We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established this model in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985. Dr. Luc Bertrand is supported in part by a postdoctoral fellowship from the American Heart Association (16POST31170002).

모든 절차는 건강 (NIH) 지침의 국립 연구소에 따라 마이애미 기관 동물 관리 및 사용위원회 (IACUC)의 대학에 의해 승인되었다. 멸균 장비 및 무균 기술의 사용이 필요합니다. 1. 폐색 봉합 준비 <ol><li> 25g과 25 사이의 마우스에 대한 0.23 mm - - 체중 35g (20) 사이의 마우스에 대해 0.21 mm 직경의 봉합사를 사용합니다. MCAO 절차에 대한 봉합사의 종류의 선택은 동물의 체중에 따라 다르다. </li><li> 실버 펜을 사용하여 실리콘 코팅 된 선단부터 9mm의 봉합사를 표시한다. 이 삽입 길이에 대한 지침을 제공합니다. </li></ol> 수술 2. 준비 <ol><li> 실험실 마취 시스템을 이용하여, 산소와 혼합하여 이소 플루 란 마취 마우스. 상업 시스템에 2에서 5와 산소의 흐름을 설정에서 이소 플루 란을 사용 (재료 표 참조). surger에 동물 이동Y면과 코 콘을 사용하여 마취를 유지 (1.5 설정을 사용 이소 플루 란 - 2 2.5과 산소의 흐름을). <ol><li> 초당 2 호흡을 헐떡없이 - 마우스 호흡 속도가 약 1이 있는지 확인합니다. 또한, 동물 수염 자극 반응과 페달 반사 (발가락 핀치)를 발생하지 않는 것을 확인합니다. , 수술 동안 적어도 매 5 분 호흡 속도와 노력을 모니터링합니다. </li></ol></li><li> 절차 동안 건조되는 것을 방지하기 위해 멸균 면봉을 사용하여 각각의 눈에 대한 안과 용 윤활제 방울을 넣어. 또한, 멸균 제약 튜브에서 눈에 안과 연고를 적용합니다. </li><li> 그 뒷면에있는 동물 플립과 절개의 영역을 면도. 그 후, 충분히 70 % 클로르헥시딘 다음 에탄올, 70 % 에탄올로 최종 면봉을 사용하여 수술 영역 소독. 실체 현미경 아래에서 따뜻한 수술 표면에 동물을 배치합니다. </li></ol> 3. 경동맥의 해부 및 내부 / External 분기 <ol><li> (- 4cm 약 3) 수술 가위와 집게를 사용하여 턱 아래에 가슴 뼈 위에서, 목에 얕은 중간 선 절개를 수행합니다. 외과 용 드레이프 비 멸균 표면과 접촉하는 수단을 방지하기 위해 수술 주변에 배치한다. 수술 드레이프가 촬영을 돕기 위해 현재 비디오 생략되었다. </li><li> 집게 신중 별도 지방 및 결합 조직을 사용하여 기관을 노출시킨다. 조직 쉽고 자연스럽게 양쪽으로 분리해야합니다. </li><li> 상기 수술 영역을 노출 네크를 확장 쥐의 목 뒤쪽에 베개 (원형 물체 직경 약 0.5 cm)을 놓는다. </li><li> 조직 트랙터 또는 후크를 사용하여 절개를 엽니 다. </li><li> 기관의 동물의 왼쪽에서 조심스럽게 왼쪽 경동맥 (CCA)를 노출하는 결합 조직을 분리 tweeze. CCA를 함께 번들로 신경 및 주요 혈관 손상되지 않도록주의하십시오. </li><li> 기본 조직에서 분리주의하면서 CCA 노출 계속 내부 대뇌 동맥 (ICA) 및 외부 대뇌 동맥 (ECA)의 분기 최고 &quot;Y&quot;를 노출합니다. CCA에서 분리 돕기 위해, 결합 조직을 관통하는 CCA에서 곡선 집게를 삽입하고 천천히 열 수 있습니다. </li></ol> MCAO 봉합 삽입을위한 CCA 4. 준비 <ol><li> CCA를 아래 길이가 약 4cm의 나일론 봉합사의 3 세그먼트를 삽입합니다. 이 크게 봉합의 삽입을 복잡하게하는 것처럼 CCA는 트위스트하지 있는지 확인하십시오. </li><li> 가장 낮은 지점 수에서 영구 매듭을 사용하여 바닥 봉합을 닫습니다. </li><li> 단지 ICA 아래 상단 봉합을 묶어 / ECA는 이동식 슬립 매듭을 사용하여 분기. </li><li> 이동식 슬립 매듭을 사용하여 중간 봉합을 묶어하지만 활짝 열려 보관하십시오. 봉합 삽입을 방해하지으로는 충분한 공간을두고하는 것이 중요하다. </li><li> 미세 절제 봄 공상을 사용하여ssors, 하단 및 중앙 봉합 사이 CCA에 절개를 수행합니다. 하단 봉합에 가까운 0.2 mm 절개를 수행합니다. </li></ol> 5. 중간 대뇌 동맥 폐색 <ol><li> 집게를 사용하여 정상에 그것을 안내는 CCA 절개로 MCAO 봉합사를 삽입합니다. CCA를 개방 응고 및 폐쇄를 방지하기 위해 4.5 단계 후 신속하게이 단계를 수행합니다. 봉합 삽입이 차단되는 경우에, 절개을 다시 포셉의 팁을 사용합니다. </li><li> 조심스럽게 주위에 혈액의 흐름을 제한하는 슬립 매듭을 사용하여 MCAO 봉합 실리콘 부분에 중간 봉합을 묶어하지만 자유롭게 이동할 수 있도록 충분히 느슨한. </li><li> 조심스럽게 MCAO 봉합 밖으로 빠져 있지 않은지 확인하고, 상단 봉합을 취소. </li><li> 몇 mm하여 ICA의 MCAO 봉합사를 삽입하고, 단계 5.2에 기재된 바와 같이 후 동일한 방법으로, 가기 봉합사를 리 클로즈. </li><li> 폐색 영역으로 MCAO 봉합을 안내합니다. 성공적으로 삽입의 표시이다실버 9mm의 마크 CCA 절개의 혈액 역류의 낮은만큼 그 증거는 CCA 절개와 ICA / ECA의 분기 사이에 위치해 있습니다. 성공적인 폐색 추가 확인은 레이저 도플러 혈류 모니터링 시스템 (15, 16)로 모니터링 방법을 사용하여 얻을 수있다. 주 : 폐색 영역 측의 중간 뇌 영역으로 혈액 흐름의 90 %의 저하가 성공적으로 폐색을 나타낸다. </li><li> 성공적인 폐쇄 후 단단히 중간과 상단 봉합을 묶어. 가짜 삽입을 수행하는 경우, 봉합 리 클로즈 대신 즉시 7.3 단계로 이동하지 않습니다. </li></ol> 6. 절개 결산 및 수술 후 케어 <ol><li> 절개 영역으로 봉합에 정력. </li><li> 트랙터 또는 조직 후크와 베개를 제거합니다. </li><li> 멸균 생리 식염수와 면봉을 사용하여 청소 봉합 영역입니다. </li><li> 나일론 봉합사 / 바늘과 집게를 사용하여 절개를 닫습니다. </li> &lt;리&gt; 관리] 항 염증 (예를 들어, 카프로 펜 (10) ㎎ / ㎏, 사우스 캐롤라이나) 및 진통제 (예, 부 프레 놀핀 0.1 ㎎ / ㎏ 사우스 캐롤라이나, 하루에 두 번) 약물은 수술 후 불편 함을 완화한다. 저체온증을 방지하고 물과 연화 식품 광고 labium 액세스 권한을 부여하기 위해 가열 패드에 배치 새장에 마우스를 놓습니다. </li><li> (- 10 분 5)과 뇌졸중의 징후 동물의 복구를 모니터링합니다. 그들은 가벼운 측면 마비에서 다양하고 반대쪽 측면 및 압연의 심한 수축에 돌고 있습니다. 여러 평가 점은 토론에 나열되어 있습니다. 호흡 곤란이나 심한 발작의 징후를 표시하는 동물은 안락사되어야한다. </li><li> 허혈 재관류 프로토콜에 따라, 120 분 이상 30 분에서 장소에 MCAO 봉합을 둡니다. 폐쇄 시간을 줄이면 사망을 방지 할 수 있습니다. </li><li> 영구적으로 폐색하지만 심각한 사망률 예상 할 수 24 시간 동안 제자리에 MCAO 봉합사를 떠난다. 이 시간 경과 후, 인트 진행페이지 7. <ol><li> 영구 폐쇄 모델을 사용하는 경우, 종점까지 새장에 마우스를 유지. 이 때, (7.8-7.1 단계) 봉합사를 제거합니다. 이 절차는 안락사 후에 수행 될 수있다. </li></ol></li></ol> 7. 재관류 <ol><li> (2.1 지침에 따라) 다시 동물을 마취하고 상처 폐쇄 봉합을 제거합니다. </li><li> 집게 및 조직 분리, 절개를 다시 열고 CCA 노출을 사용. </li><li> 조심스럽게 상단 봉합사를 제거하고 실리콘 코팅 된 부분이 중간 매듭에 위치 할 때까지 부드럽게 MCAO 봉합을 잡고 분리합니다. </li><li> (이것은 슬립 매듭 일 필요는 없다) 봉합사를 통과 흐르는 혈액을 방지하기 위해 봉합 매듭의 최상위 재실행. </li><li> 조심스럽게 중간 매듭을 취소하고 정상 매듭 과거 봉합사를 당겨하지만 CCA 내부에 보관하십시오. </li><li> 단단히 동맥 혈류를 차단 가기 매듭을 닫는다. </li><li> 완전히 MCAO 봉합을 당겨 단단히 중간 매듭을 닫습니다. </li><li> MCAO 봉합이 될 수 있습니다여러 번 재사용. 사용 후, 멸균 거즈를 사용하여 오염 물질 (예를 들면, 혈액 또는 조직)를 제거하기 위해 70 % 에탄올에 조심스럽게 세척 봉합사. 그 후, 멸균기 파우치에 봉합을 배치하고 소독. </li><li> 6.4 동물 복구를 모니터링 - 반복 6.1 단계를 반복합니다. </li></ol> 8. 조직 분석 <ol><li> 뇌졸중 볼륨을 모니터링 뇌를 해부합니다. <ol><li> 두 단계 공정을 사용하여 마우스를 안락사. 첫째, 정지 호흡 할 때까지 이소 플루 란의 과다 복용으로 동물을 노출. TTC 염색의 경우, 즉시 마우스를 목을 벨 뇌 추출로 진행합니다. 절편 뇌 (면역 염색 분석 등을 위해)의 경우, 잘린 이전에 심장 천자를 통해 관류를 수행합니다. </li><li> 두개골의 기지에서 머리를-잘라과 눈 사이까지 두개골의 상단에 목 구멍에서 두피를 잘라. 두개골을 노출하면, 두개골베이스 개도부터 양측을 따라 뼈 절단눈 소켓까지. 두개골의 넓은 부분을 따라이 상처를 확인합니다. 뇌의 손상을 방지하기 위해 가위로 너무 깊이에 도달하지 않도록주의하십시오. </li><li> 그 후, 두 눈 소켓 사이의 뼈를 잘라. </li></ol></li><li> 집게를 사용하여 뇌를 노출 두개골 상부를 들어 올려. 단지 작은 저항이 있어야합니다. 쉽게 들지 않는 경우, 두개골 절개가 불완전 할 수 있습니다. 부드럽게 무딘 곡선 집게를 사용하여 뇌를 올립니다. 뇌는 여전히 여러 가지 신경에 의해 부착 될 수 있습니다. 길을 따라 첨부 파일을 제거, 천천히 진행합니다. </li><li> , 1mm의 뇌 매트릭스에 신선한 뇌를 놓고 PBS 방울의 몇 가지를 추가하고, 면도날 또는 저온 유지 장치 블레이드를 사용하여 행렬을 따라 슬라이스. </li><li> 100mm의 접시에 옮기고 뇌 접시의 저면을 덮도록 염색 용액 (2 % 2,3,5- 트리 페닐 테트라 졸륨 클로라이드 (TTC)] PBS에 용해)을 추가한다. 두 개의 집게를 사용하여 별도의 모든 뇌 조각은 (앞면이 아래로 향하게)과 접시의 바닥에 순서대로 배치합니다. 참고 : 염색이 아닌 살아있는 조직은 흰색, 나머지 빨간색 살아있는 조직을 켜집니다. 37 ° C에 대한 해결책을 따뜻하게하는 것은 염색을 가속화 할 것이다. 행정 영역 (흰색)의 징후는 90 분 후 스트로크 빨리 볼과 7 일 이상 계속 표시 할 수 있습니다. 영상은 뇌졸중 볼륨의 계산을 할 수 있도록 통치자와 함께 수행해야합니다. </li><li> 단면 처리를위한 프로세스 해부 두뇌, 면역 염색, 단백질 분리, 또는 다른 절차 (17)의 다양한, 18, 19. </li></ol>

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M., McCabe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+gender+on+'tissue+at+risk'+in+acute+stroke:+A+diffusion-weighted+magnetic+resonance+imaging+study+in+a+rat+model+of+focal+cerebral+ischaemia.">The influence of gender on 'tissue at risk' in acute stroke: A diffusion-weighted magnetic resonance imaging study in a rat model of focal cerebral ischaemia.</a> J Cereb Blood Flow Metab. 36 (2), 381-386 (2016).</li><li>Cai, Q., 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=Co-transplantation+of+hippocampal+neural+stem+cells+and+astrocytes+and+microvascular+endothelial+cells+improve+the+memory+in+ischemic+stroke+rat.">Co-transplantation of hippocampal neural stem cells and astrocytes and microvascular endothelial cells improve the memory in ischemic stroke rat.</a> Int J Clin Exp Med. 8 (8), 13109-13117 (2015).</li><li>Cheng, Y., 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=Intravenously+delivered+neural+stem+cells+migrate+into+ischemic+brain,+differentiate+and+improve+functional+recovery+after+transient+ischemic+stroke+in+adult+rats.">Intravenously delivered neural stem cells migrate into ischemic brain, differentiate and improve functional recovery after transient ischemic stroke in adult rats.</a> Int J Clin Exp Pathol. 8 (3), 2928-2936 (2015).</li><li>Nagai, N., 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=Intravenous+Administration+of+Cilostazol+Nanoparticles+Ameliorates+Acute+Ischemic+Stroke+in+a+Cerebral+Ischemia/Reperfusion-Induced+Injury+Model.">Intravenous Administration of Cilostazol Nanoparticles Ameliorates Acute Ischemic Stroke in a Cerebral Ischemia/Reperfusion-Induced Injury Model.</a> Int J Mol Sci. 16 (12), 29329-29344 (2015).</li><li>Liu, Y., 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=Intravenous+PEP-1-GDNF+is+protective+after+focal+cerebral+ischemia+in+rats.">Intravenous PEP-1-GDNF is protective after focal cerebral ischemia in rats.</a> Neurosci Lett. 617, 150-155 (2016).</li><li>Hedna, V. 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Authors have no competing or conflicts of interest.

상술 MCAO 방식의 성공적인 이용은 뇌 혈류 해부학의 이해에 크게 의존한다. 봉합의 정확한 위치 인한 직접적인 시각적 단서의 부족으로 분별하기 어려운 때문에 반복 연습 조사 연구에 사용하기 전에 절차를 습득하는 것이 중요하다. 스트로크 볼륨은 일관된 결과를 보장하기 위해 분석되어야한다. 레이저 도플러 시스템의 첨가는 혈류의 성공적인 폐색을 결정하는 데 도움이되고 절차가 정확하게...

심혈관 질환의 연구, 뇌졸중과 같은 생체 내 모델의 사용에 의존한다. 허혈, 약물의 독성 및 / 또는 치료 가능한 의미를 이해하기 위해, 처리 군 사이의 비교 연구를 가능하게하는 질환의 적합한 표준화 된 신뢰성 및 재현성이 모델을 사용할 필요가있다. 이 논문에서는, 트랜스 제닉 마우스 표준화 평가 모델의 다수의 가용성 주어진 쥐를 사용한다. 실험 허혈성 뇌졸중과 같은 복구 다음 모터와 행동 적자를 평가하는 점수를 긁어하면, 2 1 개발되었다. 여러 허혈성 뇌졸중 모델은 전체 글로벌 대뇌 허혈, 불완전 글로벌 허혈, 다 초점 대뇌 허혈 및 국소 뇌허혈로 사용할 수 있습니다. 후자의 그룹은 또한 환자의 가장 일반적인 뇌졸중의 범주입니다. 이브의 대부분국세청은 또는 중간 대뇌 동맥 (MCA) 가까이에서 색전이나 혈전 성 폐색의 형성에 의해 시작됩니다. 이러한 매개 변수를 감안할 때, 밀접하게 제시된 모델은 인간의 뇌졸중 질환의 원인을 모방하고 관련성이 높은 3 얻어진 결과를 만든다. 그럼에도 불구하고, 인간의 질병 치료에 동물 모델에서 발견의 번역은 어려운 것으로 입증되었습니다. 지금까지 혈전 조직 플라스 미노 겐 활성제의 사용은 급성 허혈성 뇌졸중 (4)의 치료를 위해 승인되었다. 쥐에서 국소 뇌허혈 후방 대뇌 순환 뇌졸중 모델 및 대뇌 정맥 혈전증 모델의 모델마다 적용 가능성을 감소 및 분석을 수행 할 수의 범위를 제한하는 매우 침습적이다. 그러나, 예 색전증 모델 photothrombosis 모델 엔도 텔린 -1 유발 뇌졸중 모델 및 관내 봉합 중간 대뇌 동맥 폐쇄 (M 다른 기법CAO) 모델은 이러한 제한없이 사용할 수 있습니다. MCAO 모델이 프로토콜에 기재된 기술이다. 이것은 재관류 용이하고 높은 처리량 방법으로 수행 될 수있다 국소 뇌허혈을 유발하는 신뢰할 수있는 방법을 제공한다. 이 모델, 즉, ZEA - 롱가와 고이즈미 방법에 두 가지가 있습니다. 그들은 폐쇄 봉합이 혈관에 삽입하는 방식에 약간의 차이가 있습니다. ZEA-롱가 기술에서, 봉합 외부 경동맥 (5)을 통하여 삽입된다. 여기에 제시된 기술은 흡장 봉합사가 경동맥 (6)를 통해 삽입되는 고이즈미 방법에서 수정된다. MCAO 모델이 성공적으로 허혈성 뇌졸중 동안 발생하는 다양한 이벤트를 평가하기 위해 적용되었다. 재관류 후, 뇌 부종은 혈액 - 뇌 장벽의 고장에 따라 관찰 될 수있다. 피크 신경 사망은 보통 24 시간에서 관찰되고; 그러나, 다시칠일 7 후 기준선 수준으로 변합니다. 인간에서, 성별과 연령이 스트로크의 결과를 결정할 때, 이것은 또한 마우스 및 래트도 8,도 9,도 10에서 관찰되는 중요한 변수이다. 몇몇 간행물은 처리 효율 11, 12, 13, 14을 설명하기 MCAO 모델을 사용했다.

<table border="0" cellpadding="0" cellspacing="0" width="544">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MCAO suture 0.23mm</td>
			<td style="width:149px;">Doccol</td>
			<td style="width:119px;">702345PK5Re</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MCAO suture 0.21mm</td>
			<td style="width:149px;">Doccol</td>
			<td style="width:119px;">702145PK5Re</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Silver pen</td>
			<td style="width:149px;">staples</td>
			<td style="width:119px;">503205</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Anesthesia machine</td>
			<td style="width:149px;">Vetequip</td>
			<td style="width:119px;">901806</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Surgical scissors</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">14558-09</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Surgical forceps straight tip</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">00108-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Surgical forceps angled tip</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">00109-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Spring scissors</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">15000-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Nylon suture</td>
			<td style="width:149px;">Braintree scintific</td>
			<td style="width:119px;">SUT-S 104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:149px;">FisherSci</td>
			<td style="width:119px;">50-121-8005</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Brain block</td>
			<td style="width:149px;">Braintree scintific</td>
			<td style="width:119px;">BS-A 5000C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Cryostat blade</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">89202-606</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Optional:</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Periflux Laser doppler system</td>
			<td style="width:149px;">Perimed</td>
			<td style="width:119px;">Periflux 5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Monitoring unit</td>
			<td style="width:149px;">Perimed</td>
			<td style="width:119px;">PF 5010 - LDPM</td>
		</tr>
	</tbody>
</table>

induction of ischemic stroke and ischemia-reperfusion in mice using the middle artery occlusion technique and visualization of infarct area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

폐색 봉합의 삽입 경로는 그림 1에서 설명된다. MCAO 봉합사는 ICA 용 분기로, 폐색 영역으로 라우팅 될 것이다. MCA의 성공적인 폐쇄는 TTC 염색으로 표시 조직 손상으로 이어질 것입니다. 그림 2 (90 분 또는 24 시간 이후 폐쇄,도 2b에 염색) 가짜 치료 동물 (그림 2A) 및 60 분 MCAO 허혈 재관류 동물에서의 염색의 이미지를 제공합?...

Watch this Scientific Journal Video about Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area at JoVE.com

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

경색 지역의 중간 동맥 폐색 기술 및 시각화를 사용하여 마우스의 허혈성 뇌졸중 및 허혈 - 재관류 유도

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

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16.2K 조회수.  University of Miami. We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

비디오: Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

마우스의 모델링 스트로크 - 필라멘트 모델 중간 대뇌 동맥 폐색

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

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Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

중간 대뇌 동맥 폐색의 마우스 모델

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

마우스의 레이저 도플러 Flowmetry 안내와 허혈성 뇌졸중에 대한 중간 대뇌 동맥 Intraluminal 폐색 (MCAO) 모델

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

초점 대뇌 국소 빈혈에 조기 허혈성 공차를 유도하기 위해 적절한 전처리 자극으로 양측 경동맥 동맥 폐색

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

쥐의 레이저 도플러 Flowmetry 안내와 허혈성 뇌졸중에 대한 엔도 테린-1 유도 중간 뇌 동맥 폐색 모델

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

엔도 테린-1 유도 중간 뇌 동맥 폐색 동안 중동 뇌동맥을 시각화하기 위해 두개골 창 활용

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Intraluminal 중간 대뇌 동맥 폐색의 쥐 뇌졸중 모델에서 뇌 관류 모니터링에 최적화 된 시스템

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

마우스의 모델링 스트로크 : 말초 중간 대뇌 동맥의 영구 응고

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

흰쥐의 동종 혈액 혈전과 허혈성 뇌졸중에 대한 전성 중간 대뇌 동맥 폐색 (MCAO)

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

마우스의 반복 저산소 전처리 및 과도 중간 대뇌 동맥 폐쇄 후 신경 혈관 보호의 정량화

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...