按照献给Landesamt Gesundheit和Soziales，柏林，德国的准则和法规，在这篇文章中描述的所有程序进行。 手术前的程序 1。单丝的制备<ol><li>腔内长丝的制备，用8-0尼龙单丝缝合线，被切成每个长度为13mm。 </li><li>对于涂布的单丝，粘膜，Xantopren中号（贺利氏古莎公司，德国Hanau）使用激活的活化剂溶液（活化剂，通用加Heraeus公司姆古莎GmbH公司，旅游Hanau，德国）中加入一滴。 </li><li>慢慢地拉过硅氧烷的单丝，直到它被完全覆盖着它。通过这样做，单丝均匀的硅胶覆盖。避免等待太久因为激活的涂层材料将迅速成为坚定和干。 </li><li>前在硅胶开始凝固，它是建议剥离过度硅氧烷，拉放在一张纸，从灯丝。通过这样做，可以减薄硅层的近似厚度为200μm。 </li><li>该基地的有机硅覆盖的单丝刺入橡皮泥，为了让它干通宵。 </li></ol>评论：在理想的情况下，只有灭菌或消毒的单丝应至少使用，以确保在手术部位的无菌性。在实践中，灭菌或消毒的手工制作的单丝单丝的质量是非常困难的，因为可能会恶化9。因此，它可能是可取的，而不是使用市售的无菌单丝。 2。准备手术器械及动物<ol><li>所有手术器械保持在70％的乙醇。此外，每一个新的外科手术前，所有的手术器械进行了仔细清洗次用70％乙醇消毒。 </li><li>手术部位彻底消毒，用70％的乙醇在所有的动物横断前。消毒站点被允许完全干透，再进行切口。没有进行附加的裁剪的皮草，因为这可能会导致微打磨和随后的皮肤炎症9。 </li></ol>外科手术 3。 LDF探头定位和LDF监测<ol><li>麻醉诱导小鼠暴露于30％氧气，70％N 2 O，2.5％异氟醚使用的蒸发器的气体混合物组成的。维持麻醉，异氟醚的浓度降低至1.5％。小鼠的自主呼吸，通过在整个手术过程中的呼吸面罩。 </li><li>插入直肠温度探头和固定小鼠，露出顶叶头的立体框架。在手术过程中，老鼠被放在一个恒温器控制加热垫，确保了核心温度恒定为37.0±0.5°C。矢状中线切口（〜1厘米长）进行顶叶颅骨暴露。 </li><li>头骨的左侧进行了仔细的解剖和一个光纤探头直接固定到颅骨2毫米尾鳍和5毫米的横向前囟使用瞬间胶。之后，伤口边缘都经过精心改编，以避免脑低温以及感染。 </li><li>鼠标转身露出颈部腹侧。光纤探头连接通过连接探头适配器（Perimed主探头418-1耶尔费拉，瑞典）的的LDF设备（Perimed的PeriFlux系统5000耶尔费拉，瑞典）。 </li></ol>评论：光纤探头转动鼠标时左右，应小心弯曲，以避免它打破了从头骨。 <ol start="5"><li> LDF设备连接到一台笔记本电脑COM计算机。 LDF值连续拍摄发起使用PeriSoft为Windows（版本2.50，耶尔费拉，瑞典）， 图1演示的平均CBF当然，获得的从十个LDF录音期间夹闭双侧颈总动脉灌注。 </li></ol> 4。双侧颈总动脉闭塞<ol><li>麻醉维持3.1中所描述的。鼠标放置在它的后面。动物的尾巴和爪子，用胶带固定在加热垫。矢状腹正中切口（〜1cm的长度）。 </li><li>两个涎腺仔细分离和动员起来，以可视化的底层CCA的。 </li><li>两个CCA的仔细分离从各自的迷走神经及伴行静脉，不损害这些结构。迷走神经的操作可能会导致短暂或永久性的功能障碍的副交感神经系统，这有可能发生显着的心脏ARRhythmia甚至是不可逆的心脏骤停。因此，至关重要的是，以避免任何操纵的迷走神经。围绕每个CCA松动5-0丝线缝合循环。 </li><li>一个小的硅管（直径2毫米），插入在CCA和丝线，每边之间。这种管子作为一个夹板的CCA的使用，以避免损伤动脉壁的缝线收紧（ 图4）。 </li><li>两个CCA闭塞收紧丝线一分钟。只有老鼠LDF信号减少到&lt;10％的基线信号，其中包括在研究中。双方CCA的重新开放后，五分钟再灌注期间启动。 LDF一个完整的再灌注保证。再灌注不全的动物（ 即 &lt;90％恢复脑血流量相比，基线CBF），被排除在外。夹闭双侧颈总动脉一分钟和5分钟的再灌注的顺序重复两次，总的夹闭双侧颈总动脉durati上3分钟。 图1示出的平均CBF当然，10 LDF录音在BCCAO再灌注过程中获得的。在实验过程中，建议保持颈部创面用生理盐水湿。 </li><li>最后再灌注后，除去所有的管道或缝合线，以及连接到头骨的光纤探头，缝合伤口，并根据需要为利多卡因软膏（Xylocain 2％;阿斯利康GmbH公司，威德尔德国），反复施加到伤口止痛管理。 </li><li>小鼠被转移到预先加热（30℃）回收箱（MediHEAT. PecoServives有限公司布拉夫英国坎布里亚郡），直到他们重新获得完整的意识。此后，的小鼠被送回他们的动物笼舍。 </li></ol> 5。大脑中动脉闭塞<ol><li>小鼠重新麻醉，直肠温度探头插入小鼠放置在加热垫的脖子，露出腹侧。后来，他们瓦特东铁支线固定夹闭双侧颈总动脉一节中所描述的。 </li><li>一个永久使用长7-0丝线缝合结扎周围的左CCA近端和螺纹尾端轻轻拉，用蚊式钳（ 图4）。 </li><li>另一个永久结扎了周围的左外颈动脉（ECA）和该线程被被拉向动物的右侧（ 图4）。 </li><li>周围的结缔组织左ICA认真动员和舌下神经轻轻地把的远端过程中ICA得到一个很好的概述。的左侧ICA弯曲到左侧翼腭动脉（PPA），而如下的基ICA的过程中的右侧（ 图4）。 </li></ol>评论：还有另外一个被识别的转向尾方向中的右侧的动脉。这是枕动脉，无论是从CCA分叉或从公关可变起源的ECA的oximal的一部分（ 图4）。 <ol start="5"><li>一个松散的缝合环周围的近端ICA，用5-0丝线。 </li><li>使用一个小的血管钳，ICA暂时闭塞远端5-0松散的缝合环（ 图4）。 </li><li>一个小切口，在远端的一部分CCA之前CCA分叉使用微血管剪刀（ 图4）。 </li><li>预选的硅氧烷覆盖的单丝是通过小切口，引入到CCA的ICA推入，直到它停在微血管钳（ 图4）。 </li><li>小心除去微夹钳从ICA，而单 ​​丝被推进了深入的ICA，直到它停止（ 图4）。点评：在这一点上，翻转钳在左手，这样你就不会持有它像一支钢笔。这将有助于更容易抢灯丝与镊子。血管钳涂药，应在右手举行。这可能是必要的，为了防止出血从切口侧近紧固松动5-0缝合线周围的ICA。 </li><li>单丝尖端的确切位置被确定，以检查它是否被推进到ICA和不进PPA。如果单丝尖端引入近端PPA，灯丝被轻轻拉回刚刚之前的ICA / PPA分岔。有人试图再次推进到ICA的单丝，同时轻轻拉出的基ICA的位向右侧。通过这样做，ICA远端的自然过程模拟使其更容易访问的ICA。 </li><li>单丝后提前到ICA几乎完全收紧，松5-0缝线环周围的近端ICA为了保持地方灯丝。之后，所有的丝线被缩短，皮肤伤口缝合。 </li><li>在手术过程中的流体损失补货ished注入1.0毫升生理盐水IP之后，被转移到鼠标预热回收箱，直到它重新获得完整的意识。 </li><li>再灌注，老鼠再麻醉后取出仔细松开5-0丝线缝合周围的近端ICA和单丝。此后，缝合5-0再次收紧仔细，以防止出血。 </li><li>确认彻底止血后，脖子上的伤口缝合。 </li><li>反复涂抹利多卡因软膏局部皮肤需要缓解疼痛和鼠标被转移到预先加热（30℃）的回收箱，直到它重新获得完整的意识。 </li><li>作为第一个成功的提示缺血过程，顺时针盘旋的动物进行了观察。修改小鼠10使用贝德森的得分功能缺损的严重程度进行了量化。据此改性贝德森得分，神经功能评分重新分级如下：0（运动功能正常），1（当鼠标的尾巴抬起躯干前屈和对侧前肢），2（盘旋对侧的一个平坦的表面上时，鼠标的尾巴举行，但正常在休息的姿势），3（扶着到对侧休息），4（无自发运动活动或死）。 </li></ol> 6。假手术夹闭双侧颈总动脉对于夹闭双侧颈总动脉假手续，小鼠被麻醉的持续时间一个真正夹闭双侧颈总动脉PC程序，流涎腺体进行了动员和CCA的暴露。至关重要的是，假手术组颈动脉或迷走神经的直接操作过程中避免。的CCA的操作可能会损坏的内皮细胞层，这可能会导致从血管内皮细胞或诱导局部血栓形成，动脉管腔内的血管活性化合物的释放。因此，假手术后可能发生的血栓栓塞事件。这将是一个重大的混杂因素THE工作步骤。另一方面，迷走神经的操纵可能导致暂时或永久的功能障碍的副交感神经系统，该系统具有显着的心律失常的发生甚至是不可逆的心脏骤停的潜力。因此，这是至关重要的，以避免任何操纵夹闭双侧颈总动脉的迷走神经。 手术后的程序 7。梗死体积测量评估心肌梗死面积，缺血小鼠存活72小时后。后来动物深度麻醉，大脑被拆除，甲基丁快速冷冻，切片和染色梗死体积测量与苏木（使用Papanicolau协议）。脑切片在600微米间隔和脑梗死体积收集，确定使用图像分析仪（MCID核心7.0顺畅不间断，GE医疗集团尼亚加拉公司），并根据Lin 等人的方法，校正水肿。7,11 </SUP&gt;。 8。入选标准我们只包括三个夹闭双侧颈总动脉发作期间有一个LDF测量脑血流量减少90％以上的动物。理想的情况下，LDF监测应在两个脑半球在夹闭双侧颈总动脉。实用性，进行仅在左右半球，这将在稍后进行MCAO LDF监测。这种方法已经被描述过6,12。再灌注后，每个夹闭双侧颈总动脉情节必须是完整的（ 即恢复到基线值）。持续老鼠被安放在麻醉状态下的时间缩短的原因，我们没有监控CBF在缺血手术。我们用一组独立的动物，然而，在试点研究监测CBF LDF在缺血手术。在这组老鼠中，我们一贯测量脑血流量减少90％，相比前基线CCA闭塞。此外，单丝CBF除去后恢复至&gt; 90％的各CCA闭塞后的基准值。这些的LDF标准建立在一致的梗死卷7文学和结果。我们只包括动物其中有一个贝德森的得分至少1缺血后从麻醉恢复后。此外，只有动物缺血和存活72小时后显示没有颅内出血的迹象，包括在研究中。

<ol>
	<li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2011+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2011 update: a report from the American Heart Association.</a> Circulation. 123, e18-e209 (2011).</li><li>Khaja, A. M., Grotta, 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=Established+treatments+for+acute+ischaemic+stroke.">Established treatments for acute ischaemic stroke.</a> Lancet. 369, 319-330 (2007).</li><li>Foerch, C., Sitzer, M., Steinmetz, H., Neumann-Haefelin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+demographic+trends+decrease+the+proportion+of+ischemic+stroke+patients+receiving+thrombolytic+therapy:+a+call+to+set-up+therapeutic+studies+in+the+very+old.">Future demographic trends decrease the proportion of ischemic stroke patients receiving thrombolytic therapy: a call to set-up therapeutic studies in the very old.</a> Stroke. 40, 1900-1902 (2009).</li><li>Gidday, 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=Cerebral+preconditioning+and+ischaemic+tolerance.">Cerebral preconditioning and ischaemic tolerance.</a> Nat. Rev. Neurosci. 7, 437-448 (2006).</li><li>Atochin, D. N., Clark, J., Demchenko, I. T., Moskowitz, M. A., Huang, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+cerebral+ischemic+preconditioning+in+mice+deficient+in+endothelial+and+neuronal+nitric+oxide+synthases.">Rapid cerebral ischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases.</a> Stroke. 34, 1299-1303 (2003).</li><li>Cho, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obligatory+role+of+inducible+nitric+oxide+synthase+in+ischemic+preconditioning.">Obligatory role of inducible nitric oxide synthase in ischemic preconditioning.</a> J. Cereb. Blood. Flow Metab. 25, 493-501 (2005).</li><li>Kunz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+protection+by+ischemic+tolerance:+role+of+nitric+oxide+and+reactive+oxygen+species.">Neurovascular protection by ischemic tolerance: role of nitric oxide and reactive oxygen species.</a> J. Neurosci. 27, 7083-7093 (2007).</li><li>Dirnagl, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+operating+procedures+(SOP)+in+experimental+stroke+research:+SOP+for+middle+cerebral+artery+occlusion+in+the+mouse.">Standard operating procedures (SOP) in experimental stroke research: SOP for middle cerebral artery occlusion in the mouse.</a> Nature Precedings. , (2010).</li><li>Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+stroke+in+mice+-+middle+cerebral+artery+occlusion+with+the+filament+model.">Modeling stroke in mice - middle cerebral artery occlusion with the filament model.</a> J. Vis. Exp. (47), e2423 (2011).</li><li>Bederson, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>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>Kawano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iNOS-derived+NO+and+nox2-derived+superoxide+confer+tolerance+to+excitotoxic+brain+injury+through+peroxynitrite.">iNOS-derived NO and nox2-derived superoxide confer tolerance to excitotoxic brain injury through peroxynitrite.</a> J. Cereb. Blood Flow Metab. 27, 1453-1462 (2007).</li><li>Qi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublethal+cerebral+ischemia+inhibits+caspase-3+activation+induced+by+subsequent+prolonged+ischemia+in+the+C57Black/Crj6+strain+mouse.">Sublethal cerebral ischemia inhibits caspase-3 activation induced by subsequent prolonged ischemia in the C57Black/Crj6 strain mouse.</a> Neurosci. Lett. 315, 133-136 (2001).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+forebrain+ischemic+preconditioning+model+established+in+C57Black/Crj6+mice.">A forebrain ischemic preconditioning model established in C57Black/Crj6 mice.</a> J. Neurosci. Methods. 107, 101-106 (2001).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Stroke+Model+Guidelines+for+Preclinical+Stroke+Trials+(1st+Edition).">Rodent Stroke Model Guidelines for Preclinical Stroke Trials (1st Edition).</a> J. Exp. Stroke Transl. Med. 2, 2-27 (2009).</li><li>Nishio, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia-induced+ischemic+tolerance.">Hypothermia-induced ischemic tolerance.</a> Ann. N.Y. Acad. Sci. 890, 26-41 (1999).</li><li>DeBow, S., Colbourne, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+temperature+measurement+and+regulation+in+awake+and+freely+moving+rodents.">Brain temperature measurement and regulation in awake and freely moving rodents.</a> Methods. 30, 167-171 (2003).</li><li>Colbourne, F., Sutherland, G. R., Auer, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+regulating+brain+temperature+in+awake+and+freely+moving+rodents.">An automated system for regulating brain temperature in awake and freely moving rodents.</a> J. Neurosci. Methods. 67, 185-190 (1996).</li><li>Kapinya, K. J., Prass, K., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induced+prolonged+protection+against+cerebral+ischemia+in+mice:+a+redox+sensitive+mechanism?.">Isoflurane induced prolonged protection against cerebral ischemia in mice: a redox sensitive mechanism?.</a> Neuroreport. 13, 1431-1435 (2002).</li><li>Li, L., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+preconditioning+improves+short-term+and+long-term+neurological+outcome+after+focal+brain+ischemia+in+adult+rats.">Isoflurane preconditioning improves short-term and long-term neurological outcome after focal brain ischemia in adult rats.</a> Neuroscience. 164, 497-506 (2009).</li><li>Xiong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+with+isoflurane+produces+dose-dependent+neuroprotection+via+activation+of+adenosine+triphosphate-regulated+potassium+channels+after+focal+cerebral+ischemia+in+rats.">Preconditioning with isoflurane produces dose-dependent neuroprotection via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats.</a> Anesth. Analg. 96, 233-237 (2003).</li><li>Spratt, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+method+of+thread+manufacture+improves+stroke+induction+rate+and+reduces+mortality+after+thread-occlusion+of+the+middle+cerebral+artery+in+young+or+aged+rats.">Modification of the method of thread manufacture improves stroke induction rate and reduces mortality after thread-occlusion of the middle cerebral artery in young or aged rats.</a> J. Neurosci. Methods. 155, 285-290 (2006).</li><li>Zarow, G. J., Karibe, H., States, B. A., Graham, S. H., Weinstein, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endovascular+suture+occlusion+of+the+middle+cerebral+artery+in+rats:+effect+of+suture+insertion+distance+on+cerebral+blood+flow,+infarct+distribution+and+infarct+volume.">Endovascular suture occlusion of the middle cerebral artery in rats: effect of suture insertion distance on cerebral blood flow, infarct distribution and infarct volume.</a> Neurol. Res. 19, 409-416 (1997).</li></ol>

我们什么都没有透露。

在我们的研究中，我们已经表明，三个序列夹闭双侧颈总动脉，每次持续一分钟，随后再灌注五分钟，是一个足够的缺血PC刺激诱导早期的IT。 此外，我们已经证明，使用预处理协议是一个安全和未成年人的侵入性手术没有值得注意的术后并发症发生率，没有迹象表明在脑细胞凋亡和无死亡。先前6-7,12已经描述了几种不同的预处理协议。其他已经使用了6分钟夹闭双侧?...

缺血性中风是一种疾病，死亡率高和一个巨大的社会经济负担1。尽管密集的实验和临床科学纵观近十年的努力，急性缺血性脑卒中患者的治疗选择仍然非常有限2。在对比的是，在发达国家，老年人的比例的增加将显着增加缺血性中风患者的发病率和患病率与未来几十年中3。因此，迫切需要新的治疗策略与缺血性中风患者。 一种方法是应对破坏性刺激大脑内源性适应机制，以获得进一步的了解。这种脑源性的神经保护作用也被称为缺血预处理（PC）或脑缺血耐受（IT），并描述了一种现象，其中施加到大脑的非破坏性的伤害性刺激，诱导瞬态电阻对后续的损害缺血性损伤4。 IT发生在两个不同的时间窗口：早期的IT分钟到几个小时后，PC内发生延迟，这需要一个延迟了几个小时，发生4。 到目前为止，一直专注于IT研究脑延迟IT。少得多称为早期IT的机制。这项研究的目的是建立双侧颈总动脉闭塞（夹闭双侧颈总动脉），这是一个既定的刺激诱导延迟IT，作为充足PC刺激诱导早期局灶性脑缺血（大脑中动脉闭塞引起的缺血） C57BL6 / J小鼠。 在这两个手术（夹闭双侧颈总动脉缺血），通过激光多普勒血流仪（LDF）监测脑血流（CBF）进行。 夹闭双侧颈总动脉进行了在麻醉和自主呼吸小鼠。暴露双侧颈总动脉（CCA）和闭塞，持续60秒，然后通过5分钟的再灌注。此夹闭双侧颈总动脉/再灌注序列重复两次。手术后，老鼠恢复得很快，没有任何迹象表明，功能障碍。为了排除，夹闭双侧颈总动脉上述协议并未导致延迟在脑细胞的死亡，我们在执行一个TDT介导的dUTP-生物素缺口末端标记法（TUNEL）染色72小时后，夹闭双侧颈总动脉（或假手术）独立成团。 缺血诱导局灶性脑缺血啮齿动物5-7是一个广泛建立的模型。手术进行了使用标准化作业程序（SOP）8。曝光后的左CCA，硅覆盖的单丝引入的前端CCA，先进到Willis环通过内颈动脉（ICA），直到大脑前动脉（ACA），从而，中脑动脉的起源（ MCA）闭塞定义的一段时间。在这项研究中，我们用45分钟的缺血，这通常会导致的缺血性病变在马华境内涉及纹状体和大脑皮质区。 在我们的研究中，我们分析了早期使用夹闭双侧颈总动脉作为PC刺激时空的档案。我们的数据表明，夹闭双侧颈总动脉缺血的最佳时间之间的延迟引发早期的IT是30分钟。 在这段视频中，我们将给予两个外科手术， 即夹闭双侧颈总动脉缺血的示范。

<table border="1">
<tbody>
<tr>
	<td>Name of the instrument</td>
 <td>Company</td>
 <td>Catalogue number/model</td>
</tr>
<tr>
	<td>Binocular surgical microscope</td>
 <td>Zeiss</td>
 <td>Stemi 2000 C</td>
</tr>
<tr>
	<td>Light source for microscope</td>
 <td>Zeiss</td>
 <td>SteREO CL 1500 ECO</td>
</tr>
<tr>
	<td>Heating pad with rectal probe</td>
 <td>FST</td>
 <td>21061-10</td>
</tr>
<tr>
	<td>Stereotactic frame</td>
 <td>David Kopf</td>
 <td>Model 930</td>
</tr>
<tr>
	<td>Scissors</td>
 <td>FST</td>
 <td>91460-11</td>
</tr>
<tr>
	<td>Dumont forceps #5</td>
 <td>FST</td>
 <td>11251-10</td>
</tr>
<tr>
	<td>Dumont forceps #7</td>
 <td>FST</td>
 <td>11271-30</td>
</tr>
<tr>
	<td>Mircovascular clamp</td>
 <td>FST</td>
 <td>00398-02</td>
</tr>
<tr>
	<td>Clamp applicator</td>
 <td>FST</td>
 <td>00072-14</td>
</tr>
<tr>
	<td>Springscissors</td>
 <td>FST</td>
 <td>15372-62</td>
</tr>
<tr>
	<td>Needle holder</td>
 <td>FST</td>
 <td>12010-14</td>
</tr>
<tr>
	<td>Needles	</td>
 <td>Feuerstein, Suprama	</td>
 <td>BER 562-20</td>
</tr>
<tr>
	<td>5-0 silk suture</td>
 <td>Feuerstein, Suprama	</td>
 <td></td>
</tr>
<tr>
	<td>7-0 silk suture</td>
 <td>Feuerstein,Suprama	</td>
 <td></td>
</tr>
<tr>
	<td>8-0 silk suture</td>
 <td>Feuerstein, Suprama	</td>
 <td></td>
</tr>
</tbody>
</table>

bilateral common carotid artery occlusion as an adequate preconditioning stimulus to induce early ischemic tolerance to focal cerebral ischemia

有越来越多的证据，即缺血预处理 - 非伤害性脑缺血大脑的挑战 - 赋予随后破坏性的缺血性损伤的瞬态保护。我们已经建立了双侧颈总动脉闭塞C57BL6 / J小鼠局灶性脑缺血预处理刺激诱发早期缺血耐受。在这段视频中，我们将展示这项研究使用的方法。

我们夹闭双侧颈总动脉协议导致的直接和深远的（&gt; 90％）的脑血流减少时都CCA闭塞（ 图1）。重启CCA导致灌注完成，LDF（ 图1）监测。 PC，动物后没有任何迹象表明功能残疾。此外，无死亡病例。 TUNEL染色一组独立的动物进行了72小时后，夹闭双侧颈总动脉夹闭双侧颈总动脉（数据未显示）后，大脑中没有任何迹象表明，迟发性细胞死亡。 当分析夹...

Watch this Scientific Journal Video about Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia at JoVE.com

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

有越来越多的证据，即缺血预处理（PC） - 非伤害性脑缺血大脑的挑战 - 赋予随后破坏性的缺血性损伤的瞬态保护。我们建立双侧颈总动脉闭塞（夹闭双侧颈总动脉）作为预处理刺激诱导早期缺血耐受（IT）C57BL6 / J小鼠局灶性脑缺血（大脑中动脉闭塞，缺血引起的）。

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

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

bilateral-common-carotid-artery-occlusion-as-an-adequate

Research

JoVE Journal

Medicine

28.8K 次观看.  Charité - Universitätsmedizin Berlin, Germany. 有越来越多的证据，即缺血预处理（PC） - 非伤害性脑缺血大脑的挑战 - 赋予随后破坏性的缺血性损伤的瞬态保护。我们建立双侧颈总动脉闭塞（夹闭双侧颈总动脉）作为预处理刺激诱导早期缺血耐受（IT）C57BL6 / J小鼠局灶性脑缺血（大脑中动脉闭塞，缺血引起的）。

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

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 ...

科研

医学

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.

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

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 ...

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.

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

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.

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成年小鼠静脉高压型号：颈总动脉，以颈外静脉吻合。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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.

栓塞性脑梗塞（缺血）的大鼠缺血性中风与血同源凝块

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.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

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

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, ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

体内兔颈总动脉内皮的基因转移

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

颈动脉介入移植中颈静脉持久性转基因表达的家兔模型

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...