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
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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

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Tüm hayvan prosedürleri, Cincinnati Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi ve Ulusal Sağlık Enstitüleri Laboratuar Hayvanlarının Bakımı ve Kullanımı Kılavuzu uyarınca onay (NIH Yayın No. 85-23 ile yapıldı ) 1996 Revize. 1. Fare Hazırlanması <ol><li> Anestezik bir tedavi üstü doz hayvan teşhir ederek Euthanize, izofluran etkisi inhale. Bir zararlı uyarıcı olarak ayak tutam yoluyla birincil ötenazi doğrulayın. Ötenazi, diyafram kesme İkincil yöntem, yaklaşan adım ortaya çıkar. Alternatif yöntemler kullanılmıştır verilen ötenazi elde edilir edilebilir. </li><li> Sanitizie nem alanına için% 70 etanol ile, ilk kesim yapılacaktır karın bölgesi boyunca kürk püskürterek fare dış yüzeyi, böylece kürk, etanol ile ıslatılmış ve herhangi bir serbest / kuru tüyleri girmezler bölge. </li><li> Bir sur üzerinde fare dışarı LayÜst ve alt uzantıları ile yatar pozisyonda rahi kurulu dışa genişletilmiş. </li><li> Cerrahi bandı kullanarak kuruluna Güvenli uzantıları. </li></ol> Kalp ve Aort 2. izolasyonu <ol><li> Farenin doğru konumlandırma sonra, bulmak ve sternum kılıç şeklinde sürecine sadece aşağı karın cildi izole etmek forseps kullanabilir. </li><li> Düz yukarı kaldırarak cilt gerginliği oluşturun ve periton ve göğüs boşluğunun alt kısmının üstün kısmını açığa yüzeysel cilt kaldırmak için makas kullanın. </li><li> Kılıç şeklinde sürecini kaldırma ve subkostal kenarları boyunca sürecine sadece aşağı yan kesiler yaparak periton boşluğuna girin. </li><li> Kalp veya herhangi bir büyük kan damarları lacerate emin olmak, diyafram geçiyor, göğüs boşluğuna kadar parçalara ayır. </li><li> Göğüs kafesi ön kısmını kaldırmak için adım 2.3 cranially yapılan yanal kesiler uzatın. EğerGerekli, künt diseksiyon ile göğüs ön duvarından kalp ücretsiz. </li><li> Malzeme emmek için steril gazlı bez kullanılarak gereksiz kan ve sıvı göğüs boşluğunu temizleyin. Alan daha görünür sonra, daha iyi kalp ve aorta maruz akciğerleri kaldırmak. </li></ol> Kalp ve Aort 3. Perfüzyon NOT: Bu adım çok hemen önce yapmak, kalp delinerek kan elde etmek için. <ol><li> Steril buz soğuk 1x Fosfat tampon çözeltisi (PBS), 10 ml bir 10 cc şırınga doldurun ve şırıngaya bir 25 gauge iğne takılır. </li><li> Yavaşça kalbin sol ventrikül içine iğne takın. </li><li> Perfüzyon basınç birikimini hafifletmek için sağ atrium kesin. 3 dakika - 2 üzerinde fare içine şırınga içeriğini serpmek. </li><li> Perfüzyon sıvıyı emmek için sağ atrium açılışında steril gazlı bez kullanın. </li></ol> Aort 4. İzolasyon ve eksizyonu <ol><li> PE tamamlanması üzerinerfusion, göğüs boşluğu içinde görüş alanını karıştırdığın kalan sıvıyı emmek için steril gazlı bez kullanın. </li><li> Suprapubik bölgeye kesi uzanan, karın duvarından kaudal keserek gastrointestinal içeriğini Açığa. Bilateral aşağı tuş veya eksize edilebilir cilt flep oluşturmak için alt ekstremitelerde yönünde daha fazla kesi uzatın. </li><li> Daha iyi aorta görselleştirmek için karaciğer, pankreas, mide, dalak, bağırsaklar ve lobları çıkarın. Aort renal arterlerin şubelerinde yüzeysel olarak, perirenal bölgesi yakınında diseksiyon dikkatli olun. NOT: kesin bu adımı gerçekleştirin ve gastrointestinal sistem kirlenme eğilimi artırır bakteri ile zengin olarak şekilde kaydolur. </li><li> 1x PBS ile bölgeyi durulayın ve bir steril gazlı bez ile emilim bütün sıvıyı çıkarın. </li><li> Steril Microscissors ve microforceps kullanarak, omurga dorsal ve ventral yemek borusu gelen aort ayırın.Bu künt diseksiyon ve bunun için bir diseksiyon mikroskobu kullanarak kullanmak en iyisidir. NOT: aort izole iliyak bifürkasyonunda kaudal başlayacak ve kaudal diseksiyon karotis ve brakiyosefalik arterlerde cranially cranially temizlik ve kavite dorsal yüzeyinden aort ayıran taşımak veya başlatın. Her iki yöntem uygun ve araştırmacılar konfor kadar. </li><li> Aort duvarının bir kısmını kaldırmak için emin olmak ince microscissors kullanarak perivasküler yağ dokusu çıkarın. Bu aortik düz kas hücrelerinin kültür sırasında fibroblast ve kirlenme ihtimalini en aza indirmek için önemlidir. </li></ol>

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	<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>

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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.

Aort oksijenli kan ve vücut burada da benzer kısımlar ile vücut temin kardiyovasküler fonksiyon çok önemli bir rol oynar, hastalığa karşı hassastır. Ortak aort hastalıkları diyet, sigara ve sedanter yaşam tarzı 1 olmak üzere genetik ve çevresel faktörlerin sonuçları ateroskleroz ve anevrizma içerir. Ateroskleroz, tipik olarak kronik hiperlipidemi 2 olan hastalarda bulunan aort duvarının bir kalsiyum ve lipit bazlı plak birikmesidir. Anevrizma sonuçta 3 yırtılmasına neden olabilecek bir artış çapı ardından aorta ve damar duvarının incelme yapısal elemanların bozulma ile karakterize edilir. Hayvan modelleri hastalık ve potansiyel tedavilerin etkinliğinin mekanizmasını incelemek için kullanılan önemli araçlardır. Kardiyovasküler araştırmalarda yaygın olarak kullanılan hayvan modelleri, özellikle vasküler ve metabolik bozuklukların araştırılması aratmakBöyle Apolipoprotein E gen nakavt ve düşük yoğunluklu lipoprotein reseptör gen nakavt fareler 4 olarak lipid düzeylerini değiştirmek için genetiği değiştirilmiş fareler vardır. hastalık ve tedavinin etkinliğinin mekanizmasının değerlendirilmesi için yöntemlerin çoğunluğu, aort izole edilmesini ve çıkarılmasını içerir. Aort lokalize ve izole edildikten sonra, morfometrik analizi, anevrizma varlığında, tipik olarak çap 5 daha büyük bir% 50 artış olarak tarif edildiği gibi belirlenebilir. Yerinde analiz gerekli işlemi tamamlandıktan sonra, aort, ileri analiz için çıkartılır. Bir eksize edilen aort örneğin protein ve / veya gen sentezleme tahlilleri ya da moleküler çalışmalar yapmak için dondurulmuş flaş olabilir% 4 paraformaldehid ile sabitlenmiş ve sonra sarılır, bölümlere ayrılır ve histopatolojik analiz için boyanmıştır. Aort histolojik analiz gibi yapısal bozulma, plak oluşumu ve lökositlerin sızması gibi aort hastalığı ortak özellikler göstermektedir &lt;sup&gt; 6,7. Buna ek olarak, bir eksize edilen orta damar alındı, in vitro çalışmalar 7 çok çeşitli kullanılabilir endotel ve düz kas hücreleri dahil olmak üzere birincil hücre hatları izole etmek için kullanılabilir. Şu anda, aort hastalığı bu derinlemesine karakterizasyonu sağlamak yanı sıra kardiyovasküler hastalık çalışma araçları ile araştırmacılara sunmak için başka yöntemler vardır. Manyetik rezonans görüntüleme, bilgisayarlı tomografi ve ultrasonografi gibi görüntüleme yöntemleri ancak bu küçük hayvanlarda zor ve yeterli teknolojisine sahip bir aygıtı elde 8 pahalı, morfolojik aorta değerlendirmek için yakın yöntemlerdir. Ölümsüzleşmiş hücre hatları, ancak bu hücrelerin yapay doğa hücre yaşam döngüsü ve apoptoz 9 üzerindeki etkileri araştırmakla sınırlayıcı hastalık ve tedavilerin etkinliğinin potansiyel mekanizmaları, araştırmak için satın alınabilir. genel amacıBu yazının kardiyovasküler hastalık soruşturma kemirgen aorta steril izolasyon ve eksizyonu göstermektir.

<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.

Prosedürün tamamlanmasından sonra, hala (Şekil 1A) bağlı renal arter ile göğüs ve karın boşlukları içerisine inen kalp kaynaklı sağlam bir aort, orada olacak. Buradan, aort abdominal aort anevrizması (Şekil 1B ve 1C) çalışmalarında tanısal olan morfometrik değişiklikleri ölçmek için yerinde görüntülü olabilir. Daha sonra, aort, kaldırıldı sabit ve histolojik değişikliklere bakmak lekeli olabilir. Aort genel ve ortak leke hematoksilin ve eozin boyama (Şekil 2A ve 2B) &#39;dir. Buna ek olarak, aort yapısal bütünlüğü niteliksel elastin bant (Şekil 2C ve 2D) bakmak için Verhoff van Geison lekeleme kullanılarak ölçülebilir. Bunun yerine bir histolojik analiz elde, yanıp sönebilir araştırmacı sonraki protein ve ribonükleik asit Expre için aorta dondurmaksalgılanması. araştırmacı için son seçenek birincil hücre izolasyon elde etmek için aorta kullanmaktır. Bu hücreler, kültür (Şekil 3A) büyütüldü ve canlılık çalışmaları (Şekil 3B) ve protein lokalizasyonu (Şekil 3C) de dahil olmak üzere, in vitro çalışmalar çeşitli kullanılabilir. <img alt="Şekil 1," src="/files/ftp_upload/52172/52172fig1.jpg" width="700" /> Kemirgen aort yerinde temsilcisi görüntüleri Şekil 1.. (A) kalp ve böbrekler olmak üzere aort izolasyonu sonra normal fare anatomi Tasvir. (B) Normal sağlam kemirgen abdominal aorta görüntü Büyütülmüş. (C) suprarenal bir anevrizma ile hastalıklı karın aort görüntü Büyütülmüş bölge. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig1highres.jpg" target="_blank">th büyük halini görmek için buraya tıklayınız</a>rakam. <img alt="Şekil 2," src="/files/ftp_upload/52172/52172fig2.jpg" width="700" /> Kemirgen aort 2. histolojik analiz Şekil. 20X büyütmede (A) sağlıklı böbreküstü aort normal hematoksilen ve eozin leke Temsilcisi görüntü. (B) 20X büyütme hematoksilen ve eozin boyası sonrası intramural trombüs ile anevrizmal böbreküstü aort Temsilcisi görüntü. (C) Temsilcisi görüntü 10X büyütmede normal böbreküstü aortun elastin bantları vurgulayarak Verhoff van Geison leke. (D) incelmesi ve elastin bantları ve kalınlaşması çizgilenmeler tarafından belirtildiği bir böbreküstü aort gelişmekte hastalığın elastin bantları vurgulayarak Verhoff van Geison leke Temsilcisi görüntü adventisyal tabakanın. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig2highres.jpg" target="_blank">Lütfen cli</a>Bu rakamın büyük halini görmek için buraya ck. <img alt="Şekil 3," src="/files/ftp_upload/52172/52172fig3.jpg" width="700" /> Şekil 3. Primer aort myocytes aortadan kültür. (A), abdominal aort elde 20X büyütme kültürü birincil aort miyositlerin Örnek görüntüsü. (B) &#39;den elde 20X büyütmede H2O 2, 500 uM tedavi sonrası Trypan mavi dışlama tahlili esnasında birincil aort miyositlerin Örnek görüntü abdominal aort. (C) abdominal aorta birincil izole aort düz kas hücrelerinde alfa aktin için bir immünohistokimya leke Temsilcisi görüntü. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig3highres.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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.

İzolasyon ve Kemirgen Aort eksizyonu; Kardiyovasküler Hastalığı Çalışmasında Çok Yönlü Tekniği

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 Görüntüleme.  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. 

Video: 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.

Damar Hastalığı Çalışmaları Farelerde Ortotopik Aort Nakli

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

Tıp

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Farelerde Ateroskleroz Karşı miyointimal hiperplazide uyaran: İki Geçerli Modellerinin An Introduction

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

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.

Kol tekniği aort transplantasyonu - eğitici bir Video bir fare Model kullanma

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.

Modifiye Dikişsiz Manşet Tekniği Kullanılarak Murine Servikal Aort Transplantasyon Modeli

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.

Yetişkin Farelerden Atriyal ve Ventrikül Miyositlerinin Eşzamanlı İzolasyonu için Langendorff Yönteminin Modifikasyonları

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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

Biyoloji

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.

Murin Koroner Damar düz kas hücrelerinin izolasyonu

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

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.

Farelerde Transvers Aort Daralması için Modifiye Bir Teknik

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.

Atrosklerotik Fare Modelinde Aort Ark ve Kök Lezyonlarının İzolasyonu ve Analizi

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.