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

저자는 어떤 승인이 없습니다.

모든 동물의 절차는, 신시내티 대학의 기관 동물 관리 및 사용위원회와 국립 보건원 (National Institutes of Health)에서 실험 동물의 관리 및 사용을위한 설명서에 따라 승인 (NIH 공개 제 85-23으로 수행 하였다 ) 1996 개정. 1. 마우스를 준비 <ol><li> 마취의 supratherapeutic 복용량에 동물을 노출시켜 안락사, 이소 플루 란은 효과를 흡입. 유해 자극으로 발가락 핀치를 통해 주 안락사를 확인합니다. 안락사, 다이아 프램 절단의 보조 방법은, 곧 단계에서 발생합니다. 다른 방법도 사용할 부여 안락사 수득 될 수있다. </li><li> Sanitizie 수분의 포인트에 70 % 에탄올로, 초기 상처가 될 것이다 복부, 함께 모피를 분사하여 마우스의 외부 표면은되도록 모피 에탄올 젖은이며 느슨한 / 마른 머리카락은 입력하지 지역. </li><li> 쉬르에 마우스를 배치합니다상부 및 하부 부속와 앙와위에서 학적 보드 바깥쪽으로 확장. </li><li> 수술 테이프를 사용하여 보드에 대한 보안 부속. </li></ol> 심장 및 대동맥 2. 분리 <ol><li> 마우스를 적절하게 배치 한 후, 찾아 흉골의 칼 모양의 과정에 불과 열등한 복부 피부를 분리하기 위해 집게를 사용합니다. </li><li> 똑바로 피부를 들어 올려 긴장을 만들고 복막 및​​ 흉강의 열등한 부분의 우수한 부분을 노출, 표피를 제거하기 위해 가위를 사용합니다. </li><li> 칼 모양의 과정을 들어 올리고 subcostal 여백 따라 처리 단지 열등한 측면 절개하여 복강을 입력합니다. </li><li> 심장이나 주요 혈관을 괴롭히다하지 확인되고, 다이어프램을 통과, 흉강들로 해부하다. </li><li> 흉곽의 앞쪽 부분을 제거하는 단계 2.3 두개쪽으로에서 만든 횡 절개를 확장 할 수 있습니다. 면필요에 무딘 절개를 이용하여 전방 흉벽에서 심장 무료. </li><li> 물질을 흡수하는 멸균 거즈를 사용하여 외부 피와 유체의 흉강을 청소합니다. 지역이 더 눈에 보이는되면, 더 나은 심장과 대동맥을 노출 폐를 제거합니다. </li></ol> 심장 및 대동맥 3. 관류 참고 :이 단계 그래서 그냥 전에해야, 혈액을 심장 천자를 통해을 구하십시오. <ol><li> 살균 얼음 차가운 1X 인산 완충 용액 (PBS)의 10 ㎖로 10 CC 주사기를 가득 채운 후 주사기에 25 게이지 바늘을 연결합니다. </li><li> 조심스럽게 심장의 좌심실에 바늘을 삽입합니다. </li><li> 관류에서 압력 상승을 완화하기 우심방을 잘라. 3 분 - 2 위에 마우스로 주사기의 내용을 관류. </li><li> 관류 액을 흡수 우심방에서 개막식에서 멸균 거즈를 사용합니다. </li></ol> 대동맥 4. 분리 및 절제 <ol><li> PE 완료되면rfusion, 흉강 내 시야를 흐리게 남아있는 액체를 흡수하는 멸균 거즈를 사용합니다. </li><li> 치골 상부 지역에 절개를 확장, 복부 벽을 통해 꼬리 쪽 절단하여 위장 내용을 노출. 양자 아래로 고정 또는 절제 할 수 있습니다 피부 플랩을 만들 낮은 사지를 향해 더 절개를 확장합니다. </li><li> 더 나은 대동맥을 시각화 간, 췌장, 위, 비장, 창자의 로브를 제거합니다. 대동맥은 신장 동맥의 가지에서 피상적으로, perirenal 지역 근처 해부 할 때주의하십시오. 참고 : 정확한에서이 단계를 수행하고 위장관 오염에 대한 성향을 증가 세균이 풍부으로 방법을 진학. </li><li> 1X PBS와 지역을 씻어 멸균 거즈로 흡수에 의해 모든 유체를 제거합니다. </li><li> 멸균 microscissors과 microforceps를 사용하여 척추의 등쪽과 복부 식도에서 대동맥을 구분합니다.그것은 무딘 해부 및 이에 대한 해부 현미경을 이용를 사용하는 것이 가장 좋은 방법입니다. 참고 : 대동맥을 분리에서 장골 분기에 꼬리 쪽 시작하고 꼬리 쪽 해부 경동맥과 완두 동맥에서 두개쪽으로 두개쪽으로 청소 및 공동의 등면에서 대동맥을 분리 이동하거나 시작합니다. 어느 방법이 적절한 지와 연구자의 편안함에 달려있다. </li><li> 대동맥 벽의 일부를 제거하지 않도록 확인되는 미세 microscissors를 사용하여 혈관 주위 지방 조직을 제거합니다. 이것은 대동맥 평활근 세포를 배양 할 때 섬유 아세포 오염 가능성을 최소화하는 것이 필수적이다. </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>

저자가 공개하는 게 없다.

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.

대동맥 산소화 된 혈액과 신체의 다른 부분들과 유사한 몸체 공급 심혈관 기능의 중추적 인 역할을하는 질병에 민감하다. 일반 대동맥 질환 다이어트, 흡연, 앉아있는 생활 습관 하나를 포함하여 유전 적, 환경 적 요인의 결과이다 죽상 동맥 경화증과 동맥류를 포함한다. 죽상 경화증은 전형적 만성 고지혈증이있는 환자에서 발견 대동맥 벽 칼슘 및 지질 계 플라크의 침착이다. 궁극적 세 동맥류 파열로 이어질 수 대직 다음 대동맥과 용기 벽의 박막화의 구성 부품의 분해에 의해 특징 지어진다. 동물 모델은 질병과 잠재적 치료 효과의 메커니즘을 연구하는 데 중요한 도구입니다. 심혈관 연구에 사용 된 일반적인 동물 모델은, 구체적으로는 혈관 및 대사 장애를 조사 조회예컨대 아포지 단백질 E 유전자 녹아웃 및 저밀도 지단백질 수용체 유전자 녹아웃 마우스 (4)와 같은 지질 수준을 변경할 수있는 유전자 변형 마우스를 포함한다. 질병 및 치료의 효능을 평가하는 메커니즘 방법 중 대부분은 대동맥의 분리 및 절제를 포함한다. 대동맥 지역화 및 격리되면, 형태 계측 학적 분석은 동맥류의 존재하에, 일반적으로 직경 5 이상, 50 % 증가로 정의 된 바와 같이 결정될 수있다. 계내 분석 필요가 완료된 후, 대동맥을 적출 추가 분석 할 수있다. 절개 된 대동맥 단백질 및 / 또는 유전자 발현 분석법 또는 분자 연구를 수행 할 수있는 냉동 플래시 4 % 파라 포름 알데히드를 사용하여 고정 이상, 매립 단면 조직 학적 분석을 위해 염색. 대동맥의 조직 학적 분석은 구조 저하, 플라크 형성하고, 백혈구의 침투와 같은 대동맥 질환의 공통적 인 특징을 보여줄 수있는 &lt;SUP&gt; 6,7. 또한 대동맥 절개이어서 관내 연구 (7)의 다양한 사용될 수 내피 및 평활근 세포를 포함한 일차 세포주를 분리 할 수있다. 현재, 대동맥 질환이 심도있는 특성을 제공 할뿐만 아니라 추가로 심혈관 질환을 연구하는 도구와 연구자를 제공 할 다른 방법이 없습니다. 자기 공명 영상, 컴퓨터 단층 촬영과 같은 초음파 이미징을 그러나 이것은 작은 동물 어렵고 적절한 기술 취득 장치 8 비싸 형태학 대동맥을 평가하는 가장 가까운 방법이다. 불멸화 세포주는하지만이 세포의 인공 자연 세포 생존 구간 및 아폽토시스 9 효과를 연구에 제한되는 질병의 치료 효능의 잠재적 기전을 조사하기 위해 구입할 수있다. 의 전반적인 목표이 원고는 심혈관 질환의 조사에 쥐의 대동맥의 무균 분리 및 절단을 입증하는 것입니다.

<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.

절차가 완료되면, 여전히 (그림 1A)에 부착 된 신장 동맥 흉부 및 복강으로 내려 심장에서 발생하는 손상 대동맥이 될 것입니다. 여기에서, 대동맥이 복부 대 동맥류 (그림 1B 및 1C)의 연구에서 진단이다 형태 학적 변화를 정량화하기 위해 현장에서 이미징 할 수있다. 그 후, 대동맥, 제거 고정 및 조직 학적 변화를보고 염색 할 수있다. 대동맥 일반적인 공통 얼룩이 마톡 실린 및 에오신 염색 (도 2a 및도 2b)이다. 또한, 대동맥의 구조적 무결성 질적 엘라스틴 밴드 (그림 2C 및 2D)를보고 Verhoff 반 Geison 염색을 사용하여 정량화 할 수있다. 대신 조직 학적 분석을 얻기의, 깜박일 수 있습니다 연구자 후속 단백질과 리보 핵산의 expre의 대동맥을 동결ssion. 조사자에 대한 마지막 옵션은 일차 전지 격리를 획득하기 위해 대동맥을 사용하는 것이다. 이러한 세포 배양 (도 3a)에서 성장 및 생존 연구 (도 3B) 및 단백질 현지화 (도 3C)를 포함한 체외 다양한 연구에 사용될 수있다. <img alt="그림 1" src="/files/ftp_upload/52172/52172fig1.jpg" width="700" /> 쥐의 대동맥의 현장 대표 이미지에서 그림 1. (A) 심장과 신장을 포함하여 대동맥의 분리 후 정상 마우스 해부학의 묘사를. (B) 일반 그대로 쥐의 복부 대동맥의 이미지를 확대했습니다. (C) 부신에서 동맥류와 병에 걸린 복부 대동맥의 이미지 확대 지역. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig1highres.jpg" target="_blank">일의 더 큰 버전을 보려면 여기를 클릭하십시오</a>그림입니다. <img alt="그림 2" src="/files/ftp_upload/52172/52172fig2.jpg" width="700" /> 쥐의 대동맥 2. 조직 학적 분석을 그림. 20 배 배율 (A) 건강 부신 대동맥의 정상 헤 마톡 실린 및 에오신 얼룩의 대표 이미지입니다. (B) 20 배 배율에서 헤 마톡 실린 및 에오신 염색 후 교내 혈전 동맥류 부신 대동맥의 대표 이미지입니다. (C)의 대표 이미지 10 배 배율에서 정상 부신 대동맥의 탄력 밴드를 강조 Verhoff 반 Geison 얼룩. (D) 선화과 엘라스틴 밴드와 농축의 striating에 의해 언급 부신 대동맥 개발 질병의 탄력 밴드를 강조 Verhoff 반 Geison 얼룩의 대표 이미지 외막 층의. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig2highres.jpg" target="_blank">제발 CLI</a>이 그림의 더 큰 버전을 보려면 여기를 CK. <img alt="그림 3" src="/files/ftp_upload/52172/52172fig3.jpg" width="700" /> 그림 3. 주요 대동맥 근육 세포는 대동맥에서 배양. (A) 복부 대동맥으로부터 수득 20X 배율에서 배양 주 대동맥 근세포의 대표 화상. (B)로부터 얻어지는 20X 배율로 H 2 O (2)의 500 μM을 처리 한 후 트립 판 블루 배제 분석법 중 주 대동맥 근세포의 대표 화상 복부 대동맥. (C) 복부 대동맥에서 차 분리 대동맥 평활근 세포에서 알파 액틴에 대한 면역 조직 화학 염색의 대표 이미지. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig3highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

분리 및 설치류 대동맥 절제; 심혈관 질환의 연구에 다목적 기술

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

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JoVE Journal

Medicine

32.3K 조회수.  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. 

비디오: 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.

혈관 질병의 연구에 대한 마우스의 Orthotopic 대동맥 이식

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

연구

의학

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.

대동맥 이식-교육용 비디오의 마우스 모델에서 소매 기법을 사용 하 여

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.

수정된 비봉합사 커프 기술을 사용한 뮤린 자궁 경부 대동맥 이식 모델

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.

성인 마우스에서 심방 및 심실 근세포의 동시 격리를위한 랑엔도르프 방법의 수정

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.

분리 및 마우스 대동맥 내피 세포의 차 문화

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

생물학

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.

쥐 관상 동맥 혈관 평활근 세포의 분리

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.

소뇌 대동맥 파열 상해 : 효율적<em&gt; In Vivo</em&gt; 평활근 세포 증식과 내피 기능 모델

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.

생쥐의 횡방향 대동맥 수축에 대한 수정된 기술

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.

Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model(죽상경화성 마우스 모델에서 대동맥궁 및 뿌리 병변의 분리 및 분석)

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.

분리 및 설치류 대동맥 절제; 심혈관 질환의 연구에 다목적 기술

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