博士デCAESTECKERの研究室では、NIH 1RO1 HL093057-01と1RC4DK090770-01でサポートされています。博士はハリスの研究室はDK38226、DK51265、DK62794および退役軍人からの資金によってサポートされています。またバンダービルトオブライエン腎傷害センター助成1P30 DK079341が提供するマウスの腎臓の傷害手術、血清クレアチニン、および線維アッセイのサポート。

<ol><li>手術前に、すべての手術器具をオートクレーブ。一つは別のマウスで複数の手術を行っている場合、ホットビーズ滅菌器を使用して滅菌した後、使用後の楽器をすすぎ、あることに注意してください。これは、70％エタノールに浸漬するのに十分ではない。 </li><li>手術中の体液の損失を補償するために、術前術後に、すぐに0.5ミリリットルS / C無菌生理食塩水を与える。 </li><li>マウスの重量を量る。 </li><li> IPキシラジン/ケタミン混合物を用いてマウスを麻酔。それは、通常、外科的な平面麻酔に達するためにマウスで3-5分かかります。 </li><li>切開部位を汚染から髪を保つために十分な国境地帯で手術部位を剃る。これは、手術が行われる場合に、予備領域にない行う必要がある。 </li><li>手順の実行中に、乾燥を防ぐために、目に眼科潤滑軟膏を適用します。 </li><li>吸収ベンチパッドで覆われて加熱された表面上に発生しやすいマウスを置きます。手術の表面にテープ脚。 </li><li>使用している場合水浴システム、手術前に38℃で1時間に設定した熱。 </li><li>無菌調製は、ベタジンソリューション綿棒スティックを使用して：ベタジンを削除するNolvasanとスクラブが続くたびに、3倍の外周部に向かってサイトの中心からスクラブ。すべてベタジンはNolvasanですすぐことで切開する前に手術野から削除する必要があります。無菌手順手袋を開始する前に無菌になるように変更されるべきである。 </li><li>滅菌外科用ドレープと手術野をカバーしています。これは、手術野に入ることから浮遊毛を防ぎ、手術中に無菌の楽器を築くための領域を提供する。 </li><li>皮膚から腎臓の場所を触診する。 </li><li>はさみや鉗子を用いたマウス（約1.5 cm）のの正中線に沿って背部皮膚をカットします。 </li><li>はさみやピンセットを用いて鈍的切開によってこの切開を通して左右の背側面上に独立した皮膚と皮下層。 </li><li>右FLを通して小さな切開を行います腎臓上記の筋肉や筋膜をANKと右の腎臓を体外に出す。 </li><li>慎重に周囲組織から遊離腎臓の上下の極を解剖。上極の周りから脂肪組織が ​​腎臓をオフにプッシュすることができますが、明らかにマウス（ 図1）から削除されるべきではない、独自の血液供給を運ぶ副腎が含まれていることに注意してください。周囲の組織から解放腎臓後、二重手術の結び目を使用して右の腎臓の門の周りに4-0絹縫合糸を結ぶ。縫合糸の一端を残す〜あなたがカットするためにサイトを視覚化できるように、結び目に遠位切断されている限りで2が腎臓を保持する。滅菌生理食塩水で切り株を灌漑。 </li><li>吸収性縫合糸を用いて、筋層を閉じます。 </li><li>左脇腹の筋肉と腎臓上記の筋膜を通して小さな切開を行い、左腎臓（ 図2A）を体外に出す。 </li><li>腎を放出しながら、慎重に鈍鉗子を使用して腎臓を開催鉗子（ 図2B-2F）を使用して脂肪組織を取り巻くから茎。研究のために &quot;偽&quot;コントロールを設計する際に1つのニーズはこれを覚えておくように鈍鉗子を使用して、腎臓にあまり圧力が腎障害を引き起こす可能性があります。あなたが偽のコントロールが腎障害を（として血清クレアチニンの上昇によって証明）開発していることが判明した場合は、腎茎から周囲の脂肪組織を解放するために考慮することができる別の方法は優しく生理食塩水に浸した滅菌と組織を転がすことで綿先端が綿棒。我々の経験では脂肪がかなりきつく腎茎に取り付けられており、簡単に綿棒で除去することができませんので、これはあなたが使用したいと思う最初のアプローチではありません。しかし、このモデルの他の側面と同様に、これはあなたが練習と経験を自分のために確立しなければならないというものです。 </li><li>保持鉗子（クランプの配置を容易にする）を用いて、非外傷性血管クランプを使用して、左腎椎弓根をクランプする、異なるCLAのためにタイマを使用融点時間はマウス系統と（ 表1を参照）が必要傷害重症度に応じ。 </li><li>皮膚（ 図2H）と左腎をカバーしています。 </li><li>生理食塩水に浸したガーゼで皮膚切開をカバーしています。 </li><li>腎臓を体外に出す、制服薄暗い外観を確認し、指示された時間後にクランプを解除し、その薄暗い外観は腎臓全体に均一に反転（ 図2I / G）を確認します。腎臓は急速かつ均一に再灌流（ピンクアップ）されない場合は、マウスは、さらに分析から除外されるべきである。そっと後腹膜腔に戻し腎臓を押してください。 </li><li>吸収性縫合糸を使用した筋層を閉じます。 </li><li>モノフィラメントナイロン非吸収性縫合糸を使用したスキン層を閉じます。 </li><li>ブプレノルフィン鎮痛を管理します。 </li><li>彼らが目を覚ますまで、加熱パッド上に離れて手​​術野からマウスを転送します。 </li><li>動物の部屋にマウスを返します。 </li><li>密接にマウスを監視し、付加的な用量を与えるブプレノルフィンのように痛みや不快感のために、すべての8-12時間の上に示された。鎮痛薬を投与し、その結果臨床症状は、うつ病や他の行動の変化、異常な外観、またはそのようなPILO-勃起、猫背姿勢、グルーミングや食事の欠如、あるいは不動などの姿勢があります。 </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure.+II.+Experimental+models+of+acute+renal+failure:+Imperfect+but+indispensable.">Acute renal failure. II. Experimental models of acute renal failure: Imperfect but indispensable.</a> Am. J. Physiol. Renal Physiol. 278, F1-F12 (2000).</li><li>Dunn, S. R., Qi, Z., Bottinger, E. P., Breyer, M. D., Sharma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+endogenous+creatinine+clearance+as+a+measure+of+renal+function+in+mice.">Utility of endogenous creatinine clearance as a measure of renal function in mice.</a> Kidney Int. 65, 1959-1967 (2004).</li><li>Yuen, P. S., Dunn, S. R., Miyaji, T., Yasuda, H., Sharma, K., Star, R. 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+simplified+method+for+HPLC+determination+of+creatinine+in+mouse+serum.">A simplified method for HPLC determination of creatinine in mouse serum.</a> Am. J. Physiol. Renal Physiol. 286, F1116-F1119 (2004).</li><li>Hetu, P. O., Gingras, M. E., Vinet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+rapid+liquid+chromatography+isotope+dilution+tandem+mass+spectrometry+(LC-IDMS/MS)+method+for+serum+creatinine.">Development and validation of a rapid liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for serum creatinine.</a> Clin. Biochem. 43, 1158-1162 (2010).</li><li>Keppler, A., Gretz, N., Schmidt, R., Kloetzer, H. M., Groene, H. J., Lelongt, B., Meyer, M., Sadick, M., Pill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+creatinine+determination+in+mice+and+rats:+An+enzymatic+method+compares+favorably+with+a+high-performance+liquid+chromatography+assay.">Plasma creatinine determination in mice and rats: An enzymatic method compares favorably with a high-performance liquid chromatography assay.</a> Kidney Int. 71, 74-78 (2007).</li><li>Yang, L., Besschetnova, T. Y., Brooks, C. R., Shah, J. V., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+cycle+arrest+in+G2/M+mediates+kidney+fibrosis+after+injury.">Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.</a> Nat. Med. 16, 535-543 (2010).</li><li>Shanley, P. F., Rosen, M. D., Brezis, M., Silva, P., Epstein, F. H., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+focal+proximal+tubular+necrosis+after+ischemia+with+reflow+in+the+rat+kidney.">Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.</a> Am. J. Pathol. 122, 462-468 (1986).</li><li>Chawla, L. S., Amdur, R. L., Amodeo, S., Kimmel, P. L., Palant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+severity+of+acute+kidney+injury+predicts+progression+to+chronic+kidney+disease.">The severity of acute kidney injury predicts progression to chronic kidney disease.</a> Kidney Int. 79, 1361-1369 (2012).</li><li>Lo, L. J., Go, A. S., Chertow, G. M., McCulloch, C. E., Fan, D., Ordonez, J. D., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialysis-requiring+acute+renal+failure+increases+the+risk+of+progressive+chronic+kidney+disease.">Dialysis-requiring acute renal failure increases the risk of progressive chronic kidney disease.</a> Kidney Int. 76, 893-899 (2009).</li><li>Burne, M. J., Haq, M., Matsuse, H., Mohapatra, S., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+to+renal+ischemia+reperfusion+injury+revealed+in+a+murine+model.">Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model.</a> Transplantation. 69, 1023-1025 (2000).</li><li>Muller, V., Losonczy, G., Heemann, U., Vannay, A., Fekete, A., Reusz, G., Tulassay, T., Szabo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+renal+ischemia-reperfusion+injury+in+rats:+Possible+role+of+endothelin.">Sexual dimorphism in renal ischemia-reperfusion injury in rats: Possible role of endothelin.</a> Kidney Int. 62, 1364-1371 (2002).</li><li>Schmitt, R., Marlier, A., Cantley, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zag+expression+during+aging+suppresses+proliferation+after+kidney+injury.">Zag expression during aging suppresses proliferation after kidney injury.</a> J. Am. Soc. Nephrol. 19, 2375-2383 (2008).</li><li>Oxburgh, L., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+injury+of+the+mouse+kidney.">Ischemia-reperfusion injury of the mouse kidney.</a> Methods Mol. Biol. 886, 363-379 (2012).</li></ol>

著者らは、開示する利害の衝突を持っていません。

私たちは、中等度および重度の腎障害の影響を研究するためのIR-AKIの2つのモデルを記述する。これらのモデルは、私たちは、低死亡率と一貫性のある予測可能な傷害を誘発することができます。我々のプロトコルでは、伝統的にこのモデルに関連付けられた困難や落とし穴の多くを概説します。さらに、腎茎クランプの長さに応じて、モデルが不完全回復と持続的な腎線維症で、主に可逆軽...

急性腎臓損傷（AKI）の実験モデルの様々な（最近の、包括的なレビュー1リファレンスを参照してください）人間の条件の多様性と複雑さを一致させるために開発されている。これらの各モデルは、独自の長所と短所があり、それぞれが様々な効率化に対応する人間の条件を模倣しながら、どれも正確に彼らの人間の対応の病態をモデル化しません。虚血再灌流（IR）誘発AKIは、げっ歯類の急性虚血誘発性腎障害のモデルとして開発されました。このモデルで見られる尿細管傷害の重症度はほとんどその限界にもかかわらず、腎低灌流傷害患者2で観察されておらず、主な理由このモデルの比較的再現性質のため、その広範な使用が提供していると、それが期待されている間、継続します共通の基本AKIのメカニズム、修理、治療3の多くの重要な洞察を提供する。 IR手術は家族を必要とします我々は、 図1の簡略化した形で示されているマウスの腎臓の解剖とiliarity。腎臓の虚血再灌流（IR）傷害の手術を腹側（開腹）または背側（後腹膜）のアプローチを介して行うことができる。それは少ない外傷であるため、我々はより速い回復時間と生存率の改善を（最初の手順を学習する場合は特に）ができ、背のアプローチを使用しています。腎IR傷害は一方的にまたは両側行うことができます。しかし、効果的なクランプ圧（クランプ顎の間perihilar脂肪の介在に起因するもの）および/または側面間虚血、腎応答の違いの差がより変動結果につながる。これは克服できない問題ではありませんが、それはこのモデルのための主要な課題となっている実験では、間の変動性を高めることができます。片側IRは対側腎摘出術を行うことができる。これは、椎弓根間のクランプの変動を減少するための選択肢の私たちの方法であり、同時にllows片側IR独りで影響を受けません腎機能を評価するための1、。マウスでは腎機能を評価するための最も実用的な対最適な方法であるかについて議論がありました。血中尿素窒素（BUN）は、腎機能の尺度を提供し、IR傷害を含むAKIのいくつかのモデルにおいて有用 &quot;一見&quot;マーカーである。しかし、BUNレベルが回復の遅れが日手術後数の流体の経口摂取を減少させるときに、特に腹側アプローチIR損傷後の、影響を受ける可能性があるマウスの体積の状態によって影響を受けることができる。血清クレアチニンは少ない水和の状態に影響されますが、明らかに筋肉量に影響されます。血清クレアチニン測定の困難の一つは、ピクリン酸ベースの技術を使用して、マウス血清中の非クレアチニン色原体の検出に問題があった。代替として、中心の数は、このアーティファクト4によって影響されないマウスクレアチニンを定量化するHPLCに基づく方法を開発した。しかし、UNLIKE BUNと血清のみの5-10μLを必要とピクリン酸クレアチニンアッセイは、HPLC-アッセイは〜25μlのアッセイあたりの血清、重複で行った場合1アッセイあたり全血の〜100μlのが必要になります。を必要とするこれは、マウスの研究に限定することができる。いくつかのセンターでは、小さいサンプル量5、6の分析を可能にする、より高感度HPLCおよび質量分析ベースのメソッドを開発しました。しかし、これらの技術は広く利用可能ではありません。常にピクリン酸アッセイ間の代替、酵素カスケードアッセイ（血清のわずか5〜10μlのを必要とする）は、マウスおよびラットの血清サンプルで評価し、血清クレアチニンの密接並列HPLC測定することが示されているオーバー推定クレアチニン値7。このアッセイが広くAKI文献に使用されていないが、アッセイは使用するために、シンプルな市販されており、我々は、マウスのIR誘発性AKIのこのモデルでは信頼性の高い結果が得られます見つける。

<table border="1">
	<tbody>
		<tr>
			<td>Drug or solution</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ketamine (anesthetic)</td>
			<td>Webster Veterinary</td>
			<td>07-881-9413 (100 mg/ml)</td>
			<td>Final concentration 30 mg/ml Ketamine with 3 mg/ml Xylazine administered at 3-4 ml/kg (100 mg/kg Ketamine and 10 mg/kg Xylazine)</td>
		</tr>
		<tr>
			<td>Xylazine (analgesic)</td>
			<td>Webster Veterinary</td>
			<td>07-808-1939 (100 mg/ml)</td>
			<td>As above</td>
		</tr>
		<tr>
			<td>Antisedan (anti-Xylazine)</td>
			<td>Webster Veterinary</td>
			<td>07-867-7097 (5 mg/ml)</td>
			<td>Optional: to reverse effects of Xylazine in order to increase the rate of recovery after surgery Use 0.5-0.7 mg/kg</td>
		</tr>
		<tr>
			<td>Buprenorphine (analgesic)</td>
			<td>Bedford Laboratories</td>
			<td>NDC 55390-100-10 (0.3 mg/ml)</td>
			<td>Dose 0.05-0.1 &mu;g/g every 8-12 hr. Make up a 1:5 dilution in sterile normal saline preoperatively and give 2.5-5 &mu;l/g of the diluted solution.</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>NDC 17033-211-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine swab sticks</td>
			<td>Purdue Products L.P.</td>
			<td>NDC 67618-153-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nolvasan Surgical Scrub</td>
			<td>Pfizer Animal Care</td>
			<td>300253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Drugs and solutions for I/R surgery.</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Integra Miltex</td>
			<td>VWR 21909-404</td>
			<td>For skin and muscle dissection</td>
		</tr>
		<tr>
			<td>Curved iris forceps</td>
			<td>FST</td>
			<td>11065-07</td>
			<td>To hold skin and muscle, to remove peri-nephric fat</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>ROBOZ</td>
			<td>RS-5228</td>
			<td>To manipulate kidneys</td>
		</tr>
		<tr>
			<td>Vascular clamp (795 g pressure)</td>
			<td>ROBOZ</td>
			<td>RS-5459</td>
			<td>Good for C57BL/6 and BALB/c mice strains</td>
		</tr>
		<tr>
			<td>Schwartz clip applying forceps</td>
			<td>ROBOZ</td>
			<td>RS-5450</td>
			<td>For RS-5459 clamps</td>
		</tr>
		<tr>
			<td>Vascular clamp (75-85 g pressure)</td>
			<td>FST</td>
			<td>18055-02</td>
			<td>Optimal for CD1 mice strain, but also can be used for C57BL/6 and BALB/c</td>
		</tr>
		<tr>
			<td>Micro-Serrefine Clamp Applicator-14 cm with Lock</td>
			<td>FST</td>
			<td>18056-14</td>
			<td>For FST 18055-02 clamps</td>
		</tr>
		<tr>
			<td>Gayman T/Pump (water-bath heated platform)</td>
			<td>Braintree Scientific</td>
			<td>TP-650</td>
			<td>The water heater is set at 38 &deg;C 1 hr prior surgery</td>
		</tr>
		<tr>
			<td>15" W x 24" Heated platform</td>
			<td>Braintree Scientific</td>
			<td>HHP-2</td>
			<td>Connect to Gayman heated water pump</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree Scientific</td>
			<td>Model 39DP</td>
			<td>Uses microwave for heating. This is useful for warming during recovery</td>
		</tr>
		<tr>
			<td>Absorbable suture (Vicryl 5-0)</td>
			<td>ETHICON</td>
			<td>VCP834G</td>
			<td>For fascia and muscle</td>
		</tr>
		<tr>
			<td>Non-absorbable suture: monofilament nylon MONOMID 6/0 660B)</td>
			<td>CP Medical</td>
			<td>CP-B660B-03</td>
			<td>For skin</td>
		</tr>
		<tr>
			<td>Halsey needle holder: Tungsten Carbide/13 cm</td>
			<td>FST</td>
			<td>12501-13</td>
			<td>To hold suture needles</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>FST</td>
			<td>06-662-3</td>
			<td>To quantify ischemia time</td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>FST</td>
			<td>18000-45</td>
			<td>To sterilize instruments between mice if you are performing multiple surgeries (it is not sufficient to soak instruments in ethanol)</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Braintree Scientific</td>
			<td>CLP-22965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Durapore</td>
			<td>1538-0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>EXELint</td>
			<td>26044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicators</td>
			<td>Kendall</td>
			<td>8884541300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent BenchPad</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical drapes</td>
			<td>VWR</td>
			<td>21902-985</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr>
			<td>2'' x 2'' Gauze Pads</td>
			<td>Medi-First</td>
			<td>60673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Cardinal Health</td>
			<td>2D7251</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Equipment and instruments for I/R surgery.</td>
		</tr>
	</tbody>
</table>

ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice

虚血再灌流誘発される急性腎傷害（IR-AKI）が広く、マウスにおけるAKIのモデルとして使用されますが、結果は、多くの場合、分析を混乱させることが高く、頻繁に報告されていない死亡率が非常に可変であるされています。両側性腎茎クランプは、一般的にIR-AKIを誘導するために使用されますが、効果的なクランプ圧および/または腎臓の間で虚血性腎応答の違いは多くの場合、変数の結果につながるされています。また、短いクランプ時間がより変動細管障害を誘導することが知られており、より長いクランプ時間でバイラテラル損傷を受けたマウスは、より一貫した管状の損傷を開発するが、それらはしばしば重度の腎不全に起因する損傷後の最初の3日以内に死亡する。損傷後の生存率を改善し、より一貫した予測可能な結果を​​得るために、我々は、反対側の腎摘出術に続いて一方的な虚血再灌流障害の2つのモデルを開発した。両方の手術をfを得外科応力を低減する、背アプローチを使用して実行され一般的にマウスIR-AKIの手術に使用ロム腹開腹、。中程度の傷害をBALB / cマウスの誘導のために一方的には26分間腎茎のクランプ受け、また同時対腎摘出を受ける。このアプローチを使用して、マウスの50〜60％は、損傷後の適度なAKI 24時間を開発するが、マウスの90〜100％は生き残る。より厳しいAKIを誘導するために、BALB / cマウスは、8日後の傷害反対側の腎摘出術に続いて30分間クランプ腎椎弓根を受ける。これは、90〜100％の生存率と損傷後の腎回復の機能評価を可能にします。初期の損傷後尿細管障害と同様にポスト傷害線維症は、このモデルを使用して高度に一貫しています。

同時対側腎摘出術との適度なIR-誘発AKI。片側IRは、実質的結果にばらつきは減少しますが、この手順を生き残るためには、マウスのために必要な短いクランプ時間で、我々はまだ、マウスのわずか50〜60％が期待腎不全24時間を開発しましたことがわかった後に傷害（ 図3A）。実用的な面では、これは研究では、時間は腎機能を評価し、腎不全を発症しないマウスを破?...

Watch this Scientific Journal Video about Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice at JoVE.com

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

私たちは、マウスが受けている中等度および重度の虚血再灌流誘発性腎傷害のモデルを記述する一方的な腎茎それぞれ、同時または遅延対側腎摘出続いクランプ。これらのモデルは、一貫して腎機能障害や損傷後の線維化を生じさせるが、傷害の重症度と生存率は、マウス、背景、年齢や手術用機器に依存している。

マウスにおける急性腎障害とポスト傷害線維の虚血再灌流モデル

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

ischemia-reperfusion-model-acute-kidney-injury-post-injury-fibrosis

Research

JoVE Journal

Medicine

39.4K ビュー.  Vanderbilt University Medical Center. 私たちは、マウスが受けている中等度および重度の虚血再灌流誘発性腎傷害のモデルを記述する一方的な腎茎それぞれ、同時または遅延対側腎摘出続いクランプ。これらのモデルは、一貫して腎機能障害や損傷後の線維化を生じさせるが、傷害の重症度と生存率は、マウス、背景、年齢や手術用機器に依存している。

ビデオ: Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

正常体温心停止と心肺蘇生法：虚血再灌流障害のマウスモデル

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

医学

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

ランゲンドルフ灌流ラット心臓における虚血再灌流傷害の誘導と評価

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

腸管虚血 - 再灌流傷害のマウスモデル

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

ラットにおける心筋虚血再灌流後の冠動脈流量の評価

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

再建微小外科における虚血再灌流損傷の研究のための前臨床ラットモデル

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

マウスモデルにおける虚血再灌流損傷後の幹細胞の筋内内出産の遅延

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成体ゼブラフィッシュにおけるシスプラチンによる急性腎損傷モデル

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

ラット肝移植モデルにおける血球力学的変化をシミュレートすることによる臓器虚血再灌流損傷

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

温虚血/再灌流損傷のラット肺移植モデル:転帰を改善するための最適化

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

片側性腎虚血再灌流傷害の有効マウスモデル

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...