Name: murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch
Uploaded: 2015-02-10T14:00:00.000+00:00
Duration: 7 min 5 s
Description: Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director&#8217;s New Innovator Grant (1DP2 OD008716-01). The authors would like to thank the services provided by the ICMB (Institute of Cellular and Molecular Biology) core facility and TherapeUTex at University of Texas at Austin.

참고 :이 프로토콜에 표시된 모든 방법은 기관 동물 케어 및 사용위원회에 의해 승인되었습니다. 외과 표 1. 준비 <ol><li> 해부 현미경, 수술 테이블에 따뜻한 물을 재순환과 가열 패드를 설정합니다. 가열 패드 상 스테인리스 기판을 놓는다. 베이스 플레이트와 가열 패드에 멸균 흡수 패드를 놓습니다. </li><li> 인접한 멸균 흡수 패드에 멸균 된 수술 도구를 정렬합니다. 각진 끝이 뾰족한 집게 두 쌍의 각도 포셉 두 쌍의 세 지혈 겸자, 두 견인기 와이어, 작은 수술 가위, 마이크로 가위, 한 혈관 성형술 와이어 (0.15 인치 직경) 6.0 실크 봉합사의 세 6 인치 세그먼트를 수집 . </li><li> 대퇴 동맥의 곡률과 일치하도록 혈관 성형술 와이어의 원형 단부를 구부린다. 이렇게하면, 수술 중에 와이어를 전진하게된다. </li></ol> 2. 준비외과 마우스 <ol><li> 2.5 % 이소 플루 란 흡입에 의해 연속적으로 마우스를 마취. 절차를 통해 동물의 상태를 모니터링해야합니다. 완전히 마취하는지 확인하려면 마우스의 발 핀치 테스트를 수행합니다. 핀치 테스트 투여 할 때 움직이지 않는 동물을 확인합니다. </li><li> 탈수를 방지하기 위해 동물의 눈 연고 윤활 적용. 수술 테이프를 사용하여 흡수 패드에 앙와위에서 동물을 고정합니다. 코 콘을 통해 2.5 %의 이소 플루 란을 투여 계속합니다. </li><li> 제모 크림을 사용하여, 중간 선에 다리와 복부에서 모피를​​ 제거합니다. 물로 피부를 씻어. 탈모는 수술에 앞서 일일 수행 될 수있다. 피부 자극이 발생할 수 있습니다 크림과 함께 과도한 치료를합니다. </li><li> 즉시 수술 전에 피부를 소독하기 위해 면봉으로 면도 한 지역에 포비돈 - 요오드를 적용합니다. 70 % 에탄올 건조와 면도 한 피부를 씻어멸균 면봉으로. 3 번 반복합니다. </li><li> 피하 주사를 통해 카프로 펜의 5 ㎎ / ㎏의 수술 전 투여 량을 관리합니다. </li></ol> 대퇴 동맥의 3. 분리 <ol><li> 작은 수술 가위를 사용하여, 대퇴 동맥 위에 피부 절개를 만곡. 퉁명스럽게 대퇴 동맥을 찾기 위해 견인기 자기 트랙터 fixators을 사용하여 주변 조직을 해부 고정합니다. 멸균 면봉을 사용하여 식염수를 적용 관개를 위해 주기적으로 생리 식염수를 사용하여 조직을 적 십니다. </li><li> 집게를 사용하여 대퇴 동맥을 분리합니다. 부드럽게 끝이 뾰족한 집게를 사용하여 혈관 번들에서 신경을 분리합니다. 정맥을 천공하지 말고, 신경에 손상을주지 않습니다. 그것을 자극 피하려면 번들에서 신경을 밀어 넣습니다. </li><li> 조심스럽게 대퇴 분기의 위치, 대퇴 동맥에서 대퇴 정맥을 분리합니다. 분기의 지역은 해부 특히 어렵다. </li><li> 피osterior 분기에, 루프 지혈과 대퇴 동맥과 보안에서 6.0 실크 봉합사. 이 근위 봉합사 동맥 혈류를 제한하는 데 사용된다. 주 : 소작 방법 대 결찰 방법을 수행 할 때 관계에 다소 차이가있다 (도 1 및도 2 참조). </li><li> 분기에 원위부, 루프 6.0 실크 대퇴 동맥에서 봉합 및 지혈 고정합니다. 동맥의 위치에있는이 말단 봉합 보조. </li><li> 대퇴 동맥의 근육 분기 아래 루프 두 봉합, 그것들을 미리 묶어 지혈 고정합니다. 식염수로 조직을 습하게해야합니다. 와이어 부상 소작 방법을 수행하면, 하나의 루프 형 봉합사 근육 지점에 필요하다. </li></ol> 대퇴 동맥 와이어 부상 4. 성능 <ol><li> 근위 봉합사 당기는 대퇴 동맥에 혈류를 제한한다. 약간 먼 hemost 당겨지혈이 지점을 확보에서와 arteriotomy의 사이트를 노출합니다. 주위 봉합을 매서 근육 분기를 결찰. 주 : 상향 동맥 리프팅보다 효과적으로 가로 당김 형보다 혈류를 제한 할 것이다. </li><li> 두 봉합 사이의 소작으로 작은 지점을 끊다. 마이크로 가위를 사용하여 분기의 측면 지점에서 arteriotomy을 수행합니다. 마이크로 가위를 안정화 외과 테이프의 롤에 균형 집게를 사용합니다. </li><li> 끝이 뾰족한 집게를 사용 arteriotomy의 존재를 확인합니다. 부드럽게 집게로 arteriotomy의 오프닝을 들어 올립니다. 집게를 사용 arteriotomy에 와이어의 둥근 끝을 소개합니다. 와이어의 발전을 용이하게하기 위해, 주사기를 사용하여 영역 리도카인 한두 방울을 가하여. </li><li> 와이어 근위 봉합사 도달하면 봉합사를 해제하고 와이어의 전진을 방해 할 수 없도록하여 조정한다. 이 더 진행되지 때까지 와이어를 삽입합니다. 번째의 선단전자 와이어 사타구니 인대의 영역에서 중지해야합니다. </li><li> 와이어가 1 분 동안 대퇴 동맥에 남아 허용합니다. 손상과 대퇴 동맥의 내피 세포를 발가 벗기다 데 10 시간을 1 분 후에, 철회 및 절단 운동에 와이어를 진행합니다. 덜 심각한 상해, 와이어 및 동맥 뽑아 횟수를 줄이거 나 단 1 분간 동맥 선을 떠난다. </li><li> 천천히 와이어를 집어 넣으십시오. 와이어의 단부는 둥근 근위 봉합사를 경과하면, 근위 봉합사 당기는 동맥 내로 유동을 제한한다. 완전히 선을 후퇴. </li></ol> 5. 근육 지점을 결찰 <ol><li> 근육 분기에 남아있는 봉합사를 조입니다. 이 arteriotomy에서 출혈을 방지 할 수 있습니다. 근육 분기에 흐름을 반환하고, 그 혈액이 누출되지 확인합니다. 근육 분기에 봉합에서 끝을 낸다. </li></ol> 6. 다른 방법 : 현지화 된 카우Arteriotomy의 terization 주 : 대안은 근육 분기의 결찰을 피하고 큰 메인 대퇴 동맥 혈관을 통해 액세스 할 수 있도록 수행 될 수있다. <ol><li> 4.1에서 시작, 근위 봉합 당겨 대퇴 동맥에 흐름을 제한 할 수 있습니다. 말단 봉합하고 arteriotomy의 사이트 (그림 1과 그림 2) 노출 약간 근육 분기를 고정 봉합을 당깁니다. </li><li> 마이크로 가위를 사용하여, 대퇴 동맥과 근육 지점의 분기점에서 arteriotomy를 수행합니다. 절개 동맥 측 이루어진 경우, 소작하기 쉬울 수있다. 마이크로 가위를 안정화 외과 테이프의 롤에 균형 집게를 사용합니다. </li><li> 소작의 성공을 테스트하려면, 근위 봉합을 풀어 대퇴 동맥에 흐름을 복원합니다. 출혈 arteriotomy에서 발생하면, 소작을 반복한다. 소작이 성공하면, 혈류를 다시한다arteriotomy 원위부 저장됩니다. </li><li> 와이어를 소개하고 4 절에 설명 된대로 와이어 부상을 수행합니다. </li><li> 대퇴 동맥으로 혈액의 흐름을 제한 계속합니다. 적어도 6인치 마우스에서 멀리 뾰족한 소작을 가열. 소작 팁이 냉각으로, 절개를 닫습니다 arteriotomy의 다른 쪽면을 가볍게 적용됩니다. </li><li> 소작의 성공을 테스트하려면, 근위 봉합을 풀어 대퇴 동맥에 흐름을 복원합니다. 출혈 arteriotomy에서 발생하면, 소작을 반복한다. 소작이 성공하면, 혈류 arteriotomy 원위부 복원한다. 임시 근위 및 원위 관계를 제거합니다. </li></ol> 혈관 주위 약물 전달 패치 7. 주입 <ol><li> 이전의 연구 (11)에서 또는 이와 유사한 방법을 사용하여 기술 된 바와 같이 혈관 주위 약물 패치를 생성하는 생성 될 수있다. 멸균 가위를 사용하여 약물 전달 패치의 배리어 영역의 모서리를 무디게 </li><li> 약물 전달 팻 배치동맥 대향 약물 방출면이 부상 대퇴 동맥 상 CH. 필요에 따라, 패치의 위치를​​ 개선하는 집게를 사용한다. </li></ol> 8. 상처 폐쇄 및 복구 <ol><li> 광장 노트로 구성된 간단한 중단 봉합 상처를 닫습니다. 불연속 봉합사 동물 봉합사를 제거하려고 할 경우에 상처 봉합을 연장. </li><li> 마취를 끄고 설정부터 동물을 제거합니다. 동물이 온난화 패드에 복구 할 수 있습니다. </li><li> 마우스의 회복을 모니터링하기 위해 계속합니다. 이 닫혀 있는지 확인하기 위해 매일 절개 사이트를 확인하십시오. 수술 후 통증의 치료를 위해 2 일 후 매 12 시간을 수술 후 5 ㎎ / 카프로 펜마다 12 시간의 kg의 피하 주사를 관리합니다. 통증이 초기 이일 이상 계속되면, 더 진통제에 대한 방향에 대한 수의사에게 문의하십시오. </li></ol> 조직학 9. 수확 대퇴 동맥 <ol><li> 수술 후 이십팔일에서 마우스 이산화탄소 안락사를 수행합니다. 주 : 이산화탄소의 적절한 유량이 분당 챔버 10-30 %로 치환해야하며, 사용 된 챔버의 크기에 따라 달라진다. 다이어프램과 심장 천자의 절단은 안락사의 보조 방법으로 수행해야합니다. </li><li> 수술 테이프를 사용 누운 위치에 마우스를 고정합니다. 초기 수술 절개가되었다 대퇴 동맥을 통해 절개를합니다. </li><li> 손상된 동맥과 반대쪽 다리에서 손상되지 않은 동맥, 모두 퉁명스럽게 해부 대퇴 동맥을 찾기 위해 견인기 및 자기 견인기 fixators을 사용하여 주변 조직에 고정합니다. 주기적으로 관개 식염수를 사용하여 조직을 적 십니다. 멸균 면봉을 사용하여 식염수를 적용합니다. 동맥을 손상하지 않도록주의하십시오. </li><li> 대퇴 동맥 복부 대동맥에 arteriotomy 원래의 위치로부터 분리 된 후, SEG 넥타이arteriotomy의 원래 사이트 근처 실크 봉합사, 표준. 이 봉합사 대퇴 동맥의 가장 말단부를 확인하고 샘플의 처리를 완화 시키는데 도움이된다. </li><li> 마이크로 해부 가위를 사용하면 대퇴 동맥을 절제합니다. 봉합 한 절개는 원위부합니다. 복부 대동맥 옆에, 대퇴 동맥의 반대쪽에 다른 절개를합니다. </li><li> 식염수가 들어있는 유리 페트리 접시에 절제 동맥을 전송합니다. 초과 결합 조직이나 지방을 제거하기 위해 추가로 동맥을 해부하다. 부드럽게 식염수로 세척하여 루멘에서 혈액을 제거합니다. </li><li> 10 % 포르말린의 유리 병에 동맥을 전송합니다. 부드러운 48 시간 동안 락을 함께 4 ° C에서 유리 병을 저장합니다. </li><li> 가 조직학을 위해 처리 될 때까지 저장하기 위해 70 % 에탄올에 고정 동맥 전송. </li><li> 파라핀 블록과 부분 염색에 대한 블록 동맥을 포함합니다. </li><li> 부상과 내막 우울의 정도를 평가하기 위해 조직 화학 및 면역 화학 염색을 수행erplasia. 참고 : 대표 결과를 위해, 우리는 탄성 얇은 판과 다른 동맥의 구성 요소를 시각화하기 위해 핵 및 전반적인 형태 또는 Movat의 Pentachrome 얼룩을 시각화하기 위해 헤 마톡 실린 및 에오신을 사용했다. </li></ol>

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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=Paclitaxel-eluting+stents+show+superiority+to+balloon+angioplasty+and+bare+metal+stents+in+femoropopliteal+disease:+twelve-month+Zilver+PTX+randomized+study+results.">Paclitaxel-eluting stents show superiority to balloon angioplasty and bare metal stents in femoropopliteal disease: twelve-month Zilver PTX randomized study results.</a> Circ Cardiovasc Interv. 4 (5), 495-504 (2011).</li><li>Bosiers, M., 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=Results+of+the+Protege+EverFlex+200-mm-long+nitinol+stent+(ev3)+in+TASC+C+and+D+femoropopliteal+lesions.">Results of the Protege EverFlex 200-mm-long nitinol stent (ev3) in TASC C and D femoropopliteal lesions.</a> J Vasc Surg. 54 (4), 1042-1050 (2011).</li><li>Duda, S. H., 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=Drug-eluting+and+bare+nitinol+stents+for+the+treatment+of+atherosclerotic+lesions+in+the+superficial+femoral+artery:+long-term+results+from+the+SIROCCO+trial.">Drug-eluting and bare nitinol stents for the treatment of atherosclerotic lesions in the superficial femoral artery: long-term results from the SIROCCO trial.</a> J Endovasc Ther. 13 (6), 701-710 (2006).</li><li>Kumar, A., Lindner, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+with+neointima+formation+in+the+mouse+carotid+artery+after+cessation+of+blood+flow.">Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow.</a> Arterioscler Thromb Vasc Biol. 17 (10), 2238-2244 (1997).</li><li>Sata, M., 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=A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia.">A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.</a> J Mol Cell Cardiol. 32 (11), 2097-2104 (2000).</li><li>Roque, M., 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=Mouse+model+of+femoral+artery+denudation+injury+associated+with+the+rapid+accumulation+of+adhesion+molecules+on+the+luminal+surface+and+recruitment+of+neutrophils.">Mouse model of femoral artery denudation injury associated with the rapid accumulation of adhesion molecules on the luminal surface and recruitment of neutrophils.</a> Arterioscler Thromb Vasc Biol. 20 (2), 335-342 (2000).</li><li>Lindner, V., Fingerle, J., Reidy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+arterial+injury.">Mouse model of arterial injury.</a> Circ Res. 73 (5), 792-796 (1993).</li><li>Edelman, E. R., Adams, D. H., Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+adventitial+heparin+delivery+on+smooth+muscle+cell+proliferation+following+endothelial+injury.">Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury.</a> Proc Natl Acad Sci U S A. 87 (10), 3773-3777 (1990).</li><li>Stemerman, M. B., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+arteriosclerosis.+I.+Fibrous+plaque+formation+in+primates,+an+electron+microscope+study.">Experimental arteriosclerosis. I. Fibrous plaque formation in primates, an electron microscope study.</a> J Exp Med. 136 (4), 769-789 (1972).</li><li>Brouchet, L., 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=Estradiol+accelerates+reendothelialization+in+mouse+carotid+artery+through+estrogen+receptor-alpha+but+not+estrogen+receptor-beta.">Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta.</a> Circulation. 103 (3), 423-428 (2001).</li><li>Filipe, C., 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=Estradiol+accelerates+endothelial+healing+through+the+retrograde+commitment+of+uninjured+endothelium.">Estradiol accelerates endothelial healing through the retrograde commitment of uninjured endothelium.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2822-H2830 (2008).</li><li>Feuls, R., 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=Microvascular+denudation+of+the+femoral+artery+of+the+mouse+as+a+model+for+restenosis.">Microvascular denudation of the femoral artery of the mouse as a model for restenosis.</a> Rofo. 175 (7), 952-957 (2003).</li><li>Holt, A. W., Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Rat+and+Mouse+Carotid+Artery+Surgery:+Injury+&amp;+Remodeling+Studies.">Experimental Rat and Mouse Carotid Artery Surgery: Injury &amp; Remodeling Studies.</a> ISRN Minim Invasive Surg. 2013, (2013).</li><li>Iafrati, M. 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=Estrogen+inhibits+the+vascular+injury+response+in+estrogen+receptor+alpha-deficient+mice.">Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.</a> Nat Med. 3 (5), 545-548 (1997).</li><li>Sullivan, T. R., 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=Estrogen+inhibits+the+response-to-injury+in+a+mouse+carotid+artery+model.">Estrogen inhibits the response-to-injury in a mouse carotid artery model.</a> J Clin Invest. 96 (5), 2482-2488 (1995).</li><li>Yin, Y., Zhao, X., Fang, Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+artery+wire+injury+mouse+model+with+a+nonmicrosurgical+procedure.">Carotid artery wire injury mouse model with a nonmicrosurgical procedure.</a> Vascular. 18 (4), 221-226 (2010).</li><li>Nam, 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=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li><li>Nam, 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=A+model+of+disturbed+flow-induced+atherosclerosis+in+mouse+carotid+artery+by+partial+ligation+and+a+simple+method+of+RNA+isolation+from+carotid+endothelium.">A model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of RNA isolation from carotid endothelium.</a> J Vis Exp. (40), (2010).</li><li>Kuhlmann, M. T., 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=Implantation+of+a+carotid+cuff+for+triggering+shear-stress+induced+atherosclerosis+in+mice.">Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.</a> J Vis Exp. (59), (2012).</li><li>Carmeliet, P., 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=Vascular+wound+healing+and+neointima+formation+induced+by+perivascular+electric+injury+in+mice.">Vascular wound healing and neointima formation induced by perivascular electric injury in mice.</a> Am J Pathol. 150 (2), 761-776 (1997).</li></ol>

We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire inju...

Arterial injury and inflammation caused by angioplasty and stent implantation can induce neointimal hyperplasia that contributes to the thickening of the arterial wall, a process known as restenosis.1,2 The formation of restenosis is major mode of failure for interventions such as angioplasty and stenting with bare metal stents.3 Due to recent concerns with the inhibition of vascular healing in arteries treated with drug eluting stents, there is also a need to find compounds that can inhibit restenosis while maintaining vascular healing and re-endothelialization.4-7 In addition, while stents have had success in the coronary vasculature, percutaneous interventions of all types in the peripheral arteries continue to fail at a higher rate due to restenosis.8-10 Mouse models of surgical interventions allow the use of powerful genetic manipulations that can provide mechanistic insight into the mechanisms underlying the failure of clinical therapies and can provide an initial test bed for compounds to inhibit intimal hyperplasia. 
Here, we describe a mouse model of vascular injury that allows the testing of therapeutic compounds to inhibit neointimal hyperplasia and assess whether their effects on re-endothelialization following endothelial denudation. A key challenge in executing vascular injury in mice is the technical skill needed to obtain vascular access and to restore flow to the injured artery following the wire injury. For this reason, simple arterial ligation models have been used to study neointimal hyperplasia in mice that do not require endovascular manipulations but are easier to implement.11 However, this type of surgical model differs substantially from the mechanical and biological aspects of a percutaneous intervention, lacking key aspects including arterial wall stretch, endothelial denudation and luminal blood flow following injury. 
We present two methods for obtaining and closing vascular access for wire injury of the femoral artery in mice. The first technique is the conventional method described by several groups previously and uses vascular access through a side branch of the femoral artery.12-14 This method requires older, larger mice and more surgical skill to implement the endoluminal access through the smaller artery. It also requires the ligation of the muscular branch of the femoral artery following the procedure. The second method we describe uses an arteriotomy in the branch point of the main and side branch and thereby allows for a larger access to the artery for performing wire injury. In this method, the arteriotomy is closed using controlled local cauterization that leaves both branches with blood flow following the procedure. The conventional method is applicable to mice of at least 20 weeks of age while the alternative method can be used in mice of at least 15 weeks of age. In both methods, the wire creates arterial stretch and abrasion leading to injury and endothelial denudation. Following either procedure a perivascular drug delivery patch can be implanted that allows the delivery of compounds to alter the response to injury. The use of the drug delivery patch allows mice to be used as a test bed for new compounds to inhibit restenosis through perivascular therapies.15,16

<table><tbody><tr><td>Straight spring wire, 0.15&amp;rdquo; diameter</td><td>Cook</td><td>G02426</td><td></td></tr><tr><td>High Temperature cautery</td><td>Bovie Medical Corp.</td><td>HIT1</td><td></td></tr><tr><td>High-temperature fine tip for cautery</td><td>Bovie Medical Corp.</td><td>H101</td><td></td></tr><tr><td>Micro-scissors</td><td>Fine Science Tools</td><td>15000-13</td><td>For performance of arteriotomy</td></tr><tr><td>Angled fine-tipped forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>For blunt dissection of vascular bundle</td></tr><tr><td>Angled forceps</td><td>Roboz</td><td>RS-5069</td><td>For clearing tissues</td></tr><tr><td>Surgical scissors</td><td>Roboz</td><td>RS-5840</td><td>For cutting skin</td></tr><tr><td>Retractor</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Retractor wire</td><td>Fine Science Tools</td><td>18200-05</td><td>Attached to retractor</td></tr><tr><td>Base plate</td><td>Fine Science Tools</td><td>18200-03</td><td>For use with retractor</td></tr><tr><td>Magnetic retractor fixator</td><td>Fine Science Tools</td><td>18200-01</td><td></td></tr><tr><td>Needle Holder</td><td>Roboz</td><td>RS-7822</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Biomedical Research Instruments, Inc.</td><td>34-1000</td><td></td></tr><tr><td>Dissecting microscope</td><td>Meiji Techno</td><td>EMZ-5TR</td><td></td></tr><tr><td>Microscope light source</td><td>Meiji Techno</td><td>FT191</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-500</td><td>For maintaining mouse body temperature</td></tr><tr><td>Reusable heating pad</td><td>Gaymar</td><td>TP-R 22G</td><td>For maintaining mouse body temperature</td></tr><tr><td>Lidocaine</td><td>Various</td><td></td><td></td></tr><tr><td>4.0 Vicryl suture with half circle needle</td><td>Ethicon</td><td>J494G</td><td>For post-surgical wound closure</td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Puritan</td><td>25-806-2WC</td><td>For application of depilatory cream and absorbing fluids</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Various</td><td></td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>6.0 braided silk suture</td><td>Teleflex Medical</td><td>4-S</td><td>For isolation of femoral artery during surgery</td></tr><tr><td>0.9% sodium chloride</td><td>Various</td><td></td><td>For irrigating tissues</td></tr><tr><td>Gel eye lubricant</td><td>Various</td><td></td><td></td></tr><tr><td>Glass Petri dish</td><td>Pyrex</td><td>3160-60</td><td>For femoral artery harvest</td></tr><tr><td>10% buffered formalin</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>Bouin&#039;s fluid</td><td>Electron Microscopy Sciences</td><td></td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcian blue, 1%</td><td>Electron Microscopy Sciences</td><td>26385-01</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alkaline alcohol</td><td>Electron Microscopy Sciences</td><td>26385-02</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Orcein, 0.2%</td><td>Electron Microscopy Sciences</td><td>26385-03</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Hematoxylin alcoholic, 5%</td><td>Electron Microscopy Sciences</td><td>26385-04</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Ferric chloride, 10%</td><td>Electron Microscopy Sciences</td><td>26385-05</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Lugol&#039;s Iodine</td><td>Electron Microscopy Sciences</td><td>26385-06</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Woodstain scarlet-acid fuchsin working solution</td><td>Electron Microscopy Sciences</td><td>26385-07</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Acetic acid, 0.5%</td><td>Electron Microscopy Sciences</td><td>Various</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Phosphotungstic acid, 5%</td><td>Electron Microscopy Sciences</td><td>26385-09</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcoholic saffron, 6%</td><td>Electron Microscopy Sciences</td><td>26385-10</td><td>For Movat&#039;s Pentachrome staining</td></tr></tbody></table>

murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

와이어 부상에 따라, 신생 내막의 증식은 시간이 지남에 개발하고 일반적으로 14-28일 후 검사한다. 그림의 조직 학적 결과를 그림과 같이 마우스의 내막 증식증의 강력한 세대에이 작품 리드에 설명 된 기술은 3. 손상되지 않은 대퇴 동맥은 그대로 탄성 얇은 판과 일반 두께와 둘레를 시연 할 예정이다. 부상 대퇴 동맥은 내막의 증식을 보여 탄성 얇은 판을 분해하고 나중?...

Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

혈관 주위 약물 전달 패치의 주입과 대퇴 동맥 와이어 부상의 쥐 모델

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

murine-model-femoral-artery-wire-injury-with-implantation

Research

JoVE Journal

Medicine

17.9K 조회수.  University of Texas at Austin. We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis. 

비디오: Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

생분해 성 고분자 나노 입자를 사용하여 치료 혈관 신생에 대한 프로그래밍 줄기 세포

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

연구

생체공학

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

마이크로 컴퓨터 단층 촬영 영상에 대한 Microfil와 허혈성 뒷발과 혈관의 관류에 삼투의 약물 전달 (Drug Delivery)

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

의학

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

무선 원격 측정 시스템은 척수 절개 및 배아 신경 세포 이식 줄기와 흰쥐의 심장 혈관 기능을 모니터링 할 수

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

동맥 재 협착의 쥐 모델 : 대퇴 와이어 부상의 기술적 측면

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

하이드로 겔은 구축 및 쥐 심근 경색 모델에서 치료를 제공하기 위해 접착제 접근을 섬유소 기반.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

마우스에서 가속 죽상 경화증의 유도: "와이어 부상" 모델

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

쥐 대동맥 또는 하대 정맥에 혈관 성형술 패치

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

커프 기법을 통한 마우스 경동맥에 조직 공학 혈관 이식편 이식

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

쥐 뒷다리 모델에서 동맥 형성 중 백혈구 모집을 모니터링하기 위한 다광자 생체 내 이미징

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...