이 작품은 NIH 부여 NS057195, UCSF REAC 수상하고, 켄트 주립 대학 시작 기금 WH Chou의에 의해 지원되었다.

<html> 1. MCAO 방법 이 프로토콜은 실험 동물의 사용을위한 건강 지침 국립 연구소에 의해 기관 애니멀 케어 및 사용위원회 UCSF와 켄트 주립 대학에서, 그리고 abides에 의해 승인되었습니다. <ol></ol><ol><li> 20mm 세그먼트에 5-0 monofilament의 봉합을 (하버드 장치, 홀리스튼, MA) 잘라. cauterizer (브레인 트리 과학 주식 회사, 브레인 트리, MA) 근처 가열하여 각 세그먼트의 팁을 라운드. 마이크로 미터 (응용 이미지 주식, 로체스터, NY)를 사용하여 팁의 직경을 측정합니다. 우리는 25-30 G.의 체중을 가진 마우스에 대한 0.21-0.22 mm의 최종 팁 직경과 봉합을 사용하여 </li><li> autoclaving (최소 121 ° C, 15 분 15 PSI).하여 모든 수술 도구를 소독 70 % 에탄올을 사용하여 수술 테이블 및 관련 장비를 살균하십시오. </li><li> 와 8~12주 된 마우스 (25-30그램) 마취 5% 30% O 2 /에서 (Aerrane, 백스터, 디어 필드, IL) isoflurane 70% N V - 10 아나스타샤 시스템을 사용 2 O (VetEquip, Inc의 ., 플레즌턴, CA). 마취의 유도에 따라, isoflurane의 수준을 줄이고 1.5 %에 그것을 유지합니다. </li><li> 가열 패드에있는 위로 향한 위치에 마우스를 놓습니다. 직장 프로브를 삽입하고, 36.5-37.5 사이의 체온 ° C를 사용하여 TR - 200 homeothermic 온도 시스템 (좋은 과학 도구 주식 회사, 포스터 시티, CA).을 모니터링하고 유지 </li><li> 피부를 노출 전기 면도기 (브레인 트리 과학)와 복부 목 지역에있는 모피를 면도. 70 % 에탄올 세 응용 프로그램을 사용하여 외과 사이트를 소독. </li><li> 스테레오 해부 현미경 (니콘, 일본), 목을 1cm 길이 정중선 절개를합니다. 외과 분야를 노출하고 오른쪽 일반적인 경동맥 (CCA), 외부 경동맥의 동맥 (ECA), 그리고 내부 경동맥의 동맥 (ICA)을 식별 견인기 (브레인 트리 과학)를 사용하십시오. 조심스럽게 신경 및 근막 주변에서 무료로 동맥을 해부하다. </li><li> 추가 distally ECA를 해부하다 및 바이폴라 coagulator (하워드 악기 주식 회사, 터스칼루사, AL)를 사용하여 ECA와 뛰어난 갑상선 동맥 (STA) 지점을 응고. coagulated 세그먼트에서 ECA와 STA 컷. </li><li> 대충 ECA 스텀프 통해 두 8-0 실크 봉합을 묶어. ECA와 ICA에 CCA의 분기점에서 혈관 클램프 (파인 과학 도구)을 적용합니다. </li><li> Vannas 스타일 스프링 가위 (파인 과학 도구)와 ECA 유세의 끝부분에 작은 절개를합니다. 측정과 끝에 둥글게 5-0 monofilament의 봉합의 길이를 기록합니다. 절개로 봉합를 삽입하고 클램프에 미리. 충분히 인 주거 monofilament의 봉합의 이동성을 확보 아직 보존 루멘 주위 두 실크 봉합를 조이십시오. </li><li> 분기점에서 클램프를 제거합니다. 부드럽게 MCA의 출처를 막다하기 위해 CCA의 분기점 넘어 90~10밀리미터의 거리에 대한 ICA에 ECA의 루멘에서 monofilament의 치료를 사전. 수술 기간은 30-45 분 정도입니다. </li><li> 목에 절개를 봉합하고 마취에서 회복하고, 우리를 그것을 반환하는 35 ° C 간호 상자에서 마우스를 놓으십시오. 그것은 일반적으로 마취에서 회복하기 위해 생쥐를 위해 50-10 분 정도 소요됩니다. 과도 MCAO를 수행하려면, 연구자들은, 보통 사이 0.5-2 H.에 마우스를 다시 마취하고 일정 기간 후 ECA의 선거 유세에 다시 봉합을 인출할 수 </li><li> 스물 네 시간 MCAO의 유도 후 5 % isoflurane으로 마우스를 마취하고 경추 탈구하여 안락사. 마우스를 참살하고 두뇌를 수집합니다. 얼음 두뇌 매트릭스 (브레인 트리 과학)과 사 2 mm 슬라이스로 coronally 머리를 슬라이스. 2퍼센트의 뇌 조각 실온에서 20 분 1X PBS에서 2,3,5 - triphenyltetrazolium은 염화물 (TTC) (시그마 - 알드리치) 경색의 크기와 범위를 결정하는 부화. 이미징 때까지 중립은 4 포르말린 솔루션 (시그마 - 알드리치) 버퍼 10 % 뇌 조각 ° C를 수정. 조브 프로토콜 955 (에서 설명한대로 경색의 범위는 계량 수 <a href="http://www.jove.com/index/Details.stp?ID=955">http://www.jove.com/index/Details.stp?ID=955</a> ) 9. </li></ol> 2. 대표 결과 <ol></ol> MCAO에 의해 생성된 infarcts는 striatum과 dorsolateral 피질에서 볼 수 있습니다. striatum는 대뇌 피질보다 국소 빈혈에 더 민감합니다. MCAO 이상의 시간의 간격 striatum, 피질 모두를 손상시킬 수있는 동안 MCAO 서른 분 거리에 있으며, 단지 striatum의 경색을 생산합니다. 24 H 영구 MCAO 후, 총 경색의 비율은 40 ~ ± 수술 후 북반구와 사망률의 5 % ~ 10 %입니다. 과도한 출혈 수술하는 동안 발생하면 우리는 더 연구에서 생쥐를 제외, 수술 시간이 90 분 초과, 생쥐는 15 분 이내에 마취에서 회복하기 위해 실패하거나, 출혈이 뇌 조각 또는 사후 심사 중에 윌리스의 원안의 기본에서 찾을 수 있습니다 . jove_content &quot;&gt; <img src="/files/ftp_upload/2761/2761fig1.jpg" alt="그림 1" /> 영구 MCAO 24 H 후 TTC - 스테인드 뇌 조각의 그림 1. 대표 이미지 (코로나 레벨 1-4). 괴사 영역에서 이러한 효소 활동의 부재로 인해 흰색 남아 살아있는 동안에 조직 TTC는 효소, 색상에 빨간색 1,3,5 - triphenylformazan (TPF)에 dehydrogenases에 의해 감소​​됩니다. 따라서, 경색의 지역 TPF에 TTC의 변환의 부족은 백색으로 확인할 수 있습니다. 참고 : TT​​C는 다소 열 등 열과 최대한 빛으로부터 스테인드 섹션을 보호 불안정 조명입니다.

<ol>
	<li>Howard, G., Howard, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Distribution of stroke: hetrogeneity of stroke by age, and sex. , 3-12 (2004).</li><li>Thom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2006+update:+a+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">Heart disease and stroke statistics--2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 113, e85-e151 (2006).</li><li>Mohr, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Middle cerebral artery disease. , 123-166 (2004).</li><li>Longa, E. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20, 84-91 (1989).</li><li>Chou, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+protein+kinase+Cdelta+as+a+mediator+of+stroke-reperfusion+injury.">Neutrophil protein kinase Cdelta as a mediator of stroke-reperfusion injury.</a> J. Clin. Invest. 114, 49-56 (2004).</li><li>Hudgins, W. R., Garcia, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+electrocautery,+atmospheric+exposure,+and+surgical+retraction+on+the+permeability+of+the+blood-brain-barrier.">The effect of electrocautery, atmospheric exposure, and surgical retraction on the permeability of the blood-brain-barrier.</a> Stroke. 1, 375-380 (1970).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2, 396-409 (2005).</li><li>Durukan, A., Tatlisumak, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+ischemic+stroke:+overview+of+major+experimental+rodent+models,+pathophysiology,+and+therapy+of+focal+cerebral+ischemia.">Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia.</a> Pharmacol. Biochem. Behav. 87, 179-197 (2007).</li><li>Taniguchi, H., Andreasson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic-ischemic+encephalopathy+model+of+perinatal+ischemia.">The hypoxic-ischemic encephalopathy model of perinatal ischemia.</a> J Vis Exp. , (2008).</li></ol>

우리는 공개 잠재적인 충돌 관심이 없습니다.

생쥐의 MCAO는 일반적으로 인간에 초점 뇌 국소 빈혈을 모델로 사용됩니다. 뇌졸중 연구 생쥐를 사용하기 때문에 유전자 변형 및 녹아웃 종자의 가용성을 더 자주되었다. 프로토콜 참고로 몇 가지 중요한 세부 사항이 있습니다 : <ol><li> 그것은 수술하는 동안 마우스 체온을 유지하기 위해 필수적이며, 그것은 완전히 마취에서 회복하기 전에. 체온이 경색의 정도에 영향을 가지고, 저체온증의 감소와 고열은 경색의 크기를 증가시킵니다. </li><li> 노출과 CCA와 ECA를 분리하는 동안, 경색 크기 감소의 가능성을 높일 수있는 인근의 미주 신경 및 기관을 손상하지 마십시오. </li><li> 이상의 10mm의 분기점을 통과 monofilament의 봉합을 삽입하지 마십시오. 너무 멀리 삽입 봉합은 뇌출혈의 앞쪽에 대뇌 동맥 및 결과를 구멍 수 있습니다. 저항은 봉합이 분기점 포인트 너머 90-10밀리미터에 대한 고급 때 느낄 수 있습니다. 이 경우, 연구자는 봉합을 증진 중단하고 거리를 확인합니다. </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Isoflurane</td>
<td>Chemical	</td>
<td>Aerrane, Baxter</td>
<td>95045-588</td>
<td>&nbsp;</td>
<tr>
<td>V-10 Anesthesia system</td>
<td>Equipment</td>
<td>VetEquip</td>
<td>901807</td>
<td>&nbsp;</td>
<tr>
<td>TR-200 Temp Controller</td>
<td>Equipment</td>
<td>Fine Science Tools</td>
<td>21060</td>
<td>&nbsp;</td>
<tr>
<td>Electric clipper</td>
<td>Equipment</td>
<td>Braintree</td>
<td>CLP-9931</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>Equipment</td>
<td>Nikon</td>
<td>SMZ745T</td>
<td>&nbsp;</td>
<tr>
<td>Retractor system</td>
<td>Equipment</td>
<td>Braintree</td>
<td>ACD-014</td>
<td>&nbsp;</td>
<tr>
<td>Bipolar coagulator</td>
<td>Equipment</td>
<td>Howard Instrument</td>
<td>64000</td>
<td>&nbsp;</td>
<tr>
<td>Silk suture</td>
<td>Material</td>
<td>Harvard Apparatus</td>
<td>510479</td>
<td>&nbsp;</td>
<tr>
<td>Monofilament suture</td>
<td>Material</td>
<td>Harvard Apparatus</td>
<td>723351</td>
<td>&nbsp;</td>
<tr>
<td>Vascular clamp</td>
<td>Equipment</td>
<td>Fine Science Tools</td>
<td>00396-01</td>
<td>&nbsp;</td>
<tr>
<td>Vannas scissor</td>
<td>Equipment</td>
<td>Fine Science Tools</td>
<td>15000-08</td>
<td>&nbsp;</td>
<tr>
<td>Brain matrix</td>
<td>Equipment</td>
<td>Braintree</td>
<td>BS-2000C</td>
<td>&nbsp;</td>
<tr>
<td>2,3,5-triphenyltetrazolium chloride</td>
<td>Chemical</td>
<td>Sigma-Aldrich</td>
<td>T8877</td>
<td>&nbsp;</td>
<tr>
<td>10% neutral buffer formalin</td>
<td>Chemical</td>
<td>Sigma-Aldrich</td>
<td>HT5011</td>
<td>&nbsp;</td>		</tbody>
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mouse model of middle cerebral artery occlusion

뇌졸중은 미국 1 가장 흔한 치명적인 신경 질환입니다. 두뇌에있는 혈관의 막힘 (허혈성 뇌졸중) 2 스트로크의 대부분 (88 %) 결과. 대부분의 허혈성 뇌졸중 (~ 80 %) 중간 대뇌 동맥 (MCA) 3의 영토 발생 이후 개발된 많은 동물 스트로크 모델이 동맥에 집중했습니다. 중간 대뇌 동맥 폐색 (MCAO)의 intraluminal monofilament 모델은 외부 경동맥 동맥에 수술 필라멘트의 삽입과 팁은 MCA의 출처를 occludes 때까지 정지 그 결과, 내부 경동맥의 동맥 (ICA)에 기대를 스레딩을 포함 혈액 흐름과 MCA 테리토리 4 이후 뇌 경색의. 기술은 영구적인 또는 일시적인 폐색 5 모델하는 데 사용할 수 있습니다. 봉합사가 특정 간격 (30 분, 1 H, 2 H) 후 제거하면, reperfusion는 (일시적 MCAO) 이루어집니다, 필라멘트가 자리에 남아있다면 (24 H) 절차는 영구 MCAO의 모델로 적합합니다 . 이 기술은 craniectomy, intracranial 압력 및 온도 6에 영향을 미칠 수있는 두개골의 일부를 제거하는 neurosurgical 절차를 필요로하지 않습니다. 그것은 쥐 및 생쥐 7,8에 영구와 일시적인 대뇌 국소 빈혈 초점을 모방하는 데 가장 자주 사용되는 방법이되었습니다. 대뇌 경색의 범위를 평가하기 위해, 우리는 허혈성 뇌 조직 9 식별 2,3,5 - triphenyltetrazolium 염화물 (TTC)와 뇌 조각 얼룩. 이 비디오에서는, 우리는 MCAO 방법과 TTC의 얼룩에 의해 경색 크기의 결정을 보여줍니다.

Watch this Scientific Journal Video about Mouse Model of Middle Cerebral Artery Occlusion at JoVE.com

Mouse Model of Middle Cerebral Artery Occlusion

우리는 비디오 intraluminal monofilament를 사용하여 성인 마우스의 중간 대뇌 동맥 폐색을 생산하는 방법을 보여줍니다. 또한 2,3,5 - triphenyltetrazolium 염화물 (TTC) 염색법에 의해 대뇌 경색의 범위를 평가하는 방법을 보여줍니다.

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 ...

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77.3K 조회수.  Ernest Gallo Clinic and Research Center, University of California, San Francisco. 우리는 비디오 intraluminal monofilament를 사용하여 성인 마우스의 중간 대뇌 동맥 폐색을 생산하는 방법을 보여줍니다. 또한 2,3,5 - triphenyltetrazolium 염화물 (TTC) 염색법에 의해 대뇌 경색의 범위를 평가하는 방법을 보여줍니다.

비디오: Mouse Model of Middle Cerebral Artery Occlusion

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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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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

마우스에 영구 중간 대뇌 동맥 내고의 응용

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

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 ...

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 ...

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

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 ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

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 ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

P10 쥐에서 신생아 뇌졸중의 과도 중간 대뇌 동맥 폐색 모델

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...