이 책은 사우스 캐롤라이나 의과 대학에서 학술 집, NIH / NCATS 허가 번호 UL1 TR000062로, 사우스 캐롤라이나 임상 및 중개 연구 (SCTR) 연구소에 의해 지원되었다. 추가 지원은 DRM에 버지니아 우수상 BX002327-01에 의해 제공되었다. DJH는 NIH / NCATS 허가 번호 TL1 TR000061에 의해 NIH 허가 번호 T32 GM008716에 의해 지원되었다. 바다 NIH 허가 번호 T32 HL07260에 의해 지원되었다.

여기에 나열된 모든 절차는 사우스 캐롤라이나 의과 대학에서 기관 애니멀 케어 및 사용위원회에 의해 승인되었습니다. 여기에 설명 된 실험 급성 비 생존 실험이다. 따라서,이 눈 연고의 아무 소용이 없으며 멸균 운영 스위트 룸은 필요하지 않습니다. 안락사는 마음의 수확시 방혈에 의해 달성된다. 1. 실험 준비 <ol><li> 일정한 압력 또는 일정한 흐름을 Langendorff 관류 장치 (4)를 설정합니다. </li><li> 수정 크렙스 Henseleit 버퍼의 4 L 준비 (MM의 : (112)의 NaCl, 5 KCl을, 1.2 황산을 1, K 2 HPO 4, 1.25 염화칼슘 2, 25 NaHCO3를, 11 D- 글루코스, 0.2 옥 탄산, 산도 = 7.4). <ol><li> 10 배 재고 버퍼를 확인합니다. <ol><li> 3 L의 초순수에의 NaCl, KCl을, 황산, 및 K 2 HPO 4를 녹여. </li><li> 500ml의 초순수 위스콘신에 CaCl2를 녹이고1 N 염산 40 mL로 일. </li><li> 천천히 1.2.1.1에서 만든 버퍼에 염화칼슘이 솔루션을 추가 할 수 있습니다. 4 L. 총 충분히 초순수를 추가 0.8 μm의 필터를 통해 여과 전에 1 시간 동안 교반을 허용합니다. 4 ℃에서 보관하십시오. </li><li> 4 L의 초순수에 NaHCO3를 해산. 0.8 μm의 필터로를 통해 필터링하기 전에 1 시간 동안 교반을 허용합니다. 저장 4 ° C에서 </li></ol></li><li> 400 ml의 10 배 NaHCO3를 버퍼와 400 ml의 10 배 크렙스 Henseleit 버퍼를 결합합니다. 교반하면서 당과 옥 탄산을 녹이고 4 L. 총하기에 충분한 초순수를 추가합니다. 0.8 μm의 필터를 통해 필터링합니다. </li></ol></li><li> 심장을 위해 시스템을 준비합니다. <ol><li> 4 L 병에 필터링 된 버퍼의 2 L을 추가 대동맥 정맥에 붙어있는 꼭지 위의 38을 배치. 이 권장 관류 압 4 중심부에 70 ~ 80 mmHg로의 관류 압력을 제공 할 것입니다. </li><li> 병에 버퍼에 가스 분산 버블을 삽입합니다. </li><li> 튜브 내부에서 모든 거품을 제거해야되고, 시스템을 통해 버퍼를 실행합니다. </li><li> 심장은 시스템 상에 배치 될 전에 가스 (95 %, O2 / 5 % CO 2) 적어도 30 분에 돌려. 산소 포화도를 확인하기 위해 가스 유동 프로브 및 혈액 가스 분석을 사용한다. </li></ol></li><li> 수술 용품 및 풍선을 준비합니다. <ol><li> 마음의 수확, 6.75 일쌍 &quot;수술 가위, 4.5 일쌍&quot;가위, 27 G 바늘, 0 게이지 실크 봉합사의 2 개 (약 15 지혈제 이쌍, caramalt 집게 두 세트, 두 개의 주사기를 준비 길이 cm), 하나 1/16 인치 철 뉼러, 한 기관 캐뉼라. </li><li> 기관 캐뉼라를 조립하려면 Y 커넥터에 1/16 인치 직경의 플라스틱 튜브의 짧은 조각을 접착제. </li><li> 풍선 장치를 조립 : 1/16 인치 튜브의 조각에 위관 바늘을 부착 한 후 압력 변환기의 하우징에 튜브를 연결합니다. 압력 TR의 다른쪽에 부착 된 작은 주사기ansducer 하우징은 풍선의 인플레이션과 디플레이션을 수 있습니다. <ol><li> 중앙에 위관 바늘의 플라스틱 포장과 장소 팁의 사각형을 잘라. 위관 바늘 주위의 플라스틱 포장 랩. </li><li> 식염수로 풍선을 채우고 두 봉합으로 묶을. 누수가 없는지 확인하기 위해 풍선을 팽창. 팽창 된 풍선 내의 식염수의 부피는 약 200 μL해야하지만 쥐의 크기에 따라 달라질 수있다. </li><li> 또한, 플라스틱 랩 대신 라텍스 손가락 침대를 사용합니다. 손가락 침대에서 위관 바늘을 놓고 식염수로 채우고 묶을. </li></ol></li></ol></li></ol> 2. 수확 심장 <ol><li> decapicone 구속 또는 기타 수동 소자를 이용한 스프 라그 돌리 래트를 제지. 쥐의 무게를. </li><li> 쥐를 마취하는 복강 내 주사를 통해 케타민 / 자일 라진 혼합물 (0.85 ㎎ / ㎏ 케타민 0.15 ㎎ / ㎏ 자일 라진)를 관리합니다. 마취제의 용량은 충분한 마취를 제공 할 것입니다20-40 분하지만 절차는 신속히 마취가 확인 가능한 후에 완료한다. </li><li> 테스트 발가락 핀치 반사에 의한 마취의 적절한 수준을 확인합니다. </li><li> 기관 절개술 <ol><li> 절개를 만들기 위해 펠트와 가위를 상승 지혈제를 사용, 턱의 기본 중순 앞발에서 펠트를 제거합니다. 펠트 따라 절단하고 제거하는 일을 허용, 기본 근육에서 분리하는 상승 펠트를 유지합니다. </li><li> 지혈제를 사용하여, 선의 조직을 들어 올려 선의 조직 근막에 수평 절개 열등합니다. 닉하지 경정맥을 돌보는 오픈 절개를 확산 지혈제를 사용합니다. </li><li> , 지혈제를 사용하여 핀치 근육 기관을 덮는 및 사용 가위는 지혈제의 끝 아​​래 근육에 횡 절개를합니다. 지혈제를 사용하여 기관을 나타 내기 위해 따로 조직을 확산. </li><li> 조심스럽게 부드럽게 전진과 지혈을 후퇴에 의해 진행하면서 근막을 제거, 기관에서 지혈제를 삽입합니다. </li><li>지혈이 성공적으로 기관 뒤에 배치되면, 지혈제와 0-0 실크 봉합사를 꼬집어 가위의 작은 날카로운 쌍의 기관을 hemisect. </li><li> , hemisected 기관으로 기관 캐뉼라를 삽입기도 뒤에 봉합을 당겨와 외과 의사의 매듭 캐 뉼러의 Y 사타구니에 봉합사를 고정, 유관 기관 주위 봉합 넥타이. </li><li> 27 G 바늘을 사용하여 경정맥에 1000 U / kg 헤파린 주입하고 30 초 동안 순환 시키도록 허용한다. </li></ol></li><li> 개흉술 <ol><li> 지혈제로 곤란과 가위로 절단하여 중간 앞발 지역 중순 복부에서 펠트를 제거합니다. </li><li> 바로 다이어프램 아래, 복부 근육의 벽에 ¾ 인치 가로 절개를합니다. </li><li> 흉골을 따라 절단, 복부 벽과 가슴 벽까지 수직 절개를합니다. </li><li> 다시 양자 다이어프램을 절단 한 후 마음이 확산 남아 보장하는 지혈제와 클램프 흉곽을 나타 내기 위해 흉곽을 확산. </li&gt; <li> 지혈제를 사용하여 부드럽게 상행 대동맥을 노출, 흉선을 제거합니다. </li><li> 지혈제의 팁을 통해 수 있도록 끈을 확산 부드럽게 진행하고 지혈을 후퇴하고, 대동맥 루프를 통해 지혈을 애무 해줘. </li><li> , 상행 대동맥 뒤에 0-0 실크 봉합사를 당겨 느슨한 반 사각 매듭으로 봉합을 묶어 지혈을 사용합니다. </li><li> 관류 버퍼, hemisect 대동맥의 흐름을 시작하고 즉시 대동맥 루멘에 캐 뉼러를 삽입 꼭지를 돌립니다. 신속하게 반 사각 매듭을 식은 죽 먹기 철저하게 강화, 매듭을 완료합니다. </li><li> 멀리 큰 혈관에서 심장을 해부 가슴에서 심장을 제거하고 관류 열을 연결하는 가위를 사용합니다. </li><li> 초과 폐 조직을 잘라 내고 마음이 풍선을 삽입하기 전에 약 15 분 동안 열 평형 수 있습니다. 10-20 ml / 분의 중심부를 통해 버퍼의 정상 유량을 사용한다. </li></ol></li></ol> 3. 랑겐 돌프 관류 및 허혈 재관류 손상 <ol><li> LV 사전 배치ssure 풍선 <ol><li> 원뿔 모양으로 압연하는 동안 풍선을 수축 및 풍선이 수축 유지 보장하기 위해 꼭지를 켜십시오. </li><li> 소비세는 승모판 막에 대한 액세스를 노출 가위의 작은 쌍의 중앙 홀을 떠났다. </li><li> 좌심방과 좌심실에 승모판을 통해 풍선을 삽입합니다. </li><li> 압력 모니터링 소프트웨어를 시작합니다 (LabChart 프로). </li><li> 스톱 콕을 열고 부드럽게 micromanometer로부터 판독 확장기 혈압이 제로를 초과 할 때까지 풍선 식염수의 양을 증가시켜 풍선을 팽창. </li><li> 천천히 수축, 길이 긴장 응답을 확인 75 mmHg로의 이완기 압력으로 천천히 풍선을 팽창하기 위해, 한 번 반복합니다. </li><li> 풍선에 식염수의 양을 조절함으로써 10 mmHg로에 이완기 압력을 설정합니다. 인플레이션이 수준에서 풍선을 밀봉 마개를 돌려 LV 풍선과 압력 변환기 사이의 폐쇄 압력 시스템을 유지한다. </li><li> 플라스틱 가열 챔버 커버 챔버로 낮은 마음. </li></ol></li><li> 광고허혈 재관류 손상의 직무 <ol><li> 가열 된 챔버의 바닥을 연결하고 챔버 토로 버퍼에 기입 할 수 있습니다. </li><li> 버퍼가 마음을 침수되면 글로벌 허혈을 유도, 마음에 버퍼의 흐름을 중지 꼭지를 켜십시오. 허혈 시간은 실험의 목적에 따라 달라질 수있다. </li><li> 국소 빈혈의 30 분 후, 재관류를 시작, 마음에 버퍼의 흐름을 복원 꼭지를 켜십시오. </li><li> 실험을 종료하기 전에 1 시간 동안 마음의 재관류를 허용합니다. 실험의 목적에 따라 변수 재관류 시간을 사용합니다. </li></ol></li><li> 실험의 종료 및 조직의 수확 <ol><li> 꼭지의 사이드 포트를 사용하여, 10 ㎖의 속도로 마음에 4 ° C에서 인산염 완충 생리 식염수 10 ml의 관리 / 분의 마음을 체포합니다. </li><li> , 관류 열에서 마음을 제거 심방 조직을 나머지 멀리 트림. </li><li> 아빠 커버, 스테인레스 스틸 조직 슬라이스 행렬에 마음을 놓고8 분, 또는 마음까지 -80 ° C에서 rafilm 및 장소는 마시멜로 같은 일관성을 가지고있다. </li><li> 냉장고에서 블록을 제거하고 2mm의 조각으로 마음을 슬라이스 매트릭스 슬라이스로 면도날을 삽입합니다. 조직 블록은 2mm 이외의 슬라이스 크기로 구입할 수 있습니다. </li><li> 한 번에 조각 하나를 제거하고 생화학 적 연구에 동결 플래시 액체 질소에 놓습니다. 경색 염색의 정점에서 세 번째 심실 슬라이스를 유지. </li><li> 생화학 실험에 사용할 때까지 -80 ° C에서 액체 질소 저장에서 조직을 제거합니다. </li></ol></li><li> 경색 염색 <ol><li> 15 분 동안 37 ℃ / V의 트리 페닐 테트라 졸륨 클로라이드 (TTC) 1 % w 10ml에 제 심실 슬라이스 인큐베이션. 주 : TTC 인큐베이션은 효율성 15에서 변화없이 15 또는 30 분 동안 수행 될 수있다. </li><li> 10 % 포르말린에 TTC로부터 조직 절편을 전송하고, 밤새 실온에서 배양 할 수있다. 최적의 결과를 듣고,TS는 포르말린에서 꺼내어 24 시간 내에 4 군데해야합니다. </li><li> 같은 ImageJ에 같은 사진 스테인드 조각 사용 소프트웨어는 제조 업체의 프로토콜에 따라 경색 크기를 추정한다. 염료가 시간이 지남에 따라 퇴색되므로, 고정 후 가능한 한 빨리 사진을 가져 가라. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+class+I+but+not+class+IIb+histone+deacetylases+exerts+cardiac+protection+from+ischemia+reperfusion.">Selective inhibition of class I but not class IIb histone deacetylases exerts cardiac protection from ischemia reperfusion.</a> Journal of Molecular and Cellular Cardiology. 72, 138-145 (2014).</li><li>Yellon, D. 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=The+protective+role+of+heat+stress+in+the+ischaemic+and+reperfused+rabbit+myocardium.">The protective role of heat stress in the ischaemic and reperfused rabbit myocardium.</a> Journal of Molecular and Cellular Cardiology. 24 (8), 895-907 (1992).</li><li>Khaliulin, I., 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=Temperature+preconditioning+of+isolated+rat+hearts--a+potent+cardioprotective+mechanism+involving+a+reduction+in+oxidative+stress+and+inhibition+of+the+mitochondrial+permeability+transition+pore.">Temperature preconditioning of isolated rat hearts--a potent cardioprotective mechanism involving a reduction in oxidative stress and inhibition of the mitochondrial permeability transition pore.</a> The Journal of Physiology. 581 (Pt 3), 1147-1161 (2007).</li><li>Molojavyi, A., 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=Effects+of+ketamine+and+its+isomers+on+ischemic+preconditioning+in+the+isolated+rat+heart).">Effects of ketamine and its isomers on ischemic preconditioning in the isolated rat heart).</a> Anesthesiology. 94 (4), 623-629 (2001).</li><li>Asfour, 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=NADH+fluorescence+imaging+of+isolated+biventricular+working+rabbit+hearts.">NADH fluorescence imaging of isolated biventricular working rabbit hearts.</a> The Journal of Visualized Experiments. (65), (2012).</li><li>Sill, B., Hammer, P. E., Cowan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+langendorff-perfused+rat+hearts.">Optical mapping of langendorff-perfused rat hearts.</a> The Journal of Visualized Experiments. (30), (2009).</li><li>Ferrera, R., Benhabbouche, S., Bopassa, J. C., Li, B., Ovize, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+hour+reperfusion+is+enough+to+assess+function+and+infarct+size+with+TTC+staining+in+langendorff+rat+model.">One hour reperfusion is enough to assess function and infarct size with TTC staining in langendorff rat model.</a> Cardiovascular Drugs and Therapy. 23 (4), 327-331 (2009).</li><li>Sutherland, F. J., Shattock, M. J., Baker, K. E., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+isolated+perfused+heart:+Characteristics+and+cautions.">Mouse isolated perfused heart: Characteristics and cautions.</a> Clinical and Experimental Pharmacology and Physiology. 30 (11), 867-878 (2003).</li><li>Reichelt, M. E., Willems, L., Hack BA, ., Peart, J. N., Headrick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+coronary+function+in+the+langendorff-perfused+mouse+heart+model.">Cardiac and coronary function in the langendorff-perfused mouse heart model.</a> Experimental Physiology. 94 (1), 54-70 (2009).</li><li>Xie, 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=Histone+deacetylase+inhibition+blunts+ischemia/reperfusion+injury+by+inducing+cardiomyocyte+autophagy.">Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy.</a> Circulation. 129 (10), 1139-1151 (2014).</li></ol>

The authors declare that they have no competing financial interests.

절연 관류 래트 심장 성공적 허혈 재관류 손상 9 심장 전처리에 약리학 적 개입의 효과를 연구하기 위해 사용될 수있다. 그러나, 재현 가능한 결과를 보장하기 위해 표준화되어야 절차와 몇몇 중요한 단계가있다. 시스템 내에서 37.4 ℃의 온도를 유지하는 것은 심지어 가벼운 저체온증과 고열이 심장 컨디셔닝 10, 11의 원인이되므로 중요하다. 케타민에 장기간 노출이 심장 컨디셔닝...

모두 허혈 및 재관류에 심장 응답의 기초가되는 사건의 해명은 대동맥 - 클램핑 (2)를 필요로 심근 경색 (1) 심장 수술의 치료 개선에 필수적이다. 허혈 재관류 손상의 생체 내 모델이 매우 유용 종료점을 분석 할 수 있지만, 그들이 실시간으로 급성 허혈 재관류 손상의 작용 효과를 연구하기위한로서 효과적이지 않다. 생체 허혈 재관류 모델은 일반적으로 재관류시의 경색 크기에서 상당한 변동성 및 심장 약물의 직접 전달을 생산에 또한 도전. 허혈 재관류 손상을 연구 랑겐 돌프 격리하는 마음 시스템의 활용은 약물 치료, 경색 조직의 균일 한 영역, 직접 심근에 약물의 즉각적인 전달의 실시간 기능 평가를 할 수 있습니다. 먼저 B를 설명1895 년 3 Y 오스카 랑겐 돌프는 랑겐 돌프 격리하는 마음은 지난 40 년 4,5에 대한 허혈 재관류 연구에 사용 된, 허혈 재관류 손상 연구를위한 강력한 모델이다. 여기에 일부 수정 기능 분석을 위해 고립 된 마음을 최적화하기 위해 만들어집니다. 대동맥의 현장 삽관에서 심장 박동이 심장 허혈 재관류 시험 (6)의 결과를 바꿀 것 허혈 양상을 경험하지 않는 것을 보장된다. 이를 용이하게하기 위해, 기관 절개 환기 있도록 수술시 쥐의 생리 안정성 확보를 행한다. 심장이어서 크렙스 Henseleit 버퍼 ​​대동맥에 직접 역행 관류 통해 전달되는 유리 물 재킷 나선형 기둥에 부착된다. 염수로 채워진 풍선 좌심실에 삽입 및 심실 내에서 압력을 실시간으로 측정 할 수있는 압력 변환기에 부착되고여러 기능 매개 변수의 D 계산. 실험이 끝날 때, 심장 수축과 DNA, RNA 및 단백질 수준의 하류 분석을 가능하게 액체 질소에 냉동 플래시 체포 냉 식염수로 불어 낸다. 따라서 수정을 Langendorff 관류 심장 허혈 재관류 손상시 급성 언제든지 약리학 적 개입의 생리적 효과를 직접 모니터링을위한 효과적인 시스템 역할을한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td align="right">203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>RES20765-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>C2875</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Pressure Transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07521-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Easy Load Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated circulating water bath</td>
			<td>Lauda</td>
			<td>M3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Flow Module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Research Console</td>
			<td>Transonic</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline flow sensor</td>
			<td>Transonic</td>
			<td>ME2PXN</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart data acquisition software</td>
			<td>AD Instruments</td>
			<td>MLU60/8</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and assessment of ischemia-reperfusion injury in langendorff-perfused rat hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

좌심실 풍선 장치는 좌심실 수축 (도 1)에 의해 개발 된 압력의 실시간 모니터링을 허용한다. 앞서도 7과 같이,이 압접 흔적은 심실 기능 많은 매개 변수를 계산하는데 사용될 수있다. 이러한 계산은, 기준 위상뿐만 아니라 재관류 위상 이루어진 각 그룹 내에서 여러 트레이스 대하여 평균, 우리는 이전에 9했던 것처럼 약리학 적 개입, 심장 전처리에 기인 한 것인지를 ?...

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

랑겐 돌프 - 관류 쥐 마음에 유도 및 허혈 - 재관류 손상의 평가

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

induction-assessment-ischemia-reperfusion-injury-langendorff-perfused

Research

JoVE Journal

Medicine

22.6K 조회수.  Medical University of South Carolina. The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

비디오: Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

쥐 Cremaster 근육의 국소 빈혈 reperfusion의 - 상해에서 백혈구 - 내피 상호 작용의 실시간 디지털 이미징 (IRI)

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

연구

의학

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

Myocutaneous 국소 빈혈 reperfusion 상해의 쥐 모델 : 현장 가로 직근 abdominis의 Myocutaneous 플랩의

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

마우스의 급성 신장 손상 및 사후 상해 섬유증의 허혈 - 재관류 모델

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

창자 허혈 - 재관류 손상의 쥐 모델

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

쥐의 심근 허혈 재관류 후 관상 동맥 흐름 예비의 평가

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

재건 미세 수술에서 허혈 재관류 손상의 연구를위한 전 임상 쥐 모델

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

전 비보 랑엔도르프-퍼블릭 하트의 검문 및 전기생리학 연구

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

뮤린 모델에서 허혈혈 재관류 부상 후 줄기 세포의 지연 된 자궁 내 납품

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

쥐 간 이식 모형에 있는 혈역학적인 변경을 시뮬레이션하여 기관 허혈-재관류 상해

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

허혈/재관류의 돼지 모델에서 척수 미세 퍼퍼퓨전의 실시간 평가

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...