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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Alle tierischen Verfahren wurden mit Zustimmung des Institutional Animal Care und Verwenden Committee der University of Cincinnati und in Übereinstimmung mit dem Leitfaden für die Pflege und Verwendung von Labortieren durch die National Institutes of Health (NIH-Veröffentlichung Nr 85-23 durchgeführt, revised 1996). 1. Vorbereitung der Maus <ol><li> Euthanize, indem das Tier in eine supra Dosis des Anästhetikums, inhaliert Isofluran wirksam. Stellen Sie sicher, primäre Euthanasie über toe Prise als schädlichen Reiz. Sekundäre Methode der Euthanasie, Membranabbaus werden in einer der nächsten Schritt erfolgen. Alternative Methoden können verwendet erteilt Euthanasie gewonnen werden. </li><li> Sanitizie die Außenfläche der Maus durch Besprühen der Pelz entlang der Bauchregion, in der die ersten Schnitte gemacht werden, mit 70% Ethanol bis zur Feuchtigkeit, so dass das Fell nass ist mit Ethanol und lose / trockene Haare nicht eingeben die Region. </li><li> Legen Sie die Maus auf einem surgische Bord in Rückenlage mit oberen und unteren Fortsätze verlängert nach außen. </li><li> Sichere Anhängsel an den Vorstand mit chirurgischen Band. </li></ol> 2. Isolierung des Herz und Aorta <ol><li> Nach der richtigen Positionierung der Maus Verwenden einer Pinzette zu lokalisieren und zu isolieren Bauchhaut nur schlechter als die Schwertfortsatz des Brustbeins. </li><li> Spannung zu erzeugen, indem Sie den Haut gerade nach oben und mit einer Schere die oberflächliche Haut zu entfernen, indem die überlegenen Teil des Bauchfells und unteren Abschnitt der Brusthöhle. </li><li> Geben Sie die Bauchhöhle durch Anheben des Schwertfortsatz und macht seitliche Einschnitte gerade schlechter als der Prozess entlang der subkostalen Margen. </li><li> Präparieren Sie bis in die Brusthöhle, gehen durch die Membran, wobei Sie nicht, um das Herz oder keine größeren Blutgefäße zerreißen. </li><li> Erweitern Sie die seitlichen Einschnitte in Schritt 2.3 kranial, um die vorderen Teil des Brustkorbs zu entfernen. Wennnotwendig, befreien das Herz aus der vorderen Brustwand mit stumpf. </li><li> Reinigen Sie die Brusthöhle von fremden Blut und Flüssigkeit mit Hilfe steriler Gaze, um Material zu absorbieren. Sobald der Bereich ist sichtbar, entfernen Sie die Lungen, das Herz und Aorta besser aus. </li></ol> 3. Perfusion von Herz und Aorta HINWEIS: Um Blut über Herzpunktion erhalten, tun Sie dies nur vor diesem Schritt. <ol><li> Füllen Sie einen 10-ml-Spritze mit 10 ml sterilem eiskaltem 1x Phosphatpufferlösung (PBS), und fügen Sie eine 25-Gauge-Nadel an der Spritze. </li><li> Die Nadel vorsichtig in der linken Herzkammer. </li><li> Schneiden Sie den rechten Vorhof, um Druckaufbau von Blutung zu lindern. Perfuse den Inhalt der Spritze in die Maus über 2-3 min. </li><li> Sterile Gaze an der Öffnung im rechten Atrium zu Perfusionsflüssigkeit absorbieren. </li></ol> 4. Isolierung und Exzision der Aorta <ol><li> Nach Beendigung des perfusion Verwenden steriler Gaze, um alle verbleibenden Flüssigkeit trübt das Sichtfeld in der Brusthöhle zu absorbieren. </li><li> Setzen die Magen-Darm-Inhalt, indem kaudal durch die Bauchwand, Verlängerung der Inzision in die suprapubischen Bereich. Ziehen Sie die Schnittführung weiter in Richtung der unteren Extremitäten auf bilateraler Ebene zu Hautlappen, die festgenagelt oder ausgeschnitten werden können. </li><li> Entfernen Sie die Lappen der Leber, Bauchspeicheldrüse, Magen, Milz und Darm, die Aorta besser zu visualisieren. Achten Sie beim Zerlegen der Nähe des perirenalen Region, wie der Aorta ist oberflächlich in den Filialen der Nierenarterien. HINWEIS: Führen Sie diesen Schritt in einer präzisen und immatrikulieren Weise wie der Gastrointestinaltrakt ist reich an Bakterien, die die Neigung zur Verschmutzung erhöht. </li><li> Spülen Sie den Bereich mit 1x PBS und entfernen Sie alle Flüssigkeit durch Absorption mit steriler Gaze. </li><li> Mit einer sterilen Mikroschere und Mikropinzetten, trennen Sie die Hauptschlagader von der Wirbelsäule nach dorsal und ventral der Speiseröhre.Am besten ist es stumpf und Verwendung eines Dissektionsmikroskop für diesen Einsatz. HINWEIS: Bei der Isolierung der Aorta, starten kaudal an der iliakalen Bifurkation und bewegen kranial Reinigung und Trennung der Aorta von der dorsalen Oberfläche des Hohlraums oder starten kranial an den Halsschlagadern und Arterien brachiocephalica Sezieren kaudal. Jede Methode geeignet ist und bleibt dem Forscher Komfort. </li><li> Entfernen Sie mit feinen Mikroschere sicher zu sein, nicht, um einen Teil der Aortenwand entfernen Sie die perivaskulären Fettgewebe. Dies ist wesentlich, um das Potential für Fibroblasten Kontamination zu minimieren, wenn die Kultivierung glatten Muskelzellen der Aorta. </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>

Die Autoren haben nichts zu offenbaren.

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.

Die Aorta spielt eine zentrale Rolle in der kardiovaskulären Funktion Versorgung des Körpers mit Sauerstoff angereicherte Blut und ähnlich wie andere Teile des Körpers, ist anfällig für Krankheiten. Gemeinsame Aortenerkrankungen zählen Atherosklerose und Aneurysma, das die Ergebnisse der genetischen und Umweltfaktoren wie Ernährung, Rauchen und Bewegungsmangel 1 sind. Atherosklerose ist die Abscheidung eines calcium- und lipidbasierte Tafel an der Aortenwand typischerweise bei Patienten mit chronischer Hyperlipidämie 2 gefunden. Aneurysmen werden durch Abbau von Strukturkomponenten der Aorta und Verdünnung der Gefäßwand, gefolgt von einem erhöhten Durchmesser auf, der letztlich dazu führen könnte zum Bruch 3 gekennzeichnet. Tiermodelle sind wichtige Werkzeuge verwendet, um den Mechanismus der Erkrankung und die Wirksamkeit von potentiellen Behandlungen zu untersuchen. Gemeinsame Tiermodellen in der kardiovaskulären Forschung, insbesondere der Forschung untersucht Gefäß- und Stoffwechselerkrankungensind gentechnisch veränderte Mäuse Fettwerte wie Apolipoprotein E Gen-Knockout und Low Density Lipoprotein-Rezeptor-Gen-Knockout-Mäusen 4 zu ändern. Die Mehrzahl von Methoden, die Mechanismen der Erkrankung und die Wirksamkeit der Therapie zu bewerten würden die Isolierung und Entfernung der Aorta umfassen. Sobald die Aorta wird lokalisiert und isoliert ist, kann die morphometrische Analyse bestimmt werden, wie Gegenwart Aneurysma, typischerweise als eine Zunahme von mehr als 50% einen Durchmesser von 5 definiert ist. Nach in situ Analyse notwendig abgeschlossen ist, können die Aorta für die weitere Analyse herausgeschnitten werden. Ein exzidierten Aorta schockgefroren werden, um molekulare Studien wie zB Protein und / oder Gen-Expressionsassays oder Verhaltensweisen befestigt unter Verwendung von 4% Paraformaldehyd und später eingebettet, geschnitten und für eine histologische Analyse gefärbt. Die histologische Analyse der Aorta können Gemeinsamkeiten der Aorten-Krankheit so wie strukturelle Verschlechterung, Plaquebildung und Infiltration von Leukozyten zeigen &lt;sup&gt; 6,7. Zusätzlich kann ein exzidierten Aorta um primäre Zelllinien, einschließlich Endothel- und glatte Muskelzellen, die anschließend für eine Vielzahl von in vitro-Studien 7 verwendet werden können, zu isolieren. Derzeit gibt es keine anderen Methoden, um dieses eingehende Charakterisierung der Aorten-Krankheit zu liefern sowie den Forschern Werkzeuge, um Herz-Kreislauf weiter zu studieren. Bildgebungsmodalitäten, wie Magnetresonanztomographie, Computertomographie und Sonographie sind die nächsten Verfahren morphologisch bewertet die Aorta, dies ist jedoch schwierig, bei kleinen Tieren und Erhalten einer Vorrichtung mit ausreichender Technologie teuer 8. Immortalisierte Zelllinien können gekauft werden, um mögliche Mechanismen der Erkrankung und der Wirksamkeit von Therapien, die künstlichen Natur dieser Zellen bei der Untersuchung der Auswirkungen auf die Zelllebenszyklus und Apoptose 9 Begrenzungs untersuchen. Das übergeordnete Zieldiese Handschrift, die sterile Isolierung und Exzision des murinen Aorta bei der Untersuchung von Herzkreislauferkrankungen zu demonstrieren.

<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.

Nach Abschluss des Verfahrens wird es eine intakte Aorta vom Herzen stammenden Abstieg in der Brust- und Bauchhöhle mit der Nierenarterien noch angebracht (Abbildung 1A) sein. Von hier aus kann die Aorta in situ abgebildet werden, um morphometrische Änderungen, die in der diagnostischen Untersuchung von Bauchaortenaneurysmen (1B und 1C) sind quantifizieren. Anschließend kann der Aorta entfernt, fixiert und angefärbt, um auf histologische Veränderungen zu suchen. Eine allgemeine und gemeinsame Fleck der Aorta ist die Hämatoxylin-Eosin-Färbung (2A und 2B). Zusätzlich kann die strukturelle Unversehrtheit des Aorta qualitativ mit Verhoff van Geison Färbung an den Elastin Bands (2C und 2D) sucht quantifizieren. Anstatt in eine histologische Analyse, Gefriert der Ermittler kann blinken die Aorta für nachfolgende Protein und Ribonukleinsäure expression. Die letzte Option für die Forscher ist es, die Hauptschlagader verwenden, um primäre Zellisolierung erhalten. Diese Zellen können in Kultur (3A) gehalten werden und für eine Vielzahl von in vitro Studien, einschließlich Durchführbarkeitsstudien (3B) und die Lokalisierung von Proteinen (3C) eingesetzt. <img alt="Figur 1" src="/files/ftp_upload/52172/52172fig1.jpg" width="700" /> Abbildung 1. In situ repräsentative Bilder von Mäuse-Aorta. (A) Darstellung der normalen Maus Anatomie nach der Isolierung der Aorta, einschließlich des Herzens und der Nieren. (B) vergrößerte Bild eines normalen intakten murinen Bauchaorta. (C) vergrßertes Bild eines erkrankten Bauchaorta mit einem Aneurysma in der Nebennieren Region. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig1highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version zu sehen th</a>ist Figur. <img alt="Abbildung 2" src="/files/ftp_upload/52172/52172fig2.jpg" width="700" /> Abbildung 2. Die histologische Analyse von Maus-Aorta. (A) Repräsentatives Bild einer normalen Hämatoxylin und Eosin-Färbung eines gesunden Nebennieren Aorta bei 20-facher Vergrößerung. (B) Vertreter Bild einer aneurysmatischen Nebennieren Aorta mit intra-Thrombus nach Hämatoxylin und Eosin-Färbung bei 20-facher Vergrößerung. (C) Repräsentatives Bild ein Verhoff van Geison Fleck, Hervorhebung der Elastin Bands eines normalen Nebennieren Aorta bei 10-facher Vergrößerung. (D) Vertreter Bild eines Verhoff van Geison Fleck Hervorhebung der Elastin Bänder eines Nebennieren Aorta entwickelt Krankheit durch die Ausdünnung und Rastern von Elastin Bands und Verdickung festgestellt der Adventitialschicht. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig2highres.jpg" target="_blank">Bitte cli</a>ck Sie hier, um eine größere Version dieser Figur zu sehen. <img alt="Figur 3" src="/files/ftp_upload/52172/52172fig3.jpg" width="700" /> Abbildung 3. Primär Aorta Muskelzellen von Aorta kultiviert. (A) Repräsentatives Bild Primäraortamuskelzellen in Kultur bei 20-facher Vergrößerung aus der abdominalen Aorta erhalten. (B) Repräsentatives Bild Primäraortamuskelzellen während einer Trypanblau-Ausschlusstest nach einer 500 uM Behandlung von H 2 O 2 bei 20-facher Vergrößerung erhalten aus der Bauchaorta. (C) Vertreter Bild eines immunhistochemischen Färbung für alpha Aktin in Primärisolat glatten Muskelzellen der Aorta aus der Bauchaorta. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig3highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</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.

Isolierung und Exzision Murine Aorta; Eine vielseitige Technik in das Studium der Herz-Kreislauf-Krankheit

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 Aufrufe.  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. 

Serie: 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.

Orthotopen Aortic Transplantation in Mäuse für das Studium der 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. ...

Forschung

Medizin

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.

Mit Hilfe der Sleeve-Technik in einem Mausmodell der Aorta Transplantation - ein Lehr-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 ...

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.

Murine Cervical Aortic Transplantation Modell mit einer modifizierten Non-Suture Manschettentechnik

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.

Modifikationen der Langendorff-Methode zur gleichzeitigen Isolierung von atrialen und ventrikulären Myozyten aus erwachsenen Mäusen

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.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

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

Biologie

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.

Die Isolierung von murinen Coronary glatten Gefäßmuskelzellen

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.

Murine Aortic Crush Injury: Eine effiziente<em&gt; In vivo</em&gt; Modell der glatten Muskelzellproliferation und Endothelfunktion

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.

Eine modifizierte Technik zur transversalen Aortenverengung bei Mäusen

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.

Isolierung und Analyse von Aortenbogen- und Wurzelläsionen in einem atherosklerotischen Mausmodell

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.

Isolierung und Exzision Murine Aorta; Eine vielseitige Technik in das Studium der Herz-Kreislauf-Krankheit

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