Name: isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease
Uploaded: 2014-11-24T14:00:00.000+00:00
Duration: 7 min 25 s
Description: Watch this Scientific Journal Video about Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease at JoVE.com

著者は全く確認応答がありません。

全ての動物手順は、シンシナティ大学の制度的動物実験委員会の承認を得て、および国立衛生研究所（NIH公開番号85から23から実験動物の管理と使用に関する指針に従って行った）1996年改訂。 1.マウスの準備<ol><li>麻酔薬のsupratherapeutic線量に動物を暴露することにより安楽死させる、イソフルランは効果に吸入さ。侵害刺激としてつま先のピンチを経由して第一の安楽死を確認してください。安楽死、ダイヤフラム切断の二の方法は、今後のステップで発生します。代替方法が安楽死が得られる付与に使用することができる。 </li><li> Sanitizie水分のポイントに70％エタノールで、最初のカットが行われる腹部に沿って毛皮を噴霧することにより、マウスの外面は、そのように毛皮は、エタノールで濡れていると緩ん/ドライ毛は入力しないでください地域。 </li><li>シュール上でマウスをレイアウト上下の付属と仰臥位での外科ボードは、外側に延長した。 </li><li>サージカルテープを使用してボードへの安全な付属。 </li></ol>ハートと大動脈の2の分離<ol><li>マウスの適切な位置決めした後、検索し、胸骨の剣状突起のすぐ下腹部の皮膚を隔離するために鉗子を使用しています。 </li><li>まっすぐに肌を持ち上げて緊張を作成し、腹膜や胸腔の劣る部分の優れた部分を露出させ、表面的な皮膚を除去するためにハサミを使用しています。 </li><li>剣状突起を持ち上げると肋骨下の余白に沿ってプロセスのすぐ下の横切開を行うことで、腹腔を入力します。 </li><li>心臓や大きな血管をlacerateしないように注意してくださいという、横隔膜を経由、胸腔内にアップ解剖。 </li><li>胸郭の前方部分を除去するためのステップ2.3頭側で行われた横方向の切開を拡張します。もし必要に応じて、鈍的切開を使用して前胸壁から心臓を解放。 </li><li>材料を吸収するために滅菌ガーゼを使用して余分な血液や体液の胸腔を清掃してください。エリアをより見やすくしたら、より良い心と大動脈を露出させ、肺を削除します。 </li></ol>ハートと大動脈の3灌流注：このステップにとても直前に行い、心穿刺によって血液を入手します。 <ol><li>無菌の氷冷1×リン酸緩衝溶液（PBS）10mlで10 1ccシリンジに充填し、シリンジに25ゲージの針を取り付ける。 </li><li>静かに心臓の左心室に針を挿入します。 </li><li>灌流からの圧力の蓄積を軽減するために右心房をカット。 3分 - 2の上にマウスに注射器の内容物を灌流。 </li><li>灌流液を吸収するために右心房の開口部で滅菌ガーゼを使用してください。 </li></ol>大動脈の4.絶縁と切除<ol><li>ペーが完了するとrfusion、胸腔内の視野を曇り、残りの流体を吸収するために滅菌ガーゼを使用しています。 </li><li>恥骨の領域に切開を延長、腹壁を通して尾側に切断することによって胃腸の内容を公開します。二国間釘付けまたは削除することができ、皮膚のフラップを作成するために下肢に向けてさらなる切開を拡張します。 </li><li>より良い大動脈を可視化するために、肝臓、膵臓、胃、脾臓、および腸のローブを削除します。大動脈は、腎動脈の枝で表面的であるように、腎周囲領域の近く解剖ときは注意してください。 注：正確な中で、このステップを実行し、消化管の汚染の傾向を増加させ、細菌に富んでいるような方法を入学。 </li><li> 1×PBSで領域をすすぎ、滅菌ガーゼで吸収して、すべての流体を除去。 </li><li>無菌microscissorsとマイクロピンセットを使用して、背骨の背側と腹側に食道から大動脈を分離する。それは、鈍的切開し、このための解剖顕微鏡を利用した使用するのが最適です。 注：大動脈を単離する際に、腸骨分岐部に尾開始し、尾側に解剖頸動脈と腕頭動脈の頭側頭側の洗浄とキャビティの背側表面から大動脈を分離移動したり、開始します。どちらの方法が適切であると研究者の快適性に任されています。 </li><li>大動脈壁の一部を除去しないように注意してくださいという細かいmicroscissorsを使用して血管周囲の脂肪組織を削除します。これは、大動脈平滑筋細胞を培養する際に、線維芽細胞の混入の可能性を最小限に抑えることが不可欠である。 </li></ol>

<ol>
	<li>Go, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129 (3), 28-292 (2014).</li><li>Ross, R., Harker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperlipidemia+and+atherosclerosis.">Hyperlipidemia and atherosclerosis.</a> Science. 193 (4258), 1094-1100 (1976).</li><li>Clouse, W. D., 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=Acute+aortic+dissection:+population-based+incidence+compared+with+degenerative+aortic+aneurysm+rupture.">Acute aortic dissection: population-based incidence compared with degenerative aortic aneurysm rupture.</a> Mayo Clin Proc. 79 (2), 176-180 (2004).</li><li>Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc Biol. 24 (3), 429-434 (2004).</li><li>Pleumeekers, H. 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=Aneurysms+of+the+abdominal+aorta+in+older+adults.+The+Rotterdam+Study.">Aneurysms of the abdominal aorta in older adults. The Rotterdam Study.</a> Am J Epidemiol. 142 (12), 1291-1299 (1995).</li><li>Brophy, C. M., Reilly, J. M., Smit, G. J., Tilson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+inflammation+in+nonspecific+abdominal+aortic+aneurysm+disease.">The role of inflammation in nonspecific abdominal aortic aneurysm disease.</a> Ann Vasc Surg. 5 (3), 229-233 (1991).</li><li>Ray, J. L., Leach, R., Herbert, J. M., Benson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+vascular+smooth+muscle+cells+from+a+single+murine+aorta.">Isolation of vascular smooth muscle cells from a single murine aorta.</a> Methods Cell Sci. 23 (4), 185-188 (2001).</li><li>Weiss, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+murine+cardiovascular+system+with+magnetic+resonance.">Imaging the murine cardiovascular system with magnetic resonance.</a> Circ Res. 88 (6), 550-551 (2001).</li><li>Graham, E. L., Balla, C., Franchino, H., Melman, Y., del Monte, F., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+culture,+and+functional+characterization+of+adult+mouse+cardiomyoctyes.">Isolation culture, and functional characterization of adult mouse cardiomyoctyes.</a> J Vis Exp. (79), 50289 (2013).</li><li>Wassef, M., Upchurch, G. R., Kuivaniemi, H., Thompson, R. W., Tilson, M. D. 3. r. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+in+abdominal+aortic+aneurysm+research.">Challenges and opportunities in abdominal aortic aneurysm research.</a> J Vasc Surg. 45 (1), 192-198 (2007).</li></ol>

著者らは、開示することは何もない。

Aortic disease can lead to significant systemic pathology and possibly death in severe cases. Treatment options are currently limited therefore continued research in the field is necessary10. Murine models are ideal to further study disease progression and potentially therapeutic options4. Therefore, the successful completion of the method described in this manuscript will provide investigators with the tools to measure disease progression and drug efficacy. 
This technique can be modified in several ways in order to optimize the results. The use of surgical loupes or a dissecting microscope can aid in the visualization of the murine anatomy and vasculature details and when removing surrounding adipose tissue from the aorta. Additionally, the use of dedicated surgical utensils for specific tasks can increase the longevity of tools; for example, the use of scissors for cutting skin and separate scissors to cut through the ribs would be ideal. The use of a small gauged needle when perfusing the heart is preferred as this will allow the tissue to close around the puncture as opposed to a larger gauge needle where a hole will remain. 
After the aorta is successfully removed, it can be utilized for a variety of assays. The aorta can be used for molecular studies such as enzyme activity, protein expression and gene expression profiles. Additionally, the aorta can be fixed for histologic analysis where in situ assays can be utilized as well as characterization of the aortic morphology. Finally, one can isolate smooth muscle and/or endothelial cells from a freshly harvest aorta for in vitro studies with these primary isolated cells. 
The major limitation to isolation and excision of the aorta is that the animal must be sacrificed, especially in the cases of longitudinal studies. Currently, there is no single technique comparable to isolation and excision of the murine aorta. Small animal imaging technologies is becoming more advanced and approaching characterization capabilities of direct observation however it continues to have limitations including cost8. Immortalized cells lines can be used for in vitro studies however immortal cells are not as useful as primary isolated cells for in vitro assays especially for viability studies9. Isolation and excision of the murine aorta is currently the most versatile technique that can be used in the study of aortic disease.

大動脈は、酸素化された血液及び身体の他の同様の部分とボディを供給する血管機能において重要な役割を果たしている疾患に感受性である。一般的な大動脈疾患は、食事、喫煙と座りがちな生活1を含む、遺伝的要因と環境要因の結果であるアテローム性動脈硬化症や動脈瘤が含まれる。アテローム性動脈硬化症は、一般的に、慢性高脂血症2の患者で見つかった大動脈壁にカルシウムおよび脂質ベースのプラークの沈着である。動脈瘤は、最終的に3を破裂させる可能性があり拡径続い大動脈血管壁の菲薄化の構造成分の分解によって特徴付けられる。 動物モデルは、病気や潜在的な治療法の有効性のメカニズムを研究するために使用される重要なツールである。心血管の研究に使用される一般的な動物モデルは、特に、血管および代謝障害を調査研究そのようなアポリポタンパク質E遺伝子ノックアウトおよび低密度リポタンパク質受容体遺伝子ノックアウトマウス4などの脂質レベルを変化させる遺伝的に改変されたマウスを含む。疾患及び治療の有効性の機構を評価するための方法の大部分は大動脈の単離および切除を含むであろう。 大動脈は、ローカライズされ単離されると、形態学的分析は、このような動脈瘤の存在、典型的には、直径5で50％以上の増加として定義されるように、決定することができる。 in situ分析必要が完了したら、大動脈を、さらなる分析のために切り出すことができる。切除された大動脈は、タンパク質および/または遺伝子発現アッセイまたは分子的研究を行うために急速冷凍することができ、4％パラホルムアルデヒドを用いて固定し、後で、包埋し、組織学的分析のために染色した。大動脈の組織学的分析は、そのような構造劣化、プラーク形成、および白血球の浸潤として大動脈疾患の共通の特徴を示すことができる&lt;SUP&gt; 6,7。さらに、切除された大動脈は、その後in vitro試験7のさまざまな目的で使用することができ、内皮細胞および平滑筋細胞を含む初代細胞株を単離することができる。 現在、大動脈疾患のこの深いキャラクタリゼーションを提供するだけでなく、さらなる心臓血管疾患を研究するためのツールを研究者に提供するための他の方法はない。例えば、磁気共鳴画像のような画像診断法は、コンピュータ断層撮影および超音波検査は、形態学的に大動脈を評価するための最も近い方法であるが、これは、小動物では困難であり、適切な技術と装置を得ることが8に高価である。不死化細胞株は、しかしながら、これらの細胞の人工的な性質は、細胞のライフサイクルおよびアポトーシス9への影響を研究に制限されている疾患および治療 ​​の有効性の潜在的なメカニズムを調査するために購入することができる。 の全体的な目標この原稿は、心血管疾患の調査にマウス大動脈の無菌分離および切除を実証することである。

<table><tbody><tr><td>Isoflurane</td><td>Med-Vet International</td><td>#RXISO-250</td><td></td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>07-678-001</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>Sterile gauze</td><td>Dukal Corporations</td><td>1312</td><td></td></tr><tr><td>25 gauge needle</td><td>BD</td><td>305122</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td></td></tr></tbody></table>

isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

手続きが完了すると、まだ添付腎動脈と胸部と腹部の空洞（ 図1A）に下降、心から発信無傷で大動脈があるでしょう。ここからは、大動脈は、腹部大動脈瘤（ 図1Bおよび1C）の研究での診断である形態学的変化を定量化するために、その場で画像化することができる。その後、大動脈を取り出し、固定し、組織学的変化を見るために染色することができる。大動脈の一般的な共通染色はヘマトキシリンおよびエオシン染色（ 図2Aおよび2B）である。加えて、大動脈の構造的完全性を定性的エラスチンバンド（ 図2C及び2D）を見てVerhoffバンGeison染色を用いて定量することができる。代わりに、組織学的分析を得るための、点滅することが研究者は、その後のタンパク質とリボ核酸expreための大動脈を凍結するssion。研究者のための最後のオプションは、初代細胞分離を得るために、大動脈を使用することである。これらの細胞は、培養物（ 図3A）中で増殖および生存研究（ 図3B）、およびタンパク質局在化（ 図3C）を含む、 インビトロでの様々な研究のために使用することができる。 <img alt="図1" src="/files/ftp_upload/52172/52172fig1.jpg" width="700" /> マウス大動脈のその場の代表的な画像で、図1。副腎における動脈瘤と病気の腹部大動脈の（A）心臓や腎臓などの大動脈の単離後の正常マウス解剖の描写。通常の無傷のマウス腹部大動脈の（B）拡大画像。（C）拡大画像地域。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig1highres.jpg" target="_blank">番目の拡大版を表示するには、こちらをクリックしてください</a>図である。 <img alt="図2" src="/files/ftp_upload/52172/52172fig2.jpg" width="700" /> マウス大動脈の2組織学的分析図。 20X倍率で（A）健康副腎大動脈の通常のヘマトキシリン及びエオシン汚れの代表画像。（B）20X倍率でヘマトキシリン及びエオシン染色後に学内血栓と動脈瘤副腎大動脈の代表画像。（C）の代表的な画像VerhoffバンGeisonステイン、10倍の倍率で正常な副腎大動脈のエラスチンのバンドを強調している。（D）エラスチンバンドや肥厚の間伐とstriatingによって注意副腎大動脈開発疾患のエラスチンバンドを強調VerhoffバンGeison染色の代表的な画像外膜層の。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig2highres.jpg" target="_blank">してくださいCLI</a>この図の拡大版をご覧になるにはこちらをCK。 <img alt="図3" src="/files/ftp_upload/52172/52172fig3.jpg" width="700" /> 図3.プライマリ大動脈筋細胞は、大動脈から培養。 （A）腹部大動脈から得た20倍の倍率で、培養中の主要動脈の筋細胞の代表的画像。（B）から得られた20倍の倍率でのH 2 O 2の500μM処理後、トリパンブルー排除アッセイ中の主要動脈の筋細胞の代表的な画像腹部大動脈。（C）腹部大動脈からの一次分離株大動脈平滑筋細胞におけるアルファアクチンのための免疫組織化学染色の代表的なイメージ。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig3highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease at JoVE.com

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

単離とマウス大動脈の切除。心血管疾患の研究で多彩なテクニック

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

isolation-excision-murine-aorta-versatile-technique-study

Universidad San Sebastian

Research

JoVE Journal

Medicine

32.3K ビュー.  University of Cincinnati College of Medicine. Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease. 

ビデオ: Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

血管疾患の研究のためのマウスにおける同所性大動脈移植

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

医学

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

大動脈弁移植 - 教則ビデオのマウスモデルでスリーブを用いた

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

改変非縫合カフ技術を用いたマウス子宮頸動脈大動脈移植モデル

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

成体マウスからの心房と心室筋細胞の同時分離のためのランゲンドルフ法の改変

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

生物学

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

ネズミ大動脈挫傷：効率的<em&gt;インビボ</em&gt;平滑筋細胞の増殖と内皮機​​能のモデル

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

マウスにおける横行大動脈狭窄の修正技術

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study introduces a comprehensive protocol for the isolation and histological analysis of aortic arch and root lesions in a widely used atherosclerotic mouse model, low-density lipoprotein receptor knock-out (Ldlr-/-) mice. The aortic arch and root are key sites for atherosclerotic lesions, and their examination is critical for assessing the onset, progression, or regression of atherosclerosis, predicting cardiovascular event risks, and identifying potential therapeutic targets. This protocol outlines methods for quantifying atherosclerotic burden in the aortic arch and root, including tissue isolation, fixation, Oil Red O staining, aortic root sectioning, Hematoxylin and Eosin (HE) staining, Verhoeff-Van Gieson (VVG) staining, and image analysis. Oil Red O staining measures plaque area in the aortic arch, evaluating the severity of atherosclerosis, while HE staining of the aortic root reveals plaque components such as the lipid core and fibrous cap, facilitating the assessment of plaque stability and rupture risk. VVG staining can stain collagen fibers within tissues, providing further insights into plaque composition and related information. This thorough analysis offers valuable insights into the mechanisms of lesion development and may guide the creation of novel therapeutic strategies for preventing and treating atherosclerosis.

This protocol provides a comprehensive methodology for the quantitative analysis of atherosclerotic plaques to facilitate more in-depth research into the mechanisms and progression of atherosclerosis.

アテローム性動脈硬化症マウスモデルにおける大動脈弓および大動脈基部病変の単離と解析

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study ...

Surgical Aortic Debanding: A Procedure to Study Left Ventricular Reverse Remodeling in Murine Model of Aortic Constriction

Source: Goncalves-Rodrigues, P. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/60036" target="_blank">Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents</a>. J. Vis. Exp. (2021)
This video demonstrates the technique of aortic debanding, which can lead to reverse remodeling to limit the progression towards heart failure in a murine model. Post debanding, a significant reduction of ventricular afterload triggers regression of ventricular hypertrophy and fibrosis, causing a recovery in cardiac function.

単離とマウス大動脈の切除。心血管疾患の研究で多彩なテクニック

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