This work was funded by the National Institute of Health, the National Heart Lung and Blood Institute (NIH and NHLBI; HL125572-01A1) and the LSUHSC-S start up fund to F.N.E. Gavins.

비디오에보고 된이 프로토콜과 실험은 LSUHSC-S 기관 동물 관리 및 사용위원회의 승인을 NIH의 지침을 준수 하였다. 주 : 남자가 C57BL / 6 마우스 25 중량 - 29g이 연구에 사용되었다. 쥐 개별적으로 환기 케이지에서 12 시간 빛 / 어둠 사이클에서 물을 무료로 이용할 수있는 표준 우 펠릿 다이어트를 유지 하였다. 과정은 무균 기법 (예, 멸균 장갑, 멸균 기기)를 사용하여 멸균 조건 하에서 수행한다. 1. 수술 전 준비 <ol><li> 케타민 (150 ㎎ / ㎏)과 자일 라진 (10 ㎎ / ㎏)의 조합을 사용하여 마취를 유도 복강 내 (IP)을 주입. 마취가 달성되면 5 분마다 10 분 - 처음 발 핀치 때마다 (3)에 의해 마취의 깊이를 모니터링합니다. 동물이 마취 동안, 건조를 방지하기 위해 살균 안 연고를 관리. 케타민의 초기 투여 량 / 자일 라진 보통 AB 지속30-40분 아웃. 발 핀치에 대한 동물의 반응에 의해 확인되는, 필요한 경우 추가의 투여 량으로 투여 될 수있다. </li><li> 온도 조절 열 매트에 누운 위치에 마우스를 놓고 직장 프로브를 사용하여 검증 36.5 ± 0.1 ° C,에서 체온을 유지한다. </li><li> 목 면도하고 70 %의 에틸 알코올로 피부를 소독. </li></ol> MCA 2. 폐색 (그림 1) <ol><li> 아이리스 똑바로 위를 이용한 중심선 경부 절개를하고, 혈관이 노출되도록 연조직 (견인기를 사용하여) 후퇴. </li><li> 미주 신경에 손상을주지 않고 뒤몽의 집게를 사용하여 주변 조직에서 CCA와 ECA를 해부하다. </li><li> 6-0 실크를 사용하여 CCA ​​주위에 느슨한 매듭을 묶는에 의해 임시 봉합을합니다. </li><li> ECA 주위에 영구 봉합 단단히 선박합니다 (CCA의 분기점에 말초)을 결찰하여로부터 연장 작은 혈관을합니다. </li><li> 봉합 ARO 확인CCA의 분기점에 ECA의 근위 핀클. </li><li> 내부 경동맥 (ICA)와 pterygopalatine 동맥 (PPA) 주변의 미세 혈관 클립을 배치합니다. </li><li> 봄 가위를 해부 마이크로를 사용하여 ECA에 작은 절개를하고 180 μm의 실리콘 스쳐 모노 필라멘트를 삽입합니다. 필라멘트 쉽게 조작 가능한 영구 봉합에 상처가 근접합니다. </li><li> 삽입 된 필라멘트와 ECA 주위에 임시 봉합을 강화하고 미세 혈관 클립을 제거합니다. </li><li> 영구, 말초 봉합과 봄 가위를 해부 마이크로를 사용하여 필라멘트의 엔트리 포인트 사이의 ECA를 잘라. </li><li> MCA에 -합니다 (CCA의 분기점을 넘어 10 μm의. 약 9) 저항이 느껴질 때까지 ICA를 통해 필라멘트를 안내합니다. 참고 : 필라멘트의 대부분이 여전히 표시되는 동안 너무 많은 저항이 느껴지는 경우는, 필라멘트는 PPA를 입력 할 수 있습니다. 이 경우, 부드럽게 고름, 분기점으로 다시 필라멘트를 당겨전방 ICA 사용 듀 핀셋으로 H이고, 그것이 ICA에서 가시화 될 수있을 때까지, 필라멘트를 전진. </li></ol> 3. 재관류 <ol><li> 30 분 폐색 기간 후에 다시 부드럽게 당겨 듀 핀셋을 사용하여, 필라멘트를 분리하고 ECA의 개방 단부 주위에 봉합 고정. </li><li> 조심스럽게 CCA를 통해 재개 뒤몽 포셉과 혈액 흐름을 사용하여 합자를 풀어 CCA 주위에 임시 봉합을 제거합니다. </li><li> 연속 수술 봉합과 절개를 닫습니다. 피부의 폐쇄 중 연속적 또는 중단 봉합에 의해 달성 될 수있다. 스킨 스테이플은 허용 방법입니다. </li><li> 볼륨 보충으로하고 진통제 카프로 펜과 염분 피하의 1 ml의 쥐를 주입 (5 밀리그램 / kg, 사우스 캐롤라이나) 수술, 추가로 통증이 다시 웅크 리고 앉게 등이 필요하다 표시 간판, ungroomed 코트에서 통증과 불편 완화를 위해, 감소 활동, 이상자세, 식욕 감소했다. </li><li> 온도 컨트롤러를 사용 규제 열 램프를 사용하여 가열 된 30 ° C를 케이지에 마취에서 회복에 걸쳐 쥐를 관찰하고 식습관을 장려하기 위해 새장 바닥에 페트리 접시에 으깬 우를 배치합니다. 마우스는 재관류 기간 동안 케이지에 하나씩 보관됩니다. </li></ol> 4. 위장 수술 <ol><li> 모노 필라멘트의 삽입없이 동일한 방법에 따라 마우스. </li></ol> 5. 수술 후 신경 학적 점수 (표 1, 그림 2) <ol><li> 일반 신경 학적 평가를 18 점의 점수 시스템을 사용하여 적절한 재관류 기간 후에 마우스를 평가; 모터; 감각; 감수. 높은 신경 학적 점수는 신경 기능 저하에 대응한다. 참고 : 걸을 응답과없는 판단 마우스는 안락사됩니다. 인간 안락사 다른 기준보다 큰 20 %의 중량 손실을 포함 호흡 디수술 주변 스트레스, 감염. 공동이 챔버를 경추 탈구 또는 개흉술 것 죽음을 확인 안락사 물리적 방법에 사용된다. </li></ol> 측정 뇌 경색 볼륨 6. (그림 3) <ol><li> 마취가 달성되면, 5 분 및 10 분마다 - 케타민 (150 ㎎ / ㎏)과 자일 라진 (10 밀리그램 / kg) 초기 발 핀치마다 (3)에 의해 마취 IP 모니터 깊이 주입의 조합을 사용하여 마취를 유도한다. </li><li> 부정사 위치에 마우스를 놓고 조리개를 바로 가위를 사용하여 복막 뒤에 목에 복부의 피부를 잘라. </li><li> 뒤몽의 집게를 사용하여 흉골을 들어 올려 가슴을 노출 갈비뼈를 잘라. </li><li> 심장을 노출 좌측 및 지혈 두 쌍을 이용하여 상자의 오른쪽을 연다. </li><li> 좌심실에 5 ML의 주사기에 부착 된 26 G 바늘을 삽입하고 우심방를 잘라. 실온 생리 식염수 또는 포스으로 관류TE 완충 식염수 (PBS) 유체가 클리어 될 때까지 (일반적으로 3-5 분). </li><li> 아이리스에게 바로 위 및 뒤몽의 집게를 사용하여 조심스럽게 뇌에서 두개골을 제거합니다. </li><li> 후각 전구 및 뇌 행렬에 맞도록 면도날을 사용하여 소뇌를 잘라. 심지어 세그먼트로 뇌를 슬라이스이 행렬을 사용합니다. 멋진 조직을 유지하기 위해 사용하기 전에 행렬을 진정. 매트릭스로 뇌를 놓고 얼음에 설정합니다. </li><li> 두 개의 면도날을 사용하여 2mm 관상 세그먼트로 뇌를 슬라이스. 위로부터 2mm 시작 면도날을 사용하여 상기 제 절단하고 대신이 면도날을 떠난다. 제 뒤에 잘라 다른 2mm를 확인합니다. 부착 된 조직과 첫 번째 면도날을 제거합니다. 모든 조직이 분리 될 때까지이 과정을 반복합니다. 참고 : 4가 있어야합니다 - 완료되면 5 세그먼트. </li><li> 다음 얕은 물 목욕 (A)에 배치되어 2 % 2,3,5- triphenyltetrazalium 클로라이드 (TTC)를 포함하는 24 웰 플레이트에 세그먼트를 배치20 분 동안 t 37 ° C를. 개별 웰에 각각의 세그먼트를 배치하는 것은 그들이 절단 된 순서에 유지하도록 돕는다. TTC의 솔루션은 완전히 조직 세그먼트를 커버 있는지 확인합니다. 10 분 모든 조각을 넘겨 후. </li><li> 새로운 24- 웰 플레이트의 웰에 10 % 포르말린 소량을 배치하고 절단 된 순서로이 플레이트 세그먼트를 전송. </li><li> (1.57 NIH 이미지 소프트웨어) 6 컴퓨터로 세그먼트를 스캔 ImageJ에 분석 소프트웨어를 이용하여 전체 뇌 조각 (6)의 비율로 경색 크기 분석. <ol><li> 면적 측정을 생성하기 위해 경색 영역을 설명합니다. 다음 지역을 결정하기 위해 전체 반대쪽 반구를 측정한다. 반대쪽 반구의 경색 면적의 영역을 분할하고, 경색 량을 결정하기 위해 100을 곱한다. </li></ol></li></ol>

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The authors have nothing to disclose.

전에 20 년의 개념 때문에, 필라멘트의 삽입을 포함하는 인간의 뇌졸중에 대한 MCAO 모델은 연구의 거대한 숫자에 사용되어왔다. 이는 그 스트로크 (즉, 뇌졸중)의 가장 일반적인 형태로 임상 적으로 일어나는 모방한다는 사실에 주로있다. 선조체는 대뇌 피질의 허혈보다 더 민감하고, 같은 허혈성 시간의 길이는 선조체 및 배면 측면 피질 모두 영향하거나 선조체할지 여부로 번역된다. 두 경...

뇌졸중은 한 사람이 질병으로 매 4 분 죽어으로, 전 세계적으로 죽음의 다섯 번째 주요 원인이다. 80 만 이상의 미국인은 환자에 대한 파괴되지 않는 행정 매년, 고통뿐만 아니라 그 가족. 스트로크 성인 장애의 주요 원인이며, 세출이 가능한 것으로 매우 적은 치료법에도 불구 $ 36.5 억의 순서로 추정된다. 조직 플라스 미노 겐 활성제 (TPA)의 유일한 식품의 약국 (FDA)은 허혈성 뇌졸중에 대한 약물을 허가합니다. 뇌졸중의 발병에서 3~6시간 이내에 환자에게 투여하고, 이러한 경우에 환자 (2)의 4 % 혜택 그러나에만 효과적이다. 따라서, 선 재현성, 임상 적으로 중요한 동물 모델이 질환에 대한 잠재적 인 치료 전략 및 치료의 개발에 도움을 사용하는 것이 필수적이다. 체외에서 점에 유의하는 것이 중요하다 </EM&gt; 모델은 뇌 기능 장애의 특정 측면을 모델링에 유용한 반면, 뇌졸중 다음 뇌와 주변에서 발생하는 복잡한 생리 학적 상호 작용을 recapitulating 할 수 없습니다. 따라서, 생체 내 모델은 필수적이다. 뇌졸중의 가장 흔한 유형은 전체 뇌졸중의 87 %를 차지 기원 허혈성입니다. 다른 스트로크 뇌출혈 (9 %) 및 지주막 하 출혈 (4 %)이며, 중간 대뇌 동맥 (MCA)에 색전에 의해 가장 자주 발생한다. 이 파괴 될 수있는 뇌를 입력 층류 혈액의 흐름을 발생 MCA의 루트에있는 눈에 띄는 곡선 때문이다. MCA는 가로 세로 홈을 따라 내 경동맥 (ICA) 및 경로에서 발생하는 곳 분기 프로젝트 기저핵 및 전두엽, 정수리 일차 운동 및 감각 피질 포함 측두엽의 측면. 윌리스의 원이되는 후부 대뇌 동맥에 의해 생성된다대뇌 동맥과 후부 통신 동맥에 연결. MCAO의 관내 필라멘트 또는 봉합 모델은 가장 널리 스트로크 연구에서 사용 중 하나입니다. 그러나,이 모델의 다른 변형 몇 있으며, 이들은 미사 외부 경동맥에 삽입되는지 여부에 기반 3 (ECA를 상기 롱가 방법 지칭), 또는 그것은 (ICA에 삽입되어 있는지 고이즈미 지칭 방법) 4. 필라멘트는 CCA의 절개 출혈 방지하기 위해 제거되는 경우 롱가의 방법에서 영구적 5 연결되어야 ECA 인 반면 고이즈미 방법에있어서, 상기 수술 측의 경동맥 (CCA)을 영구적으로 연결되어야 . 우리는이 훨씬 우수하고 허혈성 뇌졸중의보다 임상 적으로 수술 모델 느낌으로 여기 론가 방법이 사용됩니다. 또한, 특히 롱가 법으로 규소로 만들어진 필라멘트의 사용은 매우 생산종종 불완전 흡장 및 / 또는 지주막 하 출혈 (6)을 생성 화염 무딘 모노 필라멘트, 반대로 재현 MCAO. 관내 필라멘트 방법은 영구적 또는 일시적인 흡장 4,6의 모델로서 사용될 수있다. 과도 모델을 수행하기 위해 필라멘트는 허혈 기간 후에 제거된다 (예를 들어, 30 분, 60 분, 2 시간), 재관류가 일어날 수있다. 이 모델은, 어느 정도는 인간에서 혈전 혈전을 용해시키기 위해 자연 또는 치료 개입 (예를 들어, tPA의 투여) 후 혈류의 복원을 시뮬레이션한다. 영구 모델 필라멘트 단순히 일정 기간 (예를 들면, 24 시간) 장소에 남아 있으므로 더 재관류가 발생하지 않는다. 관내 필라멘트 방법의 다른 장점은 개두술 두개골이 그대로 유지 될 수 있도록 두개 내 압력 및 온도의 변경을 방지하여, 수행되어야 할 필요가 없다는 점이다. <p c이 비디오에서 아가씨 = &quot;jove_content은&quot;&gt; 우리는 MCAO 및 재관류를 유도 할 수있는 론가 관내 필라멘트 방법을 수행하는 방법을 보여줍니다. 우리는 또한 18 포인트 신경 학적 점수를 수행하고 2,3,5- triphenyltetrazalium 클로라이드 (TTC) 염색을 사용하여 경색 볼륨을 결정하는 방법을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="1521">
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		<col />
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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Male C57BL/6 mice</td>
			<td style="width:291px;">Jackson Laboratory, Bar Harbor, ME</td>
			<td style="width:397px;">#000664</td>
			<td style="width:397px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Ketamine Hydrochloride</td>
			<td style="width:291px;">Morris &#38; Dickson, Shreveport, LA</td>
			<td>67457-108-10</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Xylazine</td>
			<td style="width:291px;">Akorn, Inc, Lake Forest, IL</td>
			<td>NADA# 139-236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">DC temperature control system</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Mini rectal thermistor probe</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-80-5D-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Heating pad</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-90-2-06</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Clippers</td>
			<td style="width:291px;">Amazon, Bellevue, WA</td>
			<td>#64800</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">70% ethanol</td>
			<td style="width:291px;">Worldwide Medical Products, Bristol, PA</td>
			<td>#51011023</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Dissecting microscope</td>
			<td style="width:291px;">Olympus, Center Valley, PA</td>
			<td>SZ40</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Iris scissors (straight)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Dumont forceps (45&#176; bent tip)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Micro vessel clip</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>18055-05</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Micro dissecting spring scissors (straight)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>14088-10</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Retractors (blunt)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>18200-11 (Helen used 17022-13)</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Cotton tipped applicators</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>23-400-100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Gauze sponges</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>#9023</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">6-0 silk braided surgical suture</td>
			<td style="width:291px;">Roboz, Gaithersburg, MD</td>
			<td>SUT-1073-11</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">0.9% sodium chloride</td>
			<td style="width:291px;">Morris &#38; Dickson, Lake Forest, IL</td>
			<td>0409-4888-20</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">6-0 medium MCAO suture (silicon rubber coated monofilament)</td>
			<td style="width:291px;">Doccol Corporation, Sharon, MA</td>
			<td>6023PKRe</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Sofsilk 6-0 silicone coated braided silk</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>SUT-14-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Carprofen</td>
			<td style="width:291px;">Pfizer, New York, NY</td>
			<td>NADA# 141-199</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Puralube</td>
			<td style="width:291px;">Dechra, Norwich, UK</td>
			<td>NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Physitemp temperature controller</td>
			<td style="width:291px;">Harvard Apparatus, Holliston, MA</td>
			<td>TCAT-2AC</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Heat lamp</td>
			<td style="width:291px;">Harvard Apparatus, Holliston, MA</td>
			<td>HL-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Laser doppler probe</td>
			<td style="width:291px;">AD Instruments, Colorado Springs, CO</td>
			<td>MSP100XP</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">24-well plates</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>#353226</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Phosphate buffered saline (PBS)</td>
			<td style="width:291px;">Life Technologies, Carlsbad, CA</td>
			<td>20012-050</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Single edge razor blades</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">2,3,5-triphenyltetrazalium chloride (TTC)</td>
			<td style="width:291px;">Sigma Aldrich, St. Louis, MO</td>
			<td>T8877-50G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Mouse brain matrix slicer</td>
			<td style="width:291px;">Braintree Scientific, Braintree, MA</td>
			<td>BS-A 5000C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Water bath</td>
			<td style="width:291px;">VWR, Radnor, PA</td>
			<td>#182</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">10% formalin</td>
			<td style="width:291px;">Sigma Aldrich, St. Louis, MO</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Image J analysis software</td>
			<td style="width:291px;">NIH, Bethesda, MD</td>
			<td>free download</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Retractor</td>
			<td style="width:291px;">Medical Device Purchase, Newcastle, CA</td>
			<td>MP-740</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical approach for middle cerebral artery occlusion and reperfusion induced stroke in mice

스트로크 전세계 주요 사망 원인이며, 장기 성인 장애의 주요 원인 중 하나가되고 있습니다. 뇌졸중의 약 87 %는 원점에서 허혈성하고 중간 대뇌 동맥 (MCA)의 영역에서 발생한다. 현재 전용 식품 의약품 안전청 (FDA)은이 치명적인 질환을 치료하기위한 약물을 승인 조직 플라스 미노 겐 활성제 (TPA)이다. 그러나 만톤 관리를위한 작은 치료 창이 있습니다 (3-6 시간)을, 실제로 그것을받을 환자의 4 % 만 효과가있다. 현재의 연구는 잠재적 인 치료 표적을 발견하기 위해 스트로크 기전을 이해하는데에 초점을 맞춘다. 따라서, 안정적인 모델은 매우 중요하고, MCA 폐색 (MCAO) 모델은 허혈성 뇌졸중의 가장 임상 적 수술 모델로 간주된다 (도 관내 필라멘트 또는 봉합사 모델 지칭) 및 비교적 비 침습적 쉽게 재현 할 수있다. 일반적 MCAO 모델 때문에 특히 생쥐와 설치류 사용이 종에 사용할 수있는 모든 유전 적 변화에. 여기에서 우리는 기술 (비디오에 존재하는) 성공적으로 신뢰성과 재현성 데이터를 생성하는 쥐 (재관류 포함) MCAO 모델을 수행하는 방법에 대해 설명합니다.

마우스는 재관류의 기간 다음에 30 분 MCAO에 의한 뇌 허혈 (그림 1) 시행 (24 시간 및 1 주당 여기에 제시,하지만 재관류의 길이는 변화 될 수있다). MCAO 동안 사망률 (약 2 %)을 최소화했다. 포스트 허혈 (제 24 시간 이내) 사망률은 약 26 %였다. 레이저 도플러 flowmetry는 전 MCAO / 재관류 후 MCA 영토에 혈액의 흐름 관류를 ?...

Watch this Scientific Journal Video about Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice at JoVE.com

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

뇌졸중의 병태 생리를 이해하기 위해, 신뢰할 수있는 모델을 사용하는 것이 중요하다. 마우스에서 가장 자주 사용되는 뇌졸중 모델 중 하나를 설명한다 본 논문의 중간대 뇌동맥 폐색 (MCAO) 모델이라 재관류 (또한 관내 또는 필라멘트 봉합사 모델 지칭).

마우스의 중간 대뇌 동맥 폐색 및 재관류 유도 스트로크에 대한 수술 적 접근

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

surgical-approach-for-middle-cerebral-artery-occlusion-reperfusion

Research

JoVE Journal

Medicine

21.4K 조회수.  Louisiana State University.  뇌졸중의 병태 생리를 이해하기 위해, 신뢰할 수있는 모델을 사용하는 것이 중요하다. 마우스에서 가장 자주 사용되는 뇌졸중 모델 중 하나를 설명한다 본 논문의 중간대 뇌동맥 폐색 (MCAO) 모델이라 재관류 (또한 관내 또는 필라멘트 봉합사 모델 지칭). 

비디오: Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

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 ...

연구

의학

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 ...