手术技术的最初收购的威廉D.山博士在乔治亚医学院的实验室。作者还要感谢大卫博士A. Rempe和兰大Prifti解剖相机使用。这项研究是由美国国立卫生研究院NS041744 NS051279，F31 NS064700和果酸30815697D支持。

该协议被批准用于对动物研究的伦理（UCAR）的大学罗切斯特委员会。在协议应遵循无菌技术。无菌手套和口罩的使用是必需的。 表1描述了所有的设备，材料，化学品，并在协议使用工具。 1。手术前准备<ol><li>注入小鼠皮下丁丙诺啡（0.05mg/kg），术前2小时后，立即手术，然后在第一个24小时后手术期每​​3-5 h。 </li><li>高压灭菌器消毒所有的手术器械，纱布海绵，棉花尖端涂药。在手术期间和无菌纱布上空气干燥之前使用的手术工具保持在70％的乙醇。喷雾用70％乙醇的工作区。 </li><li> 3％isofluorane - 20％的氧气混合气体麻醉使用麻醉蒸发器的鼠标。调整流量计的氧气量。测试脚趾或尾巴捏（他们应该反应迟钝）的麻醉水平。维持麻醉用2％（V / V）isofluorane的水平。 </li><li>应用人工泪液鼠标的眼睛和使用注意事项，以避免损坏的眼睛在手术过程中。在外侧的位置，放置在其右侧的鼠标。 </li><li>剃须面积之间的左眼和使用动物快船队的左耳朵基地的左侧。优碘溶液和70％的乙醇使用棉花尖端涂药，轻轻揉面积之间交替清洗的区域。必要时重复。 </li><li>放在鼠标连接到直肠探头体温保持在37 ° C的加热垫使用矿物油插入直肠探头。 </li><li>鼠标和定位在其右侧，在显微镜下，用胶带固定。剪下一个窗口，并在纱布覆盖手术区。 </li></ol> 2。手术程序和马华结扎<ol><li>使左眼和左耳使用细直剪基地之间的垂直切口。用弯止血，保持手术区开放。 </li><li>使用弹簧剪刀，一个横向切口的颞肌，颞肌稍分开，从头骨与钳慢慢拉。 </li><li>使用18G针，弯钳颌骨，在颧弓和鳞骨交界的一个小颞下开颅手术。 </li><li>去除骨rongeurs小块头骨暴露马华。颧弓及眶内容不应该在这个过程中损坏。如果过度干燥的组织在此过程中发生的，适用于无菌PBS棉花尖端涂药。 </li><li>通过使用一个小型船只cauterizer结扎MCA的远端部分。 </li><li>将颞肌返回到其原来的位置，并关闭切口部位，手术5-0尼龙缝线。 </li><li>停止麻醉和删除直肠探头。与第二剂量丁丙诺啡（0.05mg/kg）皮下注入鼠标。返回鼠标保持在37 ° C加热板与笼。 </li><li>密切监察任何不适（食欲减退/水的消耗，弯腰驼背的姿势，呼吸增加，pilo竖立的头发）在接下来的24 h鼠标。鼠标注入丁丙诺啡皮下注射，每3-5小时后，手术到24h。在此期间，提供回收凝胶的鼠标。 </li></ol> 3。 TTC染色和搏出量的测定<ol><li>深受二十四小时后，手术麻醉鼠标，transcardially灌注4％TTC（W / V）在磷酸盐缓冲液（PBS）为15分钟和一个10分钟的4％多聚甲醛溶液，然后通过使用鼠标中等流量的微型蠕动泵。取出大脑和过夜的4％多聚甲醛溶液放入。 </li><li>大脑切片块的位置。使用刀片的大脑切成1mm厚的片。 </li><li>将切片旁边一毫米的刻度标尺。通过使用数码相机连接到解剖显微镜的拍摄片。 </li><li>中风面积计算，减去非梗死区的总面积从对侧网站使用Image J软件（http://rsbweb.nih.gov/ij/）同侧网站。堆叠中风片22面积计算每搏输出量。 </li></ol> 4。指示使用Image J软件<ol><li>打开图像文件，通过点击"文件"菜单中的Image J软件分析。 </li><li> "直线"选项卡上单击程序。绘制一个标尺上的两个利润率之间的直线。点击"分析"菜单并选择"设置规模"。设置为1的已知距离，单位为毫米的长度。 </li><li>点击"写意圈"的标签上。画一个圆圈勾勒出对侧半球。点击"分析"菜单，选择"测量"。显示绘制的圆的面积计算窗口会弹出。 </li><li>同侧半球，但不包括中风（白）区域周围绘制另一个圆形。测量步骤4.3中所示的区域。新的值将被添加到计算窗口。第一和第二值之间的差异，这是作为在下面的公式表示梗死面积。 </li><li>中风在每片面积计算重复步骤4.2-4.4。以每片（ΣAN）计算中风面积的总和。这代表每搏输出量，知道的事实，每个切片厚度为1mm。 ΣAñ x 1毫米（每片厚度）=每搏输出量</li></ol> 5。代表性的成果: 获得永久马华结扎鼠标梗死大多皮质。但是，它有可能获得皮层下病灶，如果在MCA的豆纹分支近端结扎。马华结扎后的中风量可能会有所不同，从10mm ³到35 毫米³ 19， 23。中风卷Moyanova等染色与TTC计算1910毫米³到22毫米立方米之间，而中风的容量从23到35mm ³之间20毫米³ Filiano等MRI图像确定。这些差异可能是结扎的确切位置和使用不同的方法来衡量中风卷的可能原因。 在图1中，一个TTC染色脑24小时后永久马华在野生型C57/BL6鼠标结扎。中风的网站是在外观上（图1A，B）的白色。图1A和B，说明从不同的角度梗死的网站。图2显示了1mm厚的TTC染色前中风脑片后（图2A - G）。 24小时手术后梗死体积计算。梗死体积〜23毫米³。 <img src="/files/ftp_upload/3039/3039fig1.jpg" alt="图1"/> 图1。中风的网站。AB公司表示 ，TTC染色马华结扎后，24小时全脑图像。白色区域代表梗塞。 <img src="/files/ftp_upload/3039/3039fig2.jpg" alt="图2"/> 图2。代表性的成果 （A&gt; G），TTC染色1毫米脑切片，24 h后MCAL。由前至后的切片对齐。白色区域代表梗塞。

<ol>
	<li>Britton, M., Rafols, J., Alousi, S., Dunbar, J. 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+effects+of+middle+cerebral+artery+occlusion+on+central+nervous+system+apoptotic+events+in+normal+and+diabetic+rats.">The effects of middle cerebral artery occlusion on central nervous system apoptotic events in normal and diabetic rats.</a> Int J Exp Diabesity Res. 4, 13-20 (2003).</li><li>Ciceri, P., Rabuffetti, M., Monopoli, A., Nicosia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+leukotrienes+in+a+model+of+focal+cerebral+ischaemia+in+the+rat.">Production of leukotrienes in a model of focal cerebral ischaemia in the rat.</a> Br J Pharmacol. 133, 1323-1329 (2001).</li><li>Jin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+transplantation+of+human+neural+precursor+cells+improves+outcome+from+focal+cerebral+ischemia+in+aged+rats.">Delayed transplantation of human neural precursor cells improves outcome from focal cerebral ischemia in aged rats.</a> Aging Cell. 9, 1076-1083 (2010).</li><li>McCaig, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+GADD34+expression+after+focal+cerebral+ischaemia.">Evolution of GADD34 expression after focal cerebral ischaemia.</a> Brain Res. 1034, 51-61 (2005).</li><li>Shirotani, T., Shima, K., Chigasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+studies+of+extracellular+metabolites+in+the+striatum+after+distal+middle+cerebral+artery+occlusion+in+stroke-prone+spontaneously+hypertensive+rats.">In vivo studies of extracellular metabolites in the striatum after distal middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats.</a> Stroke. 26, 878-884 (1995).</li><li>Wu, Y. P., Tan, C. K., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Fos-like+immunoreactivity+in+the+brain+and+spinal+cord+of+rats+following+middle+cerebral+artery+occlusion.">Expression of Fos-like immunoreactivity in the brain and spinal cord of rats following middle cerebral artery occlusion.</a> Exp Brain Res. 115, 129-136 (1997).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J Cereb Blood Flow Metab. 1, 53-60 (1981).</li><li>Bederson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion:+evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.</a> Stroke. 17, 472-476 (1986).</li><li>Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+gender+influences+on+sensitivity+to+focal+cerebral+ischemia+in+the+stroke-prone+spontaneously+hypertensive+rat.">Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat.</a> Hypertension. 33, 681-685 (1999).</li><li>Mary, V., Wahl, F., Uzan, A., Stutzmann, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enoxaparin+in+experimental+stroke:+neuroprotection+and+therapeutic+window+of+opportunity.">Enoxaparin in experimental stroke: neuroprotection and therapeutic window of opportunity.</a> Stroke. 32, 993-999 (2001).</li><li>Menzies, S. A., Hoff, J. T., Betz, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+in+rats:+a+neurological+and+pathological+evaluation+of+a+reproducible+model.">Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model.</a> Neurosurgery. 31, 100-107 (1992).</li><li>Iaci, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+growth+factor+2+promotes+functional+recovery+with+treatment+initiated+up+to+7+days+after+permanent+focal+ischemic+stroke.">Glial growth factor 2 promotes functional recovery with treatment initiated up to 7 days after permanent focal ischemic stroke.</a> Neuropharmacology. 59, 640-649 (2010).</li><li>Richard, M. J. P., Khan, B. V. C. B. J., Saleh, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+by+which+lipoic+acid+confers+protection+during+the+early+stages+of+cerebral+ischemia:+A+possible+role+for+calcium.">Cellular mechanisms by which lipoic acid confers protection during the early stages of cerebral ischemia: A possible role for calcium.</a> Neuroscience Research. , (2011).</li><li>Heiss, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+derangement+of+periinfarct+viable+tissue+in+ischemic+stroke.">Progressive derangement of periinfarct viable tissue in ischemic stroke.</a> J Cereb Blood Flow Metab. 12, 193-203 (1992).</li><li>Nedergaard, M., Gjedde, A., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+ischemia+of+the+rat+brain:+autoradiographic+determination+of+cerebral+glucose+utilization,+glucose+content,+and+blood+flow.">Focal ischemia of the rat brain: autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow.</a> J Cereb Blood Flow Metab. 6, 414-424 (1986).</li><li>Nowicki, J. P., Assumel-Lurdin, C., Duverger, D., MacKenzie, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+evolution+of+regional+energy+metabolism+following+focal+cerebral+ischemia+in+the+rat.">Temporal evolution of regional energy metabolism following focal cerebral ischemia in the rat.</a> J Cereb Blood Flow Metab. 8, 462-473 (1988).</li><li>Butcher, S. P., Bullock, R., Graham, D. I., McCulloch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+amino+acid+release+and+neuropathologic+outcome+in+rat+brain+following+middle+cerebral+artery+occlusion.">Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion.</a> Stroke. 21, 1727-1733 (1990).</li><li>Arlicot, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+remote+microglial+activation+in+rodent+models+of+focal+ischaemia+using+the+TSPO+radioligand+CLINDE.">Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE.</a> Eur J Nucl Med Mol Imaging. 37, 2371-2380 (2010).</li><li>Moyanova, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+for+type+4+metabotropic+glutamate+receptors+against+ischemic+brain+damage.">Protective role for type 4 metabotropic glutamate receptors against ischemic brain damage.</a> J Cereb Blood Flow Metab. , (2010).</li><li>Ortolano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+imaging+of+new+targets+for+pharmacological+intervention+in+stroke:+real-time+tracking+of+T-cells+in+the+ischaemic+brain.">Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain.</a> Br J Pharmacol. 159, 808-811 (2010).</li><li>O'Neill, M. J., A, C. J. <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+cerebral+ischemia.">Rodent models of focal cerebral ischemia.</a> Current Protocols in Neuroscience. , 9.6.1-9.6.32 (2000).</li><li>Lin, T. N., He, Y. Y., Wu, G., Khan, M., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+brain+edema+on+infarct+volume+in+a+focal+cerebral+ischemia+model+in+rats.">Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats.</a> Stroke. 24, 117-121 (1993).</li><li>Filiano, A. J., Tucholski, J., Dolan, P. J., Colak, G., Johnson, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transglutaminase+2+protects+against+ischemic+stroke.">Transglutaminase 2 protects against ischemic stroke.</a> Neurobiol Dis. 39, 334-343 (2010).</li></ol>

永久性MCA结扎方法给予高度重复性的脑梗死体积和增加手术后的生存率率比现有的其他方法。程序的难易程度和持续时间短（约30分钟），使其更加实用。该方法被广泛用于在小鼠和大鼠。 这种方法需要一个立体显微镜下微创手术。因此，在显微镜下操作和完善一个成功的开颅手术的经验是必不可少的。最好是建立在实验室进行实验前，一致梗死。为了实现可重复性的结果是很重要结扎在马华每次相同的确切位置。马华应结扎近端豆​​纹皮质下梗死所需的分行。是完全结扎动脉闭塞是永久性的。因此，这种模式不允许再灌注通过MCA。虽然不包括在此演示，它是最好的，如果使用多普勒探头是测量血流量，以验证在受影响地区完成结扎动脉。 经营者应非常小心，不要损坏或穿刺马华，同时揭露和混凝动脉。开颅手术应具有广泛的服务，以防止任何损害颧骨。由于手术区是非常接近的梗塞面积，皮质表面造成任何损害，应避免在开颅手术或烧灼。福斯特18015-00 cateurizer单位或双极cateurizer可以使用，而不是在此演示中使用的福斯特18000-00。福斯特18015-00允许用户调整温度的cauterizer尖端发热量低，可能会阻止任何损伤皮层组织。福斯特18015-00也避免看到在电池供电烧灼工具的温度波动。在烧灼，用户应非常小心地保持氧流量的cauterizer，可以暂时关闭的O 2 / isofluorane点火，以避免风险。这不会影响鼠标的麻醉状态。为了防止这种风险，我们使用一个通风管道延伸到手术区，从而增加足够的空气流通，以及提请关闭任何多余的氧/空气isofluorane 。 永久性MCA结扎模型研究缺血性中风是非常有用的。它可用于测试神经保护剂或缺血的分子机制研究转基因小鼠模型体内。 在体内的方法使用明显的好处是，这允许缺血完整的神经网络研究和行为反应后的侮辱，除了是目前缺血性脑损伤后neuroinflammatory过程。因此，这提供了在体内中风模型的一个重要的补充办法原位模型，用来模拟在细胞培养缺血。

<table border="1"><tbody><tr><th>名称</th><th>公司联系号码</th></tr><tr><td> 答:解决方案和化学品 </td><td></td></tr><tr><td>碘伏溶液擦洗手术</td><td> Aplicare，82-209 </td></tr><tr><td> 70％的乙醇</td><td> Koptec，V1001 </td></tr><tr><td> 2,3,5 - 三苯基氯化氮唑（TTC） </td><td>西格玛，T8877 </td></tr><tr><td> 4％多聚甲醛</td><td>电子显微镜学，159SP </td></tr><tr><td>磷酸盐缓冲生理盐水，pH值:7.4 </td><td>在实验室中制造和消毒。 </td></tr><tr><td>矿物油</td><td> PML的Microbiologicals，R6570 </td></tr><tr><td> Isofluorane </td><td>巴特勒公司，029405 </td></tr><tr><td>丁丙诺啡（0.3mg/ml） </td><td> Hospira公司，NDC 0409-2012-32 </td></tr><tr><td>人工泪液</td><td>巴特勒公司，007312 </td></tr><tr><td>恢复凝胶</td><td>清除H 2 O 72-01-5022 </td></tr><tr><td> B.手术材料和设备 </td><td></td></tr><tr><td> C57/BL6鼠标，10至12周岁</td><td>殖民地大学动植物公园饲养</td></tr><tr><td> 100％的氧气罐</td><td>公司Airgas </td></tr><tr><td>麻醉蒸发器和流量计</td><td> Surgivet，型号100 </td></tr><tr><td>直肠探头的加热垫</td><td>精细的科学工具，TR200 </td></tr><tr><td>解剖显微镜</td><td> 2000年蔡司，STEMI </td></tr><tr><td>加热板</td><td> PETCO服务有限公司0307-013 </td></tr><tr><td>小血管Cauterizer集</td><td>精细的科学工具，18000-00 </td></tr><tr><td>迷你蠕动泵</td><td>哈佛器械，MPII </td></tr><tr><td> 1ml注射器</td><td>屋宇署，329650 </td></tr><tr><td> 18G针</td><td> Becton Dickinson公司，305196 </td></tr><tr><td>手术5.0尼龙缝线</td><td> Ethilon，698H </td></tr><tr><td>切片座</td><td>肯特科学，1毫米</td></tr><tr><td>单边刀片</td><td>电子显微镜学，71962 </td></tr><tr><td>标签胶带</td><td> Scienceware，倍儿最先进的产品F13463 - 2075 </td></tr><tr><td>纱布垫</td><td>肯德尔Curity 2252，约翰逊和约翰逊6415 </td></tr><tr><td>棉花提示喷枪</td><td>清教徒806 - WCL </td></tr><tr><td> C.手术工具 </td><td></td></tr><tr><td>手术弹簧剪刀</td><td>精细的科学工具15003-08 </td></tr><tr><td> Microdissecting钳角度</td><td>精细的科学工具5 / 45 </td></tr><tr><td> Microdissecting钳弯曲</td><td>精细的科学工具5钛</td></tr><tr><td>细直剪</td><td>精细的科学工具14568-12 </td></tr><tr><td>曲骨咬骨钳</td><td>精细的科学工具16021-14 </td></tr><tr><td>哈特曼止血钳弯</td><td>精细的科学工具13003-10 </td></tr></tbody></table> 表1。MCA结扎过程中使用的材料，工具和化学品清单。 A.解决方案和化学品，B.外科材料和设备，C.手术工具。

the application of permanent middle cerebral artery ligation in the mouse

局灶性脑缺血之间看到中风患者最常见的类型。由于临床意义出现了长时间的努力，制定合适的动物模型研究缺血期间开展的活动。这些技术包括短暂或永久的，局灶性或全球的许多不同的动物模型，使用最常见的啮齿类动物的缺血模型。 永久性MCA结扎的方法，这也是在文献中提到pMCAo，被广泛使用作为一个在啮齿类动物中1-6局灶性脑缺血模型。这种方法最初是老鼠田村等。在1981年7。开颅手术是在这个协议用于访问由电闭塞MCA和近端地区。梗死涉及大多是皮层和纹状体有时的地区，根据闭塞的位置。现在这项技术是建立和使用在许多实验室8-13。早期使用这种技术的定义和描述的"梗塞核心"和"半影"14日至16日，它往往是用来评估潜在的神经保护作用的化合物10，12，13， 17。虽然最初的研究是在大鼠进行的，永久的马华结扎已成功地使用在18-20稍作修改的小鼠。 这种模式产生重现梗死和增加后的存活率。在人类缺血性中风的约80％发生在MCA 面积 21，因此这是一个中风的研究非常相关的模型。目前，中风患者提供有效的治疗方法缺乏，因此需要有一个很好的模式，来测试潜在的药理的化合物和评估生理成果。这种方法也可以用于研究体内细胞缺氧反应机制。 在这里，我们目前在C57/BL6鼠标MCA结扎手术。我们描述的术前准备，马华结扎手术和2,3,5 - 三苯基氯化四氮唑（TTC）染色脑梗死体积的定量。

Watch this Scientific Journal Video about The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse at JoVE.com

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

大脑中动脉（MCA）结扎是一种技术，研究在动物模型局灶性脑缺血。在此方法中，通过开颅暴露大脑中动脉和烧灼结扎。此方法提供了高度重复性的脑梗死体积和增加手术后的生存率率比现有的其他方法。

在小鼠永久性大脑中动脉结扎术中的应用

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 ...

the-application-permanent-middle-cerebral-artery-ligation

Nanyang Technological University

Research

JoVE Journal

Medicine

19.6K 次观看.  University of Rochester. 大脑中动脉（MCA）结扎是一种技术，研究在动物模型局灶性脑缺血。在此方法中，通过开颅暴露大脑中动脉和烧灼结扎。此方法提供了高度重复性的脑梗死体积和增加手术后的生存率率比现有的其他方法。

视频: The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

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.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

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

科研

医学

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.

大脑中动脉闭塞的小鼠模型

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 ...

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.

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

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.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

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

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

永久性脑血管闭塞<em&gt;通过</em&gt;双人结扎横断

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

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.

造型中风小鼠:在远端大脑中动脉永久性混凝

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

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

左冠状动脉前降支的小鼠结扎:对心肌梗死后重塑和心衰模型

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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.

对大脑中动脉闭塞再灌注损伤诱发中风小鼠的手术方法

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.

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

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