1。准备所有的动物实验必须符合机构的指导方针，并接收由各自的动物保健委员会批准开始前。这里提出的所有程序已通过韦恩州立大学的机构动物护理和使用委员会和遵循的准则所提出的道德对待动物实验室动物护理和使用指南，向美国政府原则利用脊椎动物用于测试，研究，培训和护理。在手术开始之前，准备必要的手术材料和手术后恢复笼。本程序是一个生存手术，因此，有必要练习无菌技术。 <ol><li>为了使血管导管，切一个8英寸长的50聚乙烯（PE50）管材和迟钝的23号针头插入到另一端。导管可以单独购买预先消毒或购买在bULK与1,2 - 亚乙基二醇，然后灭菌。 </li><li>三路活塞上的一个端口，和对面的端口上放置另一个三通旋塞阀，针固定。 Heparinize活塞和导管肝素生理盐水通过运行。连接到活栓垂直和远端的导管10毫升生理盐水注射器。连接到压力传感器管的导管远端的活栓，用生理盐水填充系统，并消除任何气泡。 </li><li>按照动物使用法规设置的回收笼。恢复室应保持安静，并具有低流量开始手术前的下沉气流表或类似的气体净化系统的重要转机 。过滤蒸发异氟醚。 </li></ol> 2。手术准备 Sprague-Dawley大鼠（300-350克）用于在全球前脑缺血模型2VOH。与异氟醚麻醉诱导和维持和支持plemented与子游离剂量氯胺酮。我们观察到，异氟醚单独维持麻醉术后恢复过程中导致癫痫发作增加发生。癫痫可以促进神经损伤和死亡率，这将影响这种模式的可重复性。补充用氯胺酮麻醉允许低得多的剂量异氟醚达到手术麻醉品位，。使用一个稳态热毯，让严格监管的核心温度。手术过程需要颈动脉的缩减和隔离，动脉和股动脉切下来，插管。 注:在每一个人的手术过程中，重要的是要保持详细的记录。核心体温，血压，或前或在手术过程中发生的其他生理变量的变化可能会大大改变结果，可以分析记录，以确保个人和群体之间有程序的一致性。一致性在禁食动物在手术前，可以提高动物的生理参数。鼓励实验者判断的方法，最一致的预操作血流动力学和血中葡萄糖浓度为他们的研究结果。 <ol><li>到的感应室放置大鼠麻醉，并用30％oxygen/70％一氧化二氮的混合物在5％异氟醚。 12号导管插管，30％oxygen/70％的一氧化二氮混合物与2.5％异氟醚使用灭鼠喉镜和通风。通风率应在2.5毫升，每呼吸80次每分钟。 </li><li>给予腹膜内注射氯胺酮（20毫克/千克）在盐水中，并减少异氟醚1.5％， 重要的是必须在整个手术过程中监测麻醉水平，以确保有足够的手术的麻醉平面。要检查麻醉深度，捏之间的后肢数字，同时监测踏板反射。如果反射缺席，麻醉是足够的。股动脉插管后，血压可作为麻醉水平的指标。低至异氟醚的剂量成为可能，同时保持了手术的麻醉平面，以减少潜在的局部缺血后发作。 </li><li>将切口的颈部和骨盆区。剃须的颈部和右骨盆，大腿满足腹部。东方大鼠稳态热毯在仰卧位，然后插入直肠体温计采用手术润滑剂。应用保护眼睛的润滑剂。一个60瓦的灯泡就可以帮助调节温度。灯泡永远不应该接近从动物超过8英寸。 重要的温度高于或低于正常生理温度（37°C），可以极大地影响最终的神经损害。核心温度保持在37°C±0.5°C。 </li><li>用优碘擦洗切口区域，并用70％乙醇冲洗。重复这个重量Ø次以上。在大鼠放置一个外科领域，切孔切口暴露区域可以被孤立，而不会造成任何创伤，周围的肌肉，这将提高采收率和减少后续需要手术后镇痛重要的颈动脉和股动脉。 </li><li>等待适当的麻醉，用10号手术刀沿颈部正中切口，然后，用止血直言不讳地剖析直到达到突出的中线胸骨舌骨肌，肌肉群覆盖气管之间的唾液腺。再次使用小心钝性分离技术，从胸锁乳突肌胸骨舌骨肌主要肌肉群分开，两侧。这两个肌肉组围成一个三角形的缩进，它可以用来作为一个地标定位神经血管束，其中包含的颈动脉。的内部和外部的颈动脉颈总分支成。分离颈总动脉近端到双向分叉。 </li><li>轻轻分开，并找到这两块肌肉的颈动脉。为了帮助识别这个容器看看一个脉冲。通过了〜3-0丝线缝合船下方的5英寸长的隔离两侧的颈动脉。清除血管筋膜小心迷走神经和交感神经链都包含在颈部神经血管束，应小心分离颈动脉时，避免造成损坏。 </li><li>使用剪刀进行第二次在腹股沟切口，沿后肢大腿肌肉满足压痕腹部。下面的腹部肌肉解剖，沿大腿肌肉，直到你到达腹股沟韧带。这将会使股骨头的血管神经束。仔细分离股动脉，通过一个5英寸长的船下方3-0丝线缝合。扫清筋膜留5-7毫米暴露的容器。 </li><li>铁暴露的动脉的远端处的一个永久的结。 <st荣&gt;注意常任节应该开始摩擦结，平结（共两节）。此规则包括缝合结。将另外3-0丝线缝合周围动脉近端，并配合一个松散的结。 </li><li>应用牵引缝线的近端处的动脉阻塞血流，通过拉动缝线教。做一个小切口，用眼科剪的船只在顶部。在容器上的牵引力不足将导致的出血，这可以通过施加较重的牵引停止。在活塞关闭的导管。 </li><li>使用血管插管，插入导管进入血管，过去7-9毫米的血管切口向中线。一旦导管被放置在所需的距离，配合松散的结，周围血管和导管固定到位。从容器中取出牵引，允许它自然说谎。 </li><li>管理0.3毫升肝素（100U/ml在生理盐水），静脉注射。刷新任何血出，用少量的生理盐水的导管，以防止凝血。打开的压力监视器和校准设备。打开旋塞，使换能器，以检测血压。要获得精确的测量血压，系统的定位应该不会改变校准后。 </li><li>产生110毫米汞柱至130毫米汞柱的平均动脉压（MAP），异氟醚，应调整剂量。这张地图应该是外科手术的麻醉平面足够的指标。 </li></ol> 3。缺血协议如前所述，四艘船舶供应灌注到大脑。两个颈动脉近端闭塞不会导致脑缺血，因为椎动脉，将弥补通过Willis环。它已经表明，耦合引起的全身性低血压，颈动脉闭塞，将限制通过椎动脉，导致脑缺血再灌注。在这里，我们DESCRIBE提取血液，产生低血压和夹紧的颈动脉，生产控制，可逆性缺血的协议。请参阅图1。 血液中的氧和二氧化碳，葡萄糖浓度和pH值可以不同样品组中的其他个人之间，造成损伤的变化。监视这些生理参数，可以减少心肌梗塞的变异。如前所述，温度是很重要的另一个参数，该参数来调节，但脑温可能不对应于核心温度，灌注28实验缺血期间受到很大的限制。具体的手术设置在大脑中的温度变化，结果镜缺血时核心温度变化。然而，重要的是由经验来确定这一点与热电偶或通过其他手段程序标准化监测脑温实验者。如果核心温度不镜像大脑温度温度，重要的是脑温维持在常温下在整个过程中，核心温度无关。 随机决定，外科医生在这一点上缺血或假手术对照组随机的动物。假手术将遵循所有程序不含任何降低血压和颈内动脉闭塞缺血动物完全一样。重要的是，假手术组相同剂量的麻醉下的持续时间相匹配，缺血动物。如果有一个治疗组，这就要求麻醉前或缺血后，假手术和治疗缺血组与治疗组的麻醉剂量和疗程。 <ol><li>准备止血剪辑和剪辑涂药。的剪辑应该完全闭塞血管，而不会造成任何创伤。可诱发缺血时血流动力学已成为一致。连接10 ml肝素syri中NGE到活栓，垂直和近端导管。应为0.3毫升（30个单位）的肝素生理盐水注射器内，以防止排出的血液凝固。 </li><li>设置一个定时器，持续1分钟，打开活塞的肝素化的注射器，所以能被抽出血液。 </li><li>拉动注射器的柱塞上抽回血。如果太多吸，该船将折叠或密封导管开口，将绘制无血。它应该是可以除去在1分钟7-9毫升的血液。如果不是这种情况下，推进导管插入动脉，然后重试。我们发现7-9毫升血液，必须删除，以减少MAP近30毫米汞柱，目标血压达到缺血的重要保证抽取的血液被保持在37℃，以避免血回输过程中的冷却。 </li><li>经过一分钟的抽血（7-9毫升的血应该被撤回），适用于颈动脉止血剪辑，并开始8分钟定时器。立即检查血压。如果MAP在30毫米汞柱，注入或抽回血，慢慢达到30毫米汞柱±1毫米汞柱。继续这种做法在整个缺血维持MAP 30毫米汞柱。 重要的记录血压在整个缺血协议的。如果血压降低30毫米汞柱，可能会限制缺血。显示器核心和/或脑部温度作为温度的增加或减少回输过程中密切合作可能会影响大脑损伤。 </li><li>在8分钟结束时，开始，移除止血剪辑和回输血液缓慢，每分钟2ml灌注。 </li><li>当血液回输可以被删除从股动脉插管。为了避免任何在运算后，安全两个单独的结对动脉切口近端出血。 </li><li>具有不连续模式采用逆切割针，用5-0 VICRYL的缝合，缝合切口。这种缝合线会溶解，所以它不是Require删除。 注:在手术过程中，如果有足够的麻醉不能达到较低水平的异氟醚，管理一个额外剂量氯胺酮。 </li></ol> 4。镇痛和恢复动物福利在恢复期间，以及外科手术过程的优先级。手术后的护理，应当按照该机构的具体指导方针。进行全球性脑缺血的动物很容易发作。为了最大限度地减少癫痫发作是很重要的恢复室有最小的刺激，如噪音和视觉干扰。根据我们的动物使用协议，我们的房子就此而言，在一个僻静的房间72小时的手术后的动物。 <ol><li>已缝合切口后，老鼠可以断奶关闭呼吸机。关闭异氟醚，直到老鼠开始对呼吸机呼吸，并继续在30％oxygen/70％的一氧化二氮的通风。 </li><li>管理0.5毫克/ K克布托啡诺在5毫升生理盐水，肩胛骨之间的皮下。 </li><li>快速回输血液可能增加右心室的预紧力。这会增加肺循环压力，并可能导致肺水肿。体征包括呼吸吃力的呼吸和噼啪作响的声音。应用呼气末正压，将有助于改善肺水肿。 </li><li>当自愿控制呼吸恢复，拔管，取出温度计，关闭加热毯。在这一点上，能够将动物放置到一个回收笼。应放置回收笼，所以笼子的一半之上的循环水毯（34°C）的第一个24小时再灌注。将动物笼地板上的一部分，通过加热毯，以帮助维持温度。一旦动物开始恢复从麻醉和镇痛，它会移动在笼子里，温度调节自身。动物将被分别安置在后运。 </li><li>动物应该有无限的水。两天后运，这种水含有4毫克/公斤液体泰诺的解决方案。在除了灭鼠州城，加糖谷物早餐在笼子里，刺激的饮食和防止减肥。 </li><li>与悬垂笼盖了一半，手术后的恢复，并密切监察。 </li><li>由于动物从麻醉中恢复是很重要的监测遇险的迹象。吃力或不一致的呼吸可以是一个标志的困扰，可能引起肺水肿或气道插管时的刺激。 Butorphenol稳重的动物，减少活动，但轻度活动应在1小时内恢复。我们发现，动物们开始吃零食，喝4小时内拔管。 12小时，正常的行为和喂养活动应该可以恢复。遇险的另一个迹象是减肥;动物应该失去在任何48小时跨度不超过原体重的20％，如果过度减肥，我s者，应实施安乐死的动物。 </li><li>人性化的干预是必要的，如果出现癫痫发作。这种类型的痫性活动通常被再灌注后24至48小时。癫痫发作可引起脑损伤增加缺血侮辱本身是独立的，因而癫痫发作，应考虑在地方设置一个排除标准之前开始研究29。癫痫发作发生时，没有一个是存在的，所以重要的是认识到的迹象。床上用品会受到干扰，并可能被驱逐了鸟笼。发作后大鼠有懒洋洋的处置，快速，呼吸困难和/或撞伤或鼻出血。 </li></ol> 注意疼痛或痛苦的任何迹象，立即用止痛药缓解。如果sympotoms不是一剂止痛药缓解或坚持连续第四镇痛剂量后，将被实施安乐死的动物排除在研究之外。手术禾应检查unds适当运算后八天愈合。 5。组织收集和处理大脑是固定多聚甲醛transcardial滴注。毛切片和冷冻保护后，急速冷冻的大脑和部分在低温恒温器。这些大脑切片用甲酚紫染色，光镜下成像。 <ol><li>动物放置到的感应腔室和填充用30％氧气/ 70％的一氧化二氮，用5％异氟醚麻醉。插管动物和通风在30％氧气/ 70％的一氧化二氮的5％异氟醚。 </li><li>准备的材料的灌注过程。在一个烧杯中，用100ml的1倍磷酸盐缓冲盐水（PBS），在4℃和一个烧杯中，用100毫升的4％多聚甲醛（PFA）在4℃下大脑是用PBS冲洗PFA，其次是通过灌注泵。套管将被放置到每个烧杯中，并连接到一个由一个三通阀LLOW从PBS PFA灌注平稳过渡。迟钝的18号针头，在管道上，并用PBS填充线。确保有针和油管空气中无气泡，可能形成肺栓塞，这可能防止灌注。另外，准备一个小容器与PFA的大脑浸泡固定。 </li><li>使用流体的收集托盘，有足够的空间来容纳至少200毫升的液体。在这一点上的动物应通风足够长的时间（至少3分钟），以达到深度麻醉用异氟醚。 </li><li> 重要确保达到手术的麻醉平面。 </li><li>继续通过使切口访问通过腹部的胸腔。钳降主动脉。当心脏暴露出来，插入18号针头钝通过顶点的心脏到主动脉。做一个小切口在右耳廓允许灌流退出的动物。 </li><li>打开泵，在50毫升/分钟，用PBS开始。经过pumpi纳克100毫升的PBS，切换活塞灌注100毫升煤灰。 </li><li>取出大脑，同时小心不要损坏组织。 </li><li>将脑组织成一个矩阵，切小脑和大脑之间。第前脑;〜3毫米，从正面和背面的大脑皮质。这将产生三个部分，中间部分将包含海马。 </li><li>将脑切片成一个jar足够的煤灰完全沉浸。沉浸修复大脑整整2个小时。 </li><li>为了保护组织免受损伤引起的冻结，大脑cryoproteced更换的煤灰与30％蔗糖的PBS中。冻结保护是完整的组织标本沉沦在蔗糖溶液（24-48小时）。 </li><li>为了急速冷冻的大脑部分，一个烧杯放置在干冰，并填写与2 - 甲基丁醇为10分钟。大脑切片放置5分钟到甲基丁醇，然后转移到一个密封的容器和储存在-80°C至cryost切片。 </li><li>低温恒温器部分的大脑在20微米，收集至少3个部分，在3脑图谱坐标（大鼠脑立体定向Paxinos和Watson，板第29，31，33或毫米前囟-2.8，-3.3毫米，-3.8毫米）。的部分被放置在充电的显微镜玻片上，并使其干燥，然后储存在​​-80℃下</li><li>在pH值3.5）（0.1％甲酚紫染色脑切片，在每个脑图谱坐标。 </li><li>图片用显微镜在40倍的海马CA1区安装摄像头和插入300μm的刻度条与CA1区神经细胞的平面平行。图像保存为TIFF文件。 </li></ol> 6。脑损伤量化 ImageJ的，由美国国立卫生研究院提供一个免费的程序，可以用来计算和量化可行的神经元，这将代表脑损伤的程度。 <ol><li>打开显微镜TIFF图像ImageJ的"细胞计数器"插件。选择一个标记的颜色和计数神经元内边界的标尺。神经计算的基础上简单的纳入标准，基于神经元形态（大，锥体形状）小胶质细胞/小圆形或长管状）星形胶质细胞形态（或排除标准。 重要幻灯片之间的纳入/排除标准，和个人的神经元计数时一致。 </li><li>记录神经元计数，并保存细胞计数器窗口。 </li><li>神经元计数应完成由两个独立的人，以达到一致，准确的神经元计数。 </li><li>所有9个图像的平均值，从每个动物中，将提供一个平均的"神经元计数"，这是在统计分析中使用的海马CA1区损伤的定量测量。静态组可以比较使用单因素方差分析，随后由一个杜克的HSD 事后分析测试，或测试失败常态，秩和随后邓恩的PO单因素方差分析行列ST特别分析，统计评估组之间的差异。具体的统计分析应取决于由各个研究设计，这里介绍的可能不适合被测试的具体假设。 </li></ol>

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作者有没有经济利益冲突。

这里描述的模型产生的缺血性损伤到大脑，可发生心脏骤停，复苏的结果，提供了类似的伤害在人类身上发现的。这种方法生产全脑缺血多种协议之一。我们利用这个协议，也是最重要的，其相对较低的死亡率，恢复快，可重复的结果。心跳骤停/复苏模型可以说是临床上最相关的模型，但是技术上最难不断重现。 4VO全脑缺血模型是另一种常用的协议，有价值的事实，所有四个促成血管闭塞缺血?...

脑损伤，心脏骤停和中风的后果是死亡和长期残疾的首要原因。虽然成功心肺复苏的心脏骤停的受害者至少有60％的患者随后死在医院恢复自主循环，每年大约70,000名患者在美国7,8由于广泛的脑损伤，只有3-10％复苏的患者可以恢复他们以前的生活方式9,10。显然，了解脑损伤的机制，导致全脑缺血后设计治疗干预措施，以最大限度地减少神经创伤是非常重要的。 利用多种方法，可以模拟脑缺血。最常见的脑缺血产生的啮齿类动物的大脑，大脑中动脉闭塞的主要血管，从而产生局部缺血性中风11,12。虽然临床上重要的，局灶性脑缺血是不准确的方法来研究脑损伤所产生心脏骤停/复苏。这项临床范式建模必须整个大脑缺血后血流放归。要密切模仿这种临床表现，研究者通过实验诱发心跳骤停，心肺复苏和除颤13,14复苏。这个模型是临床相关性，但不可预知的复苏时间可以增加变化，可能使数据分析难以解释。此外，这款机型具有高死亡率相关联，进一步提高动物数量需要测试一个假设。调查更重现性好，一致性和生存能力侮辱的全脑缺血和/或再灌注损伤的脑反应可能会是首选。 全脑缺血可诱发大脑中的血流全身，同时保留了一些。这降低死亡率，同时允许侦查n的组织损伤的机制在大脑中2。要产生全球性的脑缺血，有必要中断或大大限制流量在所有四个大脑，颈内动脉和椎动脉的血管供应。这些船只供应大脑的血液流经血管结构叫做威利斯，形成一个吻合口环圈。这种血管的架构，使大脑能够保留灌注近端血管闭塞事件。因此，诱导完全缺血的大脑，血液流动必须通过所有供款船只发生。颈内动脉闭塞，可以通过使用微创腹侧颈切下来，动脉瘤夹应用所需期间。通过椎动脉血流中断可以是困难的，因为它们在横向的脊柱椎间孔incased。调查人员已经解决了这个通过electrocauterizing椎动脉24-48小时前颈闭塞和脑缺血（4VO模型）15。在这种方法中，Smith 等人开发了一种方法，通过减少全脑缺血诱导全身平均动脉压（MAP）至40毫米汞柱，以减少通过椎动脉灌注血流量丢失或大大降低到一个点。再加上颈动脉闭塞时，这种方法产生的整个前脑缺血造成的脑损伤的图案，密切模仿，心脏骤停幸存者。在此方法的进一步改进中，我们在座的模型需要在30毫米汞柱±1mHg紧MAP调节在整个8分钟的缺血。我们发现，这种改变提高了重现性脑损伤的影响该模型，Smith 等人的原始设计的技术，同时保持低的死亡率。 精确的表型的细胞死亡和整体组织损伤程度所造成的这里提出的模型是直接依赖于缺血持续时间16。 8分钟的缺血之后，CA1区神经元表现出延迟的细胞死亡，表明在再灌注阶段15,17的治疗干预，有一个时间窗口。在再灌注的发作，神经元迅速恢复功能，没有直接的细胞死亡是检测18。然而，这侮辱导致诱导细胞死亡的级联（凋亡）从线粒体释放凋亡蛋白，包括细胞色素C，4-6小时再灌注3,19之间达到高潮。 6至24小时再灌注，海马CA1区神经元的细胞消亡，凋亡的细胞死亡程序执行了19。应该指出的是负责缺血性损伤的细胞死亡表型是极具争议。早期的研究表明坏死是主要的细胞死亡表型，20,21，而其他人报告apoptOSIS的主要机制22,23。总体而言，目前的证据表明，细胞死亡的频谱范围从经典的细胞凋亡坏死的细胞死亡表型。性细胞死亡的具体模式是依赖于许多因素，各表型的贡献程度取决于侮的严重程度，除其他因素外24,25。再灌注24小时，死亡的细胞具有细胞核固缩，简明细胞质聚集细胞内容物有明确的证据，并失去功能的线粒体形态。死细胞进一步细分，由免疫细胞如巨噬细胞和/或小胶质细胞吞噬，清除从海马CA1区区域。通过再灌注4-7天，去除死细胞，所有剩下的炎症细胞和胶质细胞激活17,26。因此，7天的最佳时间再灌注海马CA1区神经元死亡的，可以量化的，使用简单，非特异性细胞污渍少量甲酚紫或复习续 - 伊红计数基于形态学纳入标准。留在这个晚期再灌注间隔可以算作存活细胞的细胞，从而提供了一个索引的脑损伤。 如果这个模型将被用来测试治疗干预，因此建议，实验设计遵循的楼梯标准（中风治疗学术产业圆桌会议）27。设计和进行研究时，应遵循这些准则，但这里不讨论。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material Name</td>
 </tr>
 <tr>
 <td>5-0 VICRYL suture, reverse cutting </td>
 <td>Ethicon</td>
 <td>J391H</td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel, No.10</td>
 <td>Swann-Morton</td>
 <td>6601</td>
 <td></td>
 </tr>
 <tr>
 <td>Gauze Sponges</td>
 <td>Fisher</td>
 <td>22-362-178</td>
 <td></td>
 </tr>
 <tr>
 <td>18G x 1 &frac12; in needle</td>
 <td>BD</td>
 <td>305201</td>
 <td></td>
 </tr>
 <tr>
 <td>23G x 1 in needle</td>
 <td>BD</td>
 <td>305145</td>
 <td></td>
 </tr>
 <tr>
 <td>26 G x 3/8 in needle</td>
 <td>BD</td>
 <td>305110</td>
 <td></td>
 </tr>
 <tr>
 <td>18 G x 1 &frac14; catheter</td>
 <td>EXEL</td>
 <td>26735</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml syringe</td>
 <td>BD</td>
 <td>309659</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml syringe</td>
 <td>BD</td>
 <td>309604</td>
 <td></td>
 </tr>
 <tr>
 <td>60 ml syringe</td>
 <td>BD</td>
 <td>309653</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgilube</td>
 <td>Henry Schein</td>
 <td>1152666</td>
 <td></td>
 </tr>
 <tr>
 <td>.9% Saline, plastic IV bag</td>
 <td>Henry Schein</td>
 <td>1537468</td>
 <td></td>
 </tr>
 <tr>
 <td>Suture 3-0 Silk</td>
 <td>Henry Schein</td>
 <td>1007842</td>
 <td></td>
 </tr>
 <tr>
 <td>Puralube Ophthalmic Ointment</td>
 <td>Henry Schein</td>
 <td>3390017</td>
 <td></td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Henry Schein</td>
 <td>6903564</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Towel Drape</td>
 <td>Moore Medical</td>
 <td>14170</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyethylene Tubing, 50</td>
 <td>Intramedic</td>
 <td>427411</td>
 <td></td>
 </tr>
 <tr>
 <td>Stopcock, 3 way</td>
 <td>Smiths medical</td>
 <td>MX9311L</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Drug Name</td>
 </tr>
 <tr>
 <td>AERRANE (isoflurane)</td>
 <td>Henry Schein</td>
 <td>2091966</td>
 <td></td>
 </tr>
 <tr>
 <td>Mapap Liquid (Tylenol)</td>
 <td>Major Pharmaceuticals</td>
 <td>1556</td>
 <td></td>
 </tr>
 <tr>
 <td>Kedavet (ketamine)</td>
 <td>Ketathesia Butney</td>
 <td>NDC 50989-996-06</td>
 <td></td>
 </tr>
 <tr>
 <td>Butorphic (butorphanol)</td>
 <td>Lloyd Labs</td>
 <td>4881</td>
 <td></td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>APP Pharmaceuticals</td>
 <td>504011</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Chemical Name </td>
 </tr>
 <tr>
 <td>Paraformaldehyde prills</td>
 <td>Elecron Microscopy Sci.</td>
 <td>19202</td>
 <td></td>
 </tr>
 <tr>
 <td>2-methylbutane</td>
 <td>Sigma</td>
 <td>270342</td>
 <td></td>
 </tr>
 <tr>
 <td>Cresyl Violet Acetate</td>
 <td>Sigma</td>
 <td>C5042</td>
 <td></td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Software</td>
 </tr>
 <tr>
 <td>ImageJ</td>
 <td>NIH</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

2-vessel occlusion/hypotension: a rat model of global brain ischemia

其次是复苏的心脏骤停，结果往往是戏剧性的脑损伤引起的大脑缺血和再灌注的。全球脑缺血产生损害特定的大脑区域是高度敏感的缺血1所示。海马神经元中有较高的灵敏度相比，其他的细胞群，具体的缺血损伤，海马CA1区是特别容易受到缺血/再灌注2。 治疗干预，或参与脑损伤的机制研究，需要设计一个模型，产生类似的临床状况和重现的方式损害。双侧颈动脉血管闭塞低血压（2VOH）的可逆的脑缺血，产生一个模型，模拟心脏骤停，复苏过程中可能发生的脑血管事件。我们描述了一个模型修改史密斯等人 。 （1984）2首次提出在其目前的形式，Sanderson 等 （2008）3，产生重复性损伤选择性脆弱的大脑区域3-6。该模型的可靠性取决于全身血压的精确控制在应用性低血压，缺血时间，接近的温度控制，一个特定的麻醉方案，手术后护理和勤奋。有8分钟的缺血性损伤产生进展超过6至24小时再灌注过程中海马CA1区神经元细胞死亡，而较脆弱的大脑区域都未能幸免。这种渐进性细胞死亡是很容易量化7-14天再灌注后，此时，一个近乎完整的CA1区神经元的损失是显而易见的。 除了这个脑损伤模型中，我们提出了一个简单而透彻，方法CA1区损伤量化的方法。重要的是，量化可以通过使用一个简单的安装在摄像机上的显微镜，达免费ImageJ的研究院（NIH）的软件插件，省却成本过高的体视软件程序进行损失评估和电动显微阶段。

全脑缺血/再灌注模型2VOH导致神经元死亡的海马CA1区。 图2表示的伤害所产生的8分钟全脑缺血再灌注后14天内处理。 图2A和2B比较海马假大脑缺血后，用甲酚紫染色。 图2A显示了表现形态正常，包括一个完整的CA1假手术大鼠海马齿状回，CA2和CA3 图2B表明海马缺血/再灌注，影响最小形态。选择性脆弱的海马CA1区神经元，不生存缺血/再灌?...

Watch this Scientific Journal Video about 2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia at JoVE.com

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

双侧颈动脉闭塞，加上全身性低血压生产全脑缺血大鼠海马损坏，导致重复性严重。受试动物损害与脑损伤预测的模式，它们方便地恢复率和死亡率相对较低。

2血管闭塞/低血压:全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

2-vessel-occlusionhypotension-a-rat-model-of-global-brain-ischemia

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Research

JoVE Journal

Medicine

19.8K 次观看.  Wayne State University School of Medicine. 双侧颈动脉闭塞，加上全身性低血压生产全脑缺血大鼠海马损坏，导致重复性严重。受试动物损害与脑损伤预测的模式，它们方便地恢复率和死亡率相对较低。

视频: 2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

科研

医学

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

在原位横向腹直肌肌皮瓣:肌缺血再灌注损伤模型大鼠

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

主动脉阻塞后脊髓缺血大鼠模型中的可再生电机缺陷

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...