemodynamic+responses+to+unilateral+renal+artery+stenosis+in+conscious+dogs.">Acute haemodynamic responses to unilateral renal artery stenosis in conscious dogs.</a> Clinical and Experimental Pharmacology and Physiology. 12 (3), 305-309 (1985).</li><li>Imanishi, 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=Critical+degree+of+renal+arterial+stenosis+that+causes+hypertension+in+dogs.">Critical degree of renal arterial stenosis that causes hypertension in dogs.</a> Angiology. 43 (10), 833-842 (1992).</li><li>Ziecina, R., Abramczyk, P., Lisiecka, A., Papierski, K., Przybylski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenal-renal+portal+circulation+contributes+to+decrease+in+renal+blood+flow+after+renal+artery+stenosis+in+rats.">Adrenal-renal portal circulation contributes to decrease in renal blood flow after renal artery stenosis in rats.</a> Journal of Physiology and Pharmacology. 49 (4), 553-560 (1998).</li><li>Johnson, J. A., Ichikawa, S., Kurz, K. D., Fowler, W. L., Payne, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressor+responses+to+vasopressin+in+rabbits+with+3-day+renal+artery+stenosis.">Pressor responses to vasopressin in rabbits with 3-day renal artery stenosis.</a> American Journal of Physiology. 240 (6), 862-867 (1981).</li><li>Eirin, 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=Changes+in+glomerular+filtration+rate+after+renal+revascularization+correlate+with+microvascular+hemodynamics+and+inflammation+in+Swine+renal+artery+stenosis.">Changes in glomerular filtration rate after renal revascularization correlate with microvascular hemodynamics and inflammation in Swine renal artery stenosis.</a> Circulation: Cardiovascular Interventions. 5 (5), 720-728 (2012).</li><li>Ma, Z., Jin, X., He, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL16+regulates+renal+injury+and+fibrosis+in+experimental+renal+artery+stenosis.">CXCL16 regulates renal injury and fibrosis in experimental renal artery stenosis.</a> American Journal of Physiology-Heart and Circulatory. 311 (3), 815-821 (2016).</li><li>Cheng, 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=Temporal+analysis+of+signaling+pathways+activated+in+a+murine+model+of+two-kidney,+one-clip+hypertension.">Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension.</a> American Journal of Physiology-Renal Physiology. 297 (4), 1055-1068 (2009).</li><li>Wiesel, P., Mazzolai, L., Nussberger, J., Pedrazzini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-kidney,+one+clip+and+one-kidney,+one+clip+hypertension+in+mice.">Two-kidney, one clip and one-kidney, one clip hypertension in mice.</a> Hypertension. 29 (4), 1025-1030 (1997).</li><li>Johns, C., Gavras, I., Handy, D. E., Salomao, A., Gavras, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+experimental+hypertension+in+mice.">Models of experimental hypertension in mice.</a> Hypertension. 28 (6), 1064-1069 (1996).</li></ol>

金銭的またはその他の利益相反は、著者によって宣言されていません。

腎動脈狭窄は、二次性または抵抗性高血圧症、および腎臓損傷の重要な原因である1,29。この2つの腎臓1つのクリップ(2K1C)ゴールドブラットモデルは、RAStenosis誘発性腎血管性高血圧症を研究するために採用されている1、17、18、19。様々な動物モデルを用...

腎動脈狭窄症(RAStenosis)は、65歳以上の人々の約6%および冠状動脈または末梢血管疾患を有する人々の最大40%に影響を及ぼす難治性の問題である1,2。この疾患の現在の治療法は限られています;したがって、RAStenosisによって誘発される腎血管性高血圧症または抵抗性高血圧症を治療するための新しい治療法を開発することが決定的に必要である。レニンアンジオテンシンアルドステロン系(RAAS)は、RAStenosis誘発性高血圧症または腎血管性高血圧症の病因に関与する重要な経路である3,4。ACE阻害剤またはアンジオテンシン受容体遮断薬などのRAASを標的とする既知の治療法は、高血圧を緩和するが、腎不全および高カリウム血症について綿密に調べる必要がある5,6,7。レニンはRAASの律速ステップを触媒する。それはアンジオテンシノーゲンをアンジオテンシンIに変換する。アテローム性動脈硬化症では、プラーク形成はレニン分泌を駆動する腎動脈の狭窄を引き起こし、腎血管性高血圧症および腎臓損傷をもたらす8。多くの研究がヒトにおける腎血管性高血圧症中の酸化ストレスのレベルの増加を報告しており、これは2つの腎臓1クリップ(2K1C)マウスモデルならびに他の高血圧動物モデル2、9、10、11、12、13、14、15、16で裏付けられた.RAStenosis誘発性腎血管性高血圧症におけるレニン発現制御の分子機構は十分に理解されておらず、さらなる研究が必要である。
RAStenosisを確実かつ再現性よく再現性よく再現する実験動物モデルは、新規治療法の開発のためにレニン発現制御の細胞・分子機構を解明する上で重要です。2K1Cマウスモデルは、腎血管性高血圧症の病因を研究するための十分に確立された実験モデルである17、18、19、20。このモデルは、クリップ17、20、21を用いた腎動脈の狭窄によって生成され、したがってレニン発現および高血圧の増加をもたらす腎動脈閉塞を生じる17、19、20、21。しかしながら、動物モデルにおいて腎動脈狭窄を発生させるための段階的な手順を記述する技術報告は入手できない。
従来のU字型銀クリップ、ポリウレタンチューブおよび他のクリップは、腎動脈狭窄を誘発するために腎動脈を狭窄させるために使用されてきた。いくつかの研究は、クリップの設計と材料が、2K1C動物モデルで信頼性が高く再現可能なデータを得るために重要であることを示しています。Lorenzらによると、従来のUデザインの銀クリップを使用すると、高血圧の成功率が低い(40〜60%)21。クリップ設計のために、腎動脈は横方向に押され、いくつかのくびれを引き起こし、腎動脈から取り除かれる可能性が高くなります。銀の可鍛性と延性により、クリップ幅の変更が可能になる場合があります。したがって、マウス間で異なる高血圧レベルを引き起こす。クリップ上の二酸化銀は、血管周囲炎症、内膜増殖、および組織肉芽形成を引き起こし、腎動脈の直径22を変化させる可能性がある。従来のUデザインシルバークリップで得られた高血圧レベルの変動性のために、WarnerらおよびLorenzらは、ラウンダーデザインポリウレタンチューブを使用してマウスの腎動脈狭窄を開始し、2つの腎臓1つのクリップ動物モデルのより信頼性が高く一貫した誘導を生成することに成功した20、21。
この報告では、腎動脈を狭窄させるためにポリウレタンチューブを使用して、マウスにおいて実験的RAStenosisを生成するための外科的プロトコールについて記載する。ポリウレタンラウンドデザインのカフは、マウスに狭窄を発生させるための、より再現性が高く、信頼性が高く、低コストのクリップです。この実験モデルの目的は、腎動脈狭窄症におけるレニン発現制御の分子および細胞機構を研究し、定義することである。我々は、レニン発現および腎障害マーカー好中球ゼラチナーゼ関連リポカリン(N-GAL)を測定することによりRAStenosisマウスモデルの成功を確認した。

<table>
	<tbody>
		<tr>
			<td>Diet Gel</td>
			<td>Clear H2O</td>
			<td>Diet-Gel 76A</td>
			<td>Surgery recovery diet</td>
		</tr>
		<tr>
			<td>EMC Heated Hard pad</td>
			<td>Hallowell</td>
			<td>000A2788B</td>
			<td>Heating pads were used to keep mice warm</td>
		</tr>
		<tr>
			<td>Ethilon Nylon Suture</td>
			<td>Ethicon</td>
			<td>662G</td>
			<td>4-0 (1.5 metric), This suture was used to close the peritoneum, and skin</td>
		</tr>
		<tr>
			<td>Ethilon Nylon Suture</td>
			<td>Ethicon</td>
			<td>2815 G</td>
			<td>8-0 (0.4 metric), This suture was used to close cuff to tie and constrict the artery</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Braintree Scientific Inc.</td>
			<td>GER 5287</td>
			<td>Sterilize surgical tools between surgeries</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Zoetis</td>
			<td>Ketofen</td>
			<td>Painkiller</td>
		</tr>
		<tr>
			<td>Polyurethane</td>
			<td>Braintree Scientific Inc.</td>
			<td>MRE-025</td>
			<td>This tube was used to initiate stenosis</td>
		</tr>
		<tr>
			<td>Povidone-iodine antiseptic swabsticks</td>
			<td>Medline</td>
			<td>MDS093901</td>
			<td>It was applied after hair removal and surgery on the skin</td>
		</tr>
		<tr>
			<td>Reflex 7 Clip Applier</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>204-1000</td>
			<td>This clip applier was used to apply clip in case one or more sutures went off</td>
		</tr>
		<tr>
			<td>Sterile towel drapes</td>
			<td>Dynarex</td>
			<td>4410</td>
			<td>It was used as a bedsheet for mice during surgery</td>
		</tr>
		<tr>
			<td>Triple antibiotic ointment</td>
			<td>Medi-First</td>
			<td>22312</td>
			<td></td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td>Stryker</td>
			<td>T/pump Professionals</td>
			<td>Used to warm and circulate water in the heating hard pad to keep mice warm during and post-surgery</td>
		</tr>
	</tbody>
</table>

a modified two kidney one clip mouse model of renin regulation in renal artery stenosis

腎動脈狭窄は、レニンアンジオテンシンアルドステロン系(RAAS)が過剰に活性化されている冠状動脈または末梢血管疾患の患者における一般的な状態である。この文脈では、RAASにおける律速プロテアーゼであるレニンの発現および放出の増加を刺激する腎動脈の狭窄がある。結果として生じるレニン発現の上昇は、腎血管性高血圧症の既知のドライバーであり、しばしば腎臓損傷および末端器官損傷に関連する。したがって、この状態に対する新規治療法の開発に大きな関心がある。腎動脈狭窄におけるレニン制御の分子および細胞機構は完全には理解されておらず、さらなる研究が必要である。マウスにおいて腎動脈狭窄を誘導するために、改変2腎臓1クリップ(2K1C)ゴールドブラットマウスモデルが開発された。右腎臓を野生型マウスで狭窄し、偽手術マウスを対照として使用した。腎動脈狭窄後、レニン発現と腎障害を判定した。腎臓を採取し、新鮮な皮質を用いてレニンのタンパク質およびmRNA発現を決定した。この動物モデルは再現性があり、腎血管性高血圧症および腎損傷に関与する病態生理学的応答、分子および細胞経路を研究するために使用することができる。

腎動脈狭窄は、対側腎臓における発現を抑制しながら、狭窄した腎臓におけるレニン発現を増加させる。狭窄の2つの腎臓1つのクリップ(2K1C)またはGoldblattモデルは、レニン発現の増加および腎臓損傷を誘導する。これは、ヒトにおける片側腎動脈狭窄の最良の代表モデルとして認識されている。
レニンおよびプロレニン(レニンの前駆体)の発現は、イムノブロッティング?...

Watch this Scientific Journal Video about A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis at JoVE.com

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

修正された2腎臓1クリップ(2K1C)ゴールドブラットマウスモデルは、腎動脈狭窄を開始し、レニン発現および腎臓損傷の増加を誘導するためにポリウレタンチューブを使用して開発された。ここでは、再現可能で一貫性のある2K1Cマウスモデルを生成するために、袖口を準備して腎動脈上に配置する詳細な手順について説明します。

腎動脈狭窄におけるレニン調節の修正された2つの腎臓1つのクリップマウスモデル

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) ...

a-modified-two-kidney-one-clip-mouse-model-renin-regulation-renal

Research

JoVE Journal

Medicine

4.5K ビュー.  Vanderbilt University Medical Center. 修正された2腎臓1クリップ(2K1C)ゴールドブラットマウスモデルは、腎動脈狭窄を開始し、レニン発現および腎臓損傷の増加を誘導するためにポリウレタンチューブを使用して開発された。ここでは、再現可能で一貫性のある2K1Cマウスモデルを生成するために、袖口を準備して腎動脈上に配置する詳細な手順について説明します。

ビデオ: A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

隔離された腎動脈閉塞のためのハンギングウェイトシステムの利用

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

医学

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

中大脳動脈閉塞のマウスモデル

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

慢性腎臓病患者における血管機能の評価

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

腎臓虚血再灌流障害：障害のマウスモデルおよび再生

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

マウスの腎臓移植​​技術

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

変更されたカフを用いた膵臓移植マウス モデル

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

糸球体疾患のマウス ・ モデルにおける腎機能の評価

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors 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 ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

腎動脈を介した腎臓への直接薬物送達

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...