이 작품은 미국 육군 그랜트 (W81XWH-08-1-0701)과 칼레 재단 병원에서 원정대에 의해 투자되었다.

절차 IACUC 프로토콜 번호 13302와 완벽한 호환성에 동물 자원의 분할에 의해 승인되었습니다. 1. 장비 준비 <ol><li> 멸균을 위해 수술 전에 사용 된 모든 비 - 일회용 악기 압력솥. </li><li> 용도 사이 유리 비드와 함께 살균 세션에서 여러 번 사용기구를 멸균. </li></ol> 2. 하이드로 겔 준비 <ol><li> 해동 및 하이드로 겔 구조의 생성에 최소 24 시간 이전에 판 줄기 세포. </li><li> 이전에 출판 (8, 18)를 상세하게 설명되었다 조형 장치를 이용하여 수술 (또는 유사 기기)에 전날 패치를 준비한다. </li><li> 하이드로 겔을 만들려면, 먼저 원하는 패치의 컴퓨터 지원 설계 (CAD) 모델을 생성 및 조형 형식 (STL)에 디자인을 수출하고 있습니다. </li><li> dissolvin에 의해 사전 겔 용액 만들기멸균 PBS 1X에 g의 폴리 (에틸렌 글리콜) 디 메타 크릴 레이트 (PEGDMA)를 20 % w / V 수용액을 얻었다. 동시에, 광개시제 1- [4- (2- 히드 록시에 톡시) 페닐] -2- 히드 록시 -2- 메틸 -1- 프로판 -1- 온 DMSO에 용해. / 0.5 V % w 마지막에 PEGDMA 솔루션 광개시제를 추가합니다. 가교 바로 전에, 세포 밀도의 원하는 100 ㎖를 추가한다. 참고 : 세포 / ㎖ 추천 6 10 × -2.0 5 10 × 2.0 사이의 세포 밀도를. </li><li> SLA 플랫폼의 중심에 접시에 프리 겔 용액을 피펫 및 이전에 업로드 STL 포맷 디자인 SLA 소프트웨어를 실행. </li><li> 장치로부터 패치를 제거하고 둘 베코 변성 이글 배지 5 % 소 태아 혈청 (FBS)으로 보충 (DMEM), 100 U / ㎖ 페니실린 하이드로 O / N을 배양하고, 세포를 조절하는 100 mg의 / ㎖ 스트렙토 마이신 있도록 패치 내의 환경. </li></ol> 3. 동물 준비 및 구강 intubati에 <ol><li> 자신의 케이지 동료와 함께 자신의 정리 습관과 상호 작용의 결정에 따라 동물, 건강에 있는지 확인하는 절차에 앞서 각 마우스를 평가합니다. 참고 : 여성 8-10주 오래 C57BL / 6J 마우스는이 절차에 사용 된, 그러나, 임의의 마우스 연령 또는 유형이 사용될 수있다. </li><li> 마취 챔버에 동물을 배치하고 1 L / 분 O 2 지원으로 5 % 이소 플루오 란에 노출. 발가락 핀치 반사에 의한 마취의 수준을 모니터링합니다. </li><li> 동물이 제대로 마취되면, 무게 삽관 스탠드에 배치합니다. </li><li> 동물의 흉강으로 광원을 직접 성대 후두 표면을 철회하고 노출 집게를 사용합니다. 조심스럽게 성대 사이에 20 G의 angiocath을 안내하고 원활 기관에 삽입합니다. 카테터는 작은 동물 인공 호흡기에 연결되면 흉강의 기계적인 움직임으로 적절한 삽입을 감지합니다. </li><li> 토륨 환기 설정을 조정합니다제조자의 지침에 기초하여 동물의 체중 E. </li><li> 삽관 후 저체온증을 방지하기 위해 가열 패드에 앙와위에서 동물을 배치합니다. 가열 패드가 40 ℃를 초과하지 않도록하십시오. 필요한 경우, 화상의 가능성을 최소화하기 위해 동물과 난로 사이의 배리어를 배치했다. </li><li> 제모 크림의 사용을 통해 수술 부위에서 머리를 제거합니다. 적절한 살균을 위해 betadine 75 % 에탄올 3 교번하여 수술 부위를 소독. 다만 수술 부위를 노출하고, 절차를 수행하는 동안 건조를 방지하기 위해 눈에 수의사 연고를 배치 드레이프를 놓습니다. </li></ol> 4. 수술 <ol><li> 개흉술 따라서, 흉골의 길이를 실행 흉골의 좌측에 피부 절개를 약 1cm을, 동물의 왼쪽에 수행된다. </li><li> 두 단계로부터 기본 흉곽 근육 층을 분리 집게를 사용한다. 델라을 준수동물의 pectoralis 주요 근육을 나타냅니다 운 전압 라인. 기본 외부 경사 근육에서 분리 및 내측 철회, 약간이 근육을 들어 올립니다. 그 후, 동일한 방식으로 기본 흉곽로부터 외부 경사 근육을 확보하고, 제 3 및 제 4 리브 unobscured보기를 제공하는 측 방향으로 후퇴. </li><li> 세 번째와 네 번째 갈비뼈 사이의 개흉술을 수행합니다. 조심스럽게 네 번째 늑골을 들어 올려 세 번째와 네 번째 갈비뼈 사이의 흉강을 엽니 다 cauterizer를 사용합니다. 더 캐비티를 열고 심장을 노출 견인기를 놓습니다. </li><li> 마음의 얇은 심낭을 파열 집게를 사용합니다. </li><li> 8-0 모노 필라멘트 나일론 봉합사와 왼쪽 관상 동맥을 결찰. 직접 좌심방의 하단부 아래, 약 4mm 마음의 정점에서 봉합사를 놓습니다. 심실의 심근 및 창백 수 투어 후 좌심방의 크기가 증가하여 적절한 봉합 위치가 결정다시 묶여있다. </li></ol> 5. 패치 배치 및 접착제 <ol><li> 사용할 때까지 CO 2 조건을 37 ℃에서 패치를 유지하고 5 %. </li><li> 부드럽게 평면 종단 주걱을 사용하여 패치를 들어 올려 부드럽게 경색의 사이트에서, 마음의 표면에 놓습니다. 주 : 좌심실 빠지는 것을 방지하기 위해, 패치 가볍게 주걱 또는 집게의 쌍의 팁을 사용하여 접촉을 유지하여 제 위치에 유지 될 수있다. </li><li> 트롬빈에 피브리노겐의 1 비율 : 4를 혼합하여 접착제를 준비합니다. 이 제제의 시작 후 통상 1 분 이내에, 두껍게하기 시작까지 반복 피펫으로 용액을 섞는다. </li><li> 용액 점도가 원하는 레벨에 도달하면, 신속하게 패치 표면에 약 10 mL로 옮긴다. 참고 : 피브린 접착제의 응고 시간이 빠르다, 접착제의 효율적인 전송을위한 기회의 작은 창을 제공한다. </li></ol> 6. 봉합 <ol><l난&gt; 6-0 모노 필라멘트 나일론 봉합사를 사용하여 모든 폐쇄 봉합을 수행합니다. </li><li> 3-4 개별 중단 봉합 리브 층을 닫습니다. </li><li> 늑간 층의 완전 폐쇄하기 전에, 폐쇄가 완료된 후 흉강을 철수하고 적절한 흉부 내 압력을 다시 절개로 PE-10의 캐 뉼러를 삽입합니다. </li><li> 3-4 개별 중단 봉합과 가슴 근육을 닫습니다. </li><li> 연속 봉합선으로 피부 층을 인감. </li><li> 완전 폐쇄 후, 캐 뉼러의 단부에 1.0 mL의 주사기를 연결하고 흉강 대피 사용. </li><li> 봉합 사이트를 강화하기 위해 절개 사이트에 조직 접착제를 적용합니다. </li></ol> 7. 후 수술 적 치료 <ol><li> 쥐에게 프레 노르 핀 (0.05-1.0 ㎎ / ㎏) 및 피하 직전 부흥 관상 동맥 결찰 및 20 분 이상 다음과 같은 카프로 펜 (2.2 ㎎ / ㎏)의 주입을 준다. 두 약물 수술 후 6 ~ 8 시간을 관리합니다다음 통증과 고통을 제어하기까지 3 일 동안 하루에 두 번 제공합니다. </li><li> 지속적으로 처음 4 시간 다음 수술을 의식하고 매 시간마다 때까지 동물을 모니터링 할 수 있습니다. 진통제의 두 번째 복용량을 관리하는 두 번째 검진에게 수술 (2-4 시간 이상)의 밤을 수행합니다. </li><li> 동물이 안정 나타날 때까지, 절차에 따라 두 번째 날 이후 매일 동물을 모니터링합니다. </li></ol> 심장 기능 및 조직학 8. 분석 <ol><li> MI는 다음의 손상 정도를 결정하는 절차에 따라, 마우스 사주에 echocardiographs 수행. </li><li> 흡입 마취의 유도를위한 마취 챔버 내에서 4-5%의 이소 플루 란과 쥐를 마취 및 유지 보수 마취 얼굴 마스크를 통해 다음 2 % 이소 플루 란. </li><li> 제모 크림 이미징 영역을 지우고 뛰는 심장의 2D M 모드 심 초음파 검사를 얻기 위해 작은 동물 초음파를 사용하여 기능 매개 변수 ASSO를 기록그것으로 ciated. </li><li> 그 다음 24 시간 동안 10 %의 포르말린 용액에 동물과 장소에서 하트를 수집 이산화탄소 챔버에서 쥐를 안락사. 24 시간 후, 70 % 에탄올에 마음을 전송하고 조직 처리 될 때까지 4 ℃에서 보관하십시오. </li><li> 마음을 처리하고 파라핀 블록을 포함. </li><li> 정점에서 시작 아트리움에서 끝나는, 심장의 심실 영역을 통해 5mm 부분을 잘라. </li><li> 얼룩은 메이슨의 트리 크롬과 헤 마톡 실린 및 표준 프로토콜을 사용하여 에오신 염색 모두 조각을 절단. </li></ol>

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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=Living'+microvascular+stamp+for+patterning+of+functional+neovessels;+orchestrated+control+of+matrix+property+and+geometry.">Living' microvascular stamp for patterning of functional neovessels; orchestrated control of matrix property and geometry.</a> Advanced Materials. 24, 58-63 (2012).</li><li>Wu, X., Ren, J., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+as+the+cell-delivery+vehicle+for+mesenchymal+stromal+cells+in+regenerative+medicine.">Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.</a> Cytotherapy. 14, 555-562 (2012).</li></ol>

The authors declare that they have no competing financial interests.

쥐의 심근 경색 모델에이 방법으로, 우리는 다른 쥐의 심근 경색 기술과 관련된 비 심근 지역에 피해를 최소화하는 시스템을 개발했다. 이 지역은 기관 절개술에 의한 손상, 근육 층의 절단 및 흉강을 노출 리브의 파손을 포함한다. 우리는 이러한 변화는 수술시로, 갈비뼈와 근육 층을 포함, 주요 구조물의 많은 손상을 유지하는 데 걸리는 치료에 전체 수?...

심근 경색 (MI)는 주요 관상 동맥의 폐색으로 인한 심장의 영역에 혈액 차단으로 정의된다. MI 인한 손상으로 인해, 또는보다 구체적으로, 좌심실은 제대로 치는 심장의 기능을 감소 비 기능적 흉터 조직으로 생존 가능한 심장 조직의 리모델링이다. 이것은 행정 용적으로 알려진 모든 심장 박동, 및 박출계수 6라고도 각 심장 박동 심장 밖으로 펌핑되어 혈액의 백분율로 본체에 전달 될 ​​수있는 혈액의 체적 감소를 초래. 이들은, 다른 기능 감소와 함께, 적절한 기능을 유지하기 위해 심장의 나머지 부분에 변형을 증가시킨다. 이 두 번째 심장 마비에서 개인 (7)의 약 10 % 본 현상을 일으키는 종종이 증가 된 균주는 매우 심각한 될 수 있습니다. 의료 행위는 치료를 위해 진화하는 동안MI의 직후, 아니 기술은 저속, 정지, 또는 조직 리모델링의 부작용을 반전하기 위해 개발 없습니다. 줄기 세포 치료가 유망한 잠재력에도 불구하고, 이러한 치료 가능한 수단으로 부상 한 줄기 세포는 임상에서 성공적으로 검증되지 않았다. 그들의 결점 일설 유익한 세포 유리한 결과를 생성하는 5 충분히 경색 부위에서 유지되도록 할 수 없다는 것이다. 그것은 단순히 경색 부위에 주입 세포의 더 이상의 24 %가 생존하지 않고 손상된 사이트 1 일 인도 후 2에 남아 있음을 보였다. 세포의 보존이 문제를 해결하기위한 가능한 전망은 손상된 세포 또는 사이트로 전달 될 수있는 치료제, 하나를 캡슐화하는 생체 적합성 히드로 겔 시스템을 개발하는 것이다. 이 프로토콜의 선택의 하이드로 겔 인해 전지 밀봉 PR에 이전에 사용 된 폴리 (에틸렌 글리콜) 디 메타 크릴 레이트 인ocedures 그러나 캡슐화 할 수있는 임의의 하이드로 겔 8을 사용할 수있다. 직접 부상에 패치의 전달은 세포가 심근 근본적인 요인에 유익을 제공 할 수있는 시간의 길이를 증가, 장기간에 걸쳐 세포 간 조직의 접촉을 보장한다. 패치 접근법 병목 심장 표면에 패치를 부착하는 어려움이다. 많은 그룹은 기술, 심장 표면 9,10에 구조를 묶는 간단한 봉합되는 가장 널리 다양한 통해이를 극복했다. 이 구조는 강성 재료로 제조되는 경우의 수가 성공적으로 입증하지만 인해 높은 물 농도 패치 구조의 섬세한 특성상 하이드로 시스템에서 시도 할 때 실패했다. 이것을 극복하기 위해, 우리는 혈전 형성의 화학을 모방 피브린 접착제 외부 접착 시스템을 이용했다. 섬유소 접착제는 INC, 수많은 의료 수술에 사용되어왔다상처 밀봉 11-13로 제품의 생체 적합성을 강조, 경질 눈물, 기관지 누공, 각막 이식을 luding. 또한 섬유소하지만, 심장 패치 부착 접착제로서의 사용은 일반적으로 14 ~ 17 사용하지 좌심실 파열 및 관상 동맥 우회 수술 수술을 포함한 심장 다양한 목적을 위해 사용되어왔다. 심장 작용에 패치를 보장하기 위해 가능한 부착 시스템을 제공하는 단계, 외부 심장 패치의 외부에 직접 배치 될 수있는 생체 적합성 접착제 피브리노겐과 트롬빈 결과의 간단한 제제.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

수술하는 동안, 좌측 관상 동맥의 결찰이 폐색 된 동맥의 하류 희게 표시된 의해 식별 될 수있다. 매듭을 묶는 전에 테스트로, 봉합사는 적절한 위치에 있는지 확인하기 위해 잠시 동안 강화 될 수있다. 또한, 적절히 이길 좌심방의 능력에 거의 순간적인 감소 동맥 폐색의 결과 때문에, 좌심방이 시스템 내의 혈액의 역류에 응답하여 확대된다. M 모드 심 초음파 측정 후 경색이...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

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

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 ...

a-hydrogel-construct-fibrin-based-glue-approach-to-deliver

Research

JoVE Journal

Medicine

8.6K 조회수.  University of Illinois at Urbana-Champaign. 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.

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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

고양이에서 급성 심근 경색

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

연구

의학

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

경색의 Murine 모델에서 관상 동맥 내고과 Intramyocardial 사출

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

심근 국소 빈혈 및 Reperfusion의 Murine 폐쇄 가슴 모델

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

돼지의 심근 경색 및 기능적 결과의 평가

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

급성 심근 경색의 돼지 모델의 주요 결과 평가

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

신생아 쥐의 심근 경색, 심장 재생의 모델

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

왼쪽 전방 내림차순 관상 동맥의 영구 결찰을 사용하는 뮤린 심근 경색 모델

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

마우스에서 심근 경색을 연구하는 극저온 손상 모델

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

줄기 세포 치료를위한 심근 경색 심부전의 돼지 모델 설립

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine 모델에서 심근 경색 후 MSC 로딩 주사용 하이드로겔의 자궁 내 이식

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...