この作品は、健康のペンシルバニア部からの助成金によって支えられて。 博士ミゲルガルシアトスカーノはこの調査中にアルフォンソマルティンエスクデロ財団が資金提供テンプル大学でポスドクだった。 我々は、ビデオの製作のためにIliya YordanovとケビンKotredesに感謝したいと思います。

1。ヒト敗血症のマウスモデルとして盲腸結紮穿刺この手順のC57BL / 6マウスのために（7-9週齢）に使用されています。 <ol><li>腹腔内に1時01分ケタミン（75mg/kg）とキシラジン（15mg/kg）の溶液を注入することにより、マウスを麻酔。参考として、20グラムのマウスに1:1溶液30μlを注入する。また、マウスは、麻酔薬の気化器を用いて吸入イソフルランで麻酔をすることができます。 </li><li>マウスの腹部をひげをそると最初の70％アルコールに浸した綿棒（下の写真の順序で番号1）で拭い続いbetadineのソリューションを適用することによって領域を消毒する。クロルヘキシジンは、代替の防腐剤として使用することができます。オプション：滅菌ドレープがきれいな部分を維持するために使用されることがあります。 </li><li>無菌条件下では、1〜2センチメートル正中開腹術を練習し、隣接する腸（2と3）と盲腸を公開。 </li><li>盲腸はしっかり6.0絹縫合糸（6-0 PROLENE、8680G、エチコン）と連結する回腸盲腸弁下に、そのベースにある、と同じで穴あき一度か二度19ゲージ針（4と5）となります盲腸の側面。ライゲーションのポイントに盲腸の先端からの距離として定義されて連結された盲腸の長さは、重症度を決定することに注意してください。 ≤1cmの距離が中〜低学年の敗血症を生成しながら&gt; 1cmの距離は、高品位の敗血症を生成します。また、ここに示されて盲腸穿孔のメソッドを使用しても、（盲腸を通して針を導入する）を介して穿刺の技術を利用して、標準とは異なることに注意してください。どちらのメソッドも同じ敗血症の予後を確実に生成する。しかし、根っからの技術の欠点の1つは、盲腸が絞られているときに押し出された糞の量を制御すること容易にしないことです。 </li><li>盲腸は、その後、静かに穿孔部位（6）から糞の少量を押し出すために圧迫されています。盲腸を腹腔内に返され、腹膜が6.0絹縫合糸で閉じられます。 （7）。 </li><li>皮膚は、リフレックス7ミリメートルクリップ（RS - 9258、Roboz手術器具）やミシェルの創傷クリップ（7ミリメートル、RS - 9270）（8）で閉じられます。 </li><li> 25G針を用いて予め温めておいた0.9％生理食塩水の皮下1mlを注入することにより、マウスを復活させる。この流体の蘇生措置は、敗血症の亢進相を誘導する。この重要なステップは、議論のセクションで詳しく説明されています。 </li><li>オプション：術後鎮痛のために皮下ブプレノルフィン（0.05mg / kg体重）またはトラマドールの（20mg/kg体重）注入。これらの麻薬は、呼吸や運動を抑制することができることをお勧めすると、これらの効果は、敗血症の兆候として誤って解釈することができます。 </li><li>動物は、加熱パッドを一時的に置くか、彼らは麻酔から回復するまで交互に150Wの赤外線加熱ランプへの曝露とのケージに直ちに返されます。回復時間は30分〜1時間からです。 </li><li>ケージの底部に配置食べ物と水（ハイドロゲル）への無料アクセスを提供します。 </li><li>マウスは1週間から2週間生存のために12時間ごとに監視したり、様々なパラメータの解析のために異なる時点で安楽死されています。 </li></ol>実験的な設計のための対照として、偽の動物は結紮穿刺することなく開腹術のテクニックに従う。 外科的処置のマウスの後の6〜12時間は、無気力になり、発熱、立毛、下痢、拠り所、および倦怠感を開発し、敗血症のすべての症状。非常に重症敗血症のマウスは、ほとんど死亡前に移動し、体温の劇的な低下を示すことができる。このステップのマウスでの長時間の痛みと苦しみを避けるために安楽死されるべきである。 <img src="/files/ftp_upload/2860/2860fig0.jpg" alt="図0" /> 2。最も一般的な分析されたパラメータ手続きの結果を評価するために、異なるパラメータが臓器、細胞抽出物または体液で分析することができます。サンプルは、外科的処置後3時間から一週間に異なるタイムポイントで収集することができます。 <ol><li>マウスの生存率。 </li><li>血清、腹腔内および臓器抽出物におけるサイトカインとケモカイン。 <ol><li> インターロイキン6：単球、樹状細胞、マクロファージ、B細胞、T細胞、顆粒球、マスト細胞と、より多くの細胞型により産生され、放出。それは急性相反応と炎症において重要な役割を果たしている。 </li><li> 腫瘍壊死因子-α：炎症およびアポトーシスにおいて中心的な役割を果たすPleiotrophicサイトカイン。単球、マクロファージ、好中球、樹状細胞と線維芽細胞によって産生さ。 </li><li> nterleukin -1β：単球、NK細胞、樹状細胞、B細胞とT細胞により産生される。発熱や急性期タンパク質の合成を誘導する。 </li><li> インターロイキン10：貪食取り込みおよびTh2応答を促進するがantig抑制ENプレゼンテーションとTh1の炎症性応答。 </li><li> インターロイキン10：貪食取り込みおよびTh2応答を促進するが、抗原の提示とTh1の炎症反応を抑制します。 </li><li> 単球走化性タンパク質-1（もCCL2とも呼ばれる）：新兵の単球、メモリーT細胞、および組織損傷や感染症のサイトへの樹状細胞。 </li><li> KC（CXCL1）：マクロファージと血管内皮細胞により産生される。マウスKCは、強力な好中球の誘引及び活性剤です。 </li><li> RANTES：単球走化性。それは、非刺激CD4 + / CD45RO +メモリーT細胞と刺激CD4 +およびNaのVEとメモリの表現型を有するCD8 + T細胞を化学的に誘引することができます。 </li><li> インターフェロン-γ：Th1細胞、細胞傷害性T細胞、樹状細胞とナチュラルキラー（NK）細胞から分泌される。マクロファージの抗原提示と溶解活性を増加させ、Th2細胞の活性を抑制する。白血球遊走に必要な接着との結合を促進し、NK細胞活性を促進する。 </li><li> 高移動度群B - 1蛋白質：単球では、敗血症の後期段階における炎症性メディエーターの放出を活性化する、事前の糖化最終産物の細胞受容体（RAGE）に結合することにより、核因子カッパB活性化因子である。 </li></ol></li><li>好中球浸潤の指標としての臓器におけるミエロペルオキシダーゼの定量。ミエロペルオキシダーゼは（MPO）好中球顆粒球の中で最も豊富に存在するペルオキシダーゼ酵素です。それは好中球のアズール顆粒に格納されたリソソーム蛋白質である。 MPOは、膿や粘液のいくつかの形態として、好中球の豊富な分泌物で、その緑の色を、引き起こすヘム色素を持っています。 </li><li>臓器や血流中の細菌負荷、ミリリットル当たりコロニー形成単位の数として測定する。 </li></ol> 3。代表的な結果 CLPの手順は、最初にC57BL / 6マウスの系統で実施した。我々は図1と図2に示すように針のライゲーションと厚さの長さを変えることによって敗血症の重症度を調節するためにいくつかのパラメータをテスト。これら二つの因子の中で、ライゲーションの長さは、生存率を変更するために、針の太さよりも効果的と思われる。 図1に示すように、1センチメートル以上でライゲーションの長さを増加させると≤1cmのライゲーションを有するマウスに比べて100％の死亡率の増加を引き起こす。針の太さを大きくすると、2つの穴を開けて100％（22G針を使用して）から55％（19G針を使用して）に生存率を減少させた。我々はまた、異なるマウス系統は、CLP -誘導敗血症に類似または異なる感受性を表示するかどうかを判断するためにC57BL / 6と129SvJマウスにCLPの効果を試験した。図3と同じ条件の下で、129SvJマウスは増加パーセントの死亡率で示されたC57BL / 6、より感染を受けやすくなったことを示している。 <img src="/files/ftp_upload/2860/2860fig1.jpg" alt="図1" /> 図1。動物の生存に盲腸の結紮の長さの効果。CLPは、盲腸結紮二つの異なる長さを使用して、C57BL / 6マウスで行われた。この単一のパラメータは、劇的に1cmの約のライゲーションの面積を有する動物の群と比較して3日未満で死亡した1cm未満長いライゲーションエリアと群の動物のすべて以来、動物の生存に影響を与えます。 （N = 8）。 <img src="/files/ftp_upload/2860/2860fig2.jpg" alt="図2" /> 図2。生存に対する針板厚の影響は。CLPは、19Gおよび22Gつの異なる針サイズを、使用してC57BL / 6マウスで行われた。 19G針を用いて穿孔盲腸とマウスは、55〜60パーセントの生存率を示した。彼らは最初の3-4日間は炎症の典型的な症状を経験したものの、これとは対照的に、22G針を用いて穿孔盲腸とマウスは100％の生存率を示したが、これは（n = 8）を4日後に消失した。 <img src="/files/ftp_upload/2860/2860fig3.jpg" alt="図3" /> 図3。二つの異なるマウス系統間で敗血症の感受性の比較。CLPモデルは、同じ条件で129SvJとC57BL / 6で行われた。評価期間の終了時に129SvJ株の生存率は、複数菌感染により誘導される炎症に高い感受性を示し、C57BL / 6系統に比べて25％低かった。 （N = 6）。

<ol>
	<li>Angus, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+severe+sepsis+in+the+United+States:+analysis+of+incidence,+outcome,+and+associated+costs+of+care.">Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.</a> Crit Care Med. 29, 1303-1310 (2001).</li><li>Riedemann, N. C., Guo, R. F., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigma+of+sepsis.">The enigma of sepsis.</a> J Clin Invest. 112, 460-467 (2003).</li><li>Eichacker, P. Q., Gerstenberger, E. P., Banks, S. M., Cui, X., Natanson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+acute+lung+injury+and+acute+respiratory+distress+syndrome+trials+testing+low+tidal+volumes.">Meta-analysis of acute lung injury and acute respiratory distress syndrome trials testing low tidal volumes.</a> Am J Respir Crit Care Med. 166, 1510-1514 (2002).</li><li>Buras, J. A., Holzmann, B., Sitkovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis:+setting+the+stage.">Animal models of sepsis: setting the stage.</a> Nat Rev Drug Discov. 4, 854-865 (2005).</li><li>Doi, K., Leelahavanichkul, A., Yuen, P. S., 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=Animal+models+of+sepsis+and+sepsis-induced+kidney+injury.">Animal models of sepsis and sepsis-induced kidney injury.</a> J Clin Invest. 119, 2868-2878 (2009).</li><li>Godshall, C. J., Scott, M. J., Peyton, J. C., Gardner, S. A., Cheadle, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+susceptibility+during+murine+septic+peritonitis.">Genetic background determines susceptibility during murine septic peritonitis.</a> J Surg Res. 102, 45-49 (2002).</li><li>De Maio, A., Torres, M. B., Reeves, R. 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+determinants+influencing+the+response+to+injury,+inflammation,+and+sepsis.">Genetic determinants influencing the response to injury, inflammation, and sepsis.</a> Shock. 23, 11-17 (2005).</li><li>Diodato, M. D., Knoferl, M. W., Schwacha, M. G., Bland, K. I., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+the+inflammatory+response+and+survival+following+haemorrhage+and+subsequent+sepsis.">Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis.</a> Cytokine. 14, 162-169 (2001).</li><li>Turnbull, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+age+on+mortality+and+antibiotic+efficacy+in+cecal+ligation+and+puncture.">Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture.</a> Shock. 19, 310-313 (2003).</li><li>Miyaji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethyl+pyruvate+decreases+sepsis-induced+acute+renal+failure+and+multiple+organ+damage+in+aged+mice.">Ethyl pyruvate decreases sepsis-induced acute renal failure and multiple organ damage in aged mice.</a> Kidney Int. 64, 1620-1631 (2003).</li><li>Torres, M. B., De Maio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exaggerated+inflammatory+response+after+CLP+correlates+with+a+negative+outcome.">An exaggerated inflammatory response after CLP correlates with a negative outcome.</a> J Surg Res. 125, 88-93 (2005).</li><li>Doi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AP214,+an+analogue+of+alpha-melanocyte-stimulating+hormone,+ameliorates+sepsis-induced+acute+kidney+injury+and+mortality.">AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.</a> Kidney Int. 73, 1266-1274 (2008).</li><li>Wilson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation+attenuates+early+cytokine+mRNA+expression+after+peritonitis.">Fluid resuscitation attenuates early cytokine mRNA expression after peritonitis.</a> J Trauma. 41, 622-627 (1996).</li><li>Zanotti-Cavazzoni, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation+influences+cardiovascular+performance+and+mortality+in+a+murine+model+of+sepsis.">Fluid resuscitation influences cardiovascular performance and mortality in a murine model of sepsis.</a> Intensive Care Med. 35, 748-754 (2009).</li><li>Hugunin, K. M., Fry, C., Shuster, K., Nemzek, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tramadol+and+buprenorphine+on+select+immunologic+factors+in+a+cecal+ligation+and+puncture+model.">Effects of tramadol and buprenorphine on select immunologic factors in a cecal ligation and puncture model.</a> Shock. 34, 250-260 (2010).</li></ol>

利害の衝突は宣言されません。<br /&gt;動物実験は、ケアと実験動物の使用のための健康ガイドの国立研究所にしたがって行われ、テンプル大学で動物のケアと使用に関する委員会によって承認された。

ここでは、マウスにCLPモデルを実行する方法を詳細に示し、重症度を調節している。 敗血症の他の動物モデルと比較すると、CLPは、異なる年齢と性別のいずれかのマウス系統で行うことができます。それは比較的簡単で安価な手術です。このモデルでは、重症度のグレードは、生存の割合に直接影響を与えます。ライゲーションの長さ、針の太さとパンクの数は、CLPの?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>ケタミン</td><td> Ketaset </td><td> 0856-2013-01 </td><td></td></tr><tr><td>キシラジン</td><td> AnaSed </td><td> NADA＃139から236 </td><td></td></tr><tr><td>インスリン注射器の29G </td><td>エクセル</td><td> 26028 </td><td></td></tr><tr><td>絹縫合糸、6から0 PROLENE </td><td>エチコン</td><td> 8680G </td><td></td></tr><tr><td> 19Gと25Gニードル</td><td> BD </td><td> 305186 </td><td></td></tr><tr><td>シェーバー</td><td>一般的な供給</td><td>一般的な供給</td><td></td></tr><tr><td>赤外線加熱ランプ</td><td>一般的な供給</td><td>一般的な供給</td><td></td></tr><tr><td>ミシェルは、クリップ7mmの巻き</td><td> Roboz手術InStr関数</td><td> RS - 9270 </td><td></td></tr><tr><td>耳ループマスク</td><td>フィッシャーサイエンティフィック</td><td> 19-130-4181 </td><td></td></tr><tr><td>解剖はさみ</td><td> Roboz手術InStr関数</td><td> RS - 6702 </td><td></td></tr><tr><td> Betadineソリューション</td><td> VWR </td><td> 63410-992 </td><td></td></tr><tr><td>手術用鉗子</td><td> Roboz手術InStr関数</td><td> RS - 5135 </td><td></td></tr><tr><td> 70％のイソプロピルアルコールパッド</td><td>フィッシャーサイエンティフィック</td><td> 22-031-350 </td><td></td></tr><tr><td>ブプレノルフィン</td><td>ベッドフォード研究所</td><td> 55390-100-10 </td><td></td></tr><tr><td>トラマドール</td><td>シグマアルドリッチ</td><td> 42965 </td><td></td></tr></tbody></table>

cecal ligation puncture procedure

人間の敗血症は、ローカルホストによって含まれていることに失敗した集中的かつ大規模な感染に応答して、全身反応のセットによって特徴付けられる。現在、米国集中治療室1の死亡率のトップ10に名の間で敗血症のランク。敗血症時には重複する可能性が確立された2つの血行動態のフェーズがあります。初期段階は（亢進）マクロファージと血管透過性（低血圧につながる）、心機能に影響を及ぼし、組織の壊死や臓器不全​​で最高潮に達する代謝の変化を誘発する好中球による炎症性サイトカインや活性酸素種の大規模な生産として定義されています。その結果、死亡率の最も一般的な原因は、急性腎障害である。第2フェーズが（hypodynamic）改変された単球、抗原提示を含む抗炎症プロセスであり、リンパ球の増殖および機能とアポトーシスの増加を減少させた。免疫抑制や免疫抑うつと呼ばれるこの状態は急激にnocosomial感染し、最終的に、死のリスクを増大させる。これらの病態生理学的プロセスのメカニズムは十分に特徴付けされていません。敗血症の両方のフェーズでは不可逆的と取り返しのつかない損傷を与える可能性があるので、それは患者の免疫学的および生理学的状態を判断するために不可欠です。これは、多くの治療薬が失敗した主な理由です。敗血症のさまざまな段階で与えられた同じ薬剤は、治療またはその他の有害な場合または効果2,3がないかもしれません。さまざまなレベルで敗血症を理解するには、疾患の臨床経過を再現した適切かつ包括的な動物モデルを持つことが重要です。敗血症時に発生する病態生理学的メカニズムを特徴づけるし、潜在的な治療薬をテストするためのモデルの条件を制御することが重要です。 人間の敗血症の研究者の病因を研究するために別の動物モデルを開発しました。最も広く使用されている臨床モデルでは、盲腸結紮穿刺（CLP）です。 CLPモデルでは、腹腔内に糞便材料のリリースでは、複数菌感染により誘導される悪化免疫応答を生成できるように盲腸の穿孔から構成されています。このモデルは、臨床的に関連している人間の条件を満たしています。人間のように、流体蘇生でCLPを受けるマウスは、時間に秒（後期）hypodynamic段階に進行する最初（初期）亢進相を示す。さらに、サイトカインプロフィールは、リンパ球アポトーシスを（4,5でレビューさ）が増大されている場所、人間の敗血症で見られるものと似ています。敗血症に関与し、複数のと重複するメカニズムにより、研究者が一貫して再現性のある結果を得るために制御された重篤度の適切な敗血症モデルを必要としています。

Watch this Scientific Journal Video about Cecal Ligation Puncture Procedure at JoVE.com

Cecal Ligation Puncture Procedure

人間の敗血症の研究のための貴重なツールとして、盲腸結紮穿刺のマウスモデル。

盲腸結紮穿刺手順

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the ...

Research

JoVE Journal

Medicine

54.5K ビュー.  Temple University. 人間の敗血症の研究のための貴重なツールとして、盲腸結紮穿刺のマウスモデル。

ビデオ: Cecal Ligation Puncture Procedure

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

心筋梗塞のマウスモデルにおける冠動脈結紮および心筋内注入

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

医学

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

マウスの永久中大脳動脈結紮の応用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

マウスにおける冠動脈結紮のために修飾されたテクニック

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

マウスにおける胆管結紮：閉塞性胆汁うっ滞による炎症性肝障害と線維症の誘発

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

肝のパーティションを関連付けると段階的な肝切除 (ALPPS) プロシージャの門脈結紮ラットモデル

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

虹彩ルベオーシスのマウス モデルとして虹彩新生血管を穿刺誘発

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

ラットの血液サンプル コレクションの代替方法として鎖骨下静脈穿刺

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

マウスの鎖骨下静脈穿刺による血液採取

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

左前皮下冠動脈の永久的なライゲーションを用いたマウス心筋梗塞モデル

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

ラット中の僧帽弁逆流による制御容積過負荷の画像誘導型僧帽弁リーフレット穿刺モデル

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...