Name: continuous iv infusion is the choice treatment route for arginine-vasopressin receptor blocker conivaptan in mice to study stroke-evoked brain edema
Uploaded: 2016-09-01T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema at JoVE.c

我们感谢瑞典医疗中心提供资金和设施。我们也感谢克雷格医院的大量使用的实验室空间。

实验是按照美国国立卫生研究院的照顾和使用动物研究的指导方针进行，由瑞典医学中心的动物护理和使用委员会批准。所有的程序都用适当的无菌技术进行的。这项研究使用的实验动物均为男性，3个月大，体重野生型C57小鼠25日至27克 1. 在体内诱导中风<ol><li>预涂层与树脂牙灯丝此前的手术。 <ol><li>剪切从7-0尼龙线12毫米长片丝。混合2份该树脂与硬化剂的1份，然后立即浸长丝的前端到混合物，以覆盖整个长度的约1/3。快速清除并验证它覆盖的牙科树脂的表面光滑的外套。 </li><li>风干灯丝在使用前2小时。 </li></ol></li><li>将鼠标进入Ñ​​麻醉诱导室（2L体积），并设置在2升/分钟进入腔室1.5％异氟烷在25％的富氧室内空气的流动。 </li><li>允许小鼠留在感应室中直至完全麻醉，8由缺乏响应于尾捏确定。 </li><li>将鼠标走上手术台用加热垫，并设定1％的异氟醚浓度维持通过鼻锥自然娩出麻醉。 </li><li>夹上的前部和颈部的背毛，并用酒精喷颈部。在两只眼睛应用兽医眼药膏。 </li><li>连续监测体温，用在手术过程中连接到数字显示直肠温度探头。 </li><li>鼠标放置在仰卧位，应用碘伏10％溶液擦洗消毒，用无菌悬垂盖，然后使沿颈部正中切口，大小10手术刀刀片。 </li> &lt;利&gt;暂时结扎左颈总动脉（CCA）以6.0缝合丝线。 </li><li>放置在左侧颈外动脉（ECA）的另一个领带。 </li><li>放置在颈内动脉（ICA）微血管剪辑。 </li><li>削减与微血管剪刀颈外动脉一小的孔。插入来自涂上树脂牙将在ECA开幕7-0尼龙线做灯丝，并cephalically推进它，同时消除夹，直到感到有阻力（从CCA分叉处约5-7毫米）。 </li><li>放置一个临时粘结到所述内颈动脉，以确保灯丝中的位置。参考下图， 图1A。收用丝线缝合皮肤，并渗透在颈部的前部的伤口用0.2ml 0.5％布比卡因，如在步骤2.6。 </li><li>让鼠标从麻醉中苏醒并恢复意识在恢复室中60分钟。不要离开无人看管的动物，直到它已经恢复足够的意识，以保持胸骨靠着。 </li><li>测试鼠标的神经功能缺损评分（NDS）如下:0 =运动功能正常，躯干和尾板对侧前肢1 =前屈，2 =盘旋至对侧，但在休息，3正常坐姿=偏向对侧侧静止的，4 =无自发运动活性。4,9 <ol><li>对于NDS测试，尾巴和地点在一个平面上进行观察（2-3分钟）抬起鼠标。另外，观察在恢复室​​老鼠。排除表现出比NDS 2从实验降低因MCA闭塞不足小鼠。 注意:激光多普勒血流仪技术可以被用来确认MCA闭塞9-11。 </li></ol></li><li>重新麻醉如上所述动物后在感应腔室恢复60分钟，再消毒用10％聚维酮碘溶液擦洗先前切开皮肤，并重新打开在手术伤口通过切割和去除丝线缝合颈部的前面。 </li><li>从ECA取出长丝执行以下步骤: <ol><li>广场上的ICA微血管剪辑。解开对ICA的缝线拉出长丝的同时抱夹打开。 </li><li>关闭剪辑和近端放置领带到非洲经委会晋级。 </li><li>除去夹子和从CCA除去结扎以恢复血流到大脑。参考下图， 图1B。 </li></ol></li></ol> 2.安装静脉导管进入颈静脉连续48小时处理注:步骤1.16.3没有鼠标醒后立即进行安装导管。 <ol><li>使用柔性管的两种不同的尺寸:插入所述内部管道（外径0.94毫米;​​以包含药物被注入）到外管（外径3.18毫米;对于内管的保护）。 </li><li> P花边鼠标在俯卧位，适用10％聚维酮碘溶液擦洗消毒，并在脖子上的，大小10手术刀刀片后面有1厘米的切口。转小鼠成仰卧位，重新消毒用10％聚维酮碘溶液擦洗颈部的前部，并重新打开在颈部的前部的外科手术伤口。隧道从背面到颈部的前所作的切口在皮肤下的管道，并从打开的伤口1厘米exteriorize它。 </li><li>从颈部的前部横向约1cm距离中线的左边除去皮下脂肪组织。找到左侧颈内静脉，将两条领带沿静脉相距5mm，并且略微拉长静脉。就微血管剪刀两个关系之间的颈静脉一个小孔;插入内管5 mm深尾端尖（朝心脏）。 </li><li>固定管与两个联系的颈静脉。关闭皮肤在颈部的前端与3-0丝线缝合。参考下图， 图1C。 </li><li>固定油管的颈部与缝合背面的皮肤，并通过一个旋转连接管连接到一个微注射静脉泵。旋转允许在笼子里面有完全访问食物和水的小鼠自由流动。 </li><li>渗透前面的皮肤切口和颈部用0.2ml 0.5％布比卡因防止手术后疼痛的背面。 注:根据机构特定动物的协议，动物可能与阿片类药物，NSAIDs类药物或抗生素前和手术后进行治疗。然而，提及的治疗可能会改变缺血性脑卒中结局结果 （见讨论部分 ）11-14 </li><li>让动物在回收室唤醒，然后安置动物在一个单独的笼子，直到实验结束点。不要让动物无人看管，直到它已经恢复了足够的意识，以保持胸骨靠着。 </li><li>以1.5ml / kg /小时，4设置为连续48小时静脉治疗考尼伐坦的（0.14毫克/毫升，在5％右旋糖）或车辆输注速度和开始输注。 注意:四输注考尼伐坦的总量应该大约为0.2毫克/小鼠/天，1.44毫升总体积。 注意:为0.2毫克总量在1.44毫升5％右旋糖，每天两次执行考尼伐坦腹腔（IP）注射。实现IP注射如前所述。15通过抓住主体和尾部的后表面的皮肤抑制的小鼠。注入考尼伐坦或车辆进入使用26 G / 1 /寸毫针腹部的左下腹。15 </li><li>观察动物每天两次的实验（48小时）的整个持续时间。让动物充分获得食物和水。如果动物表现出痛苦或呼吸困难的迹象，请教兽医。如果安乐死是必要的严重DIST动物RESS参考以下步骤3.1，并排除在研究动物。 </li></ol>在终点中风诱发脑水肿3.评估<ol><li>通过用5％异氟醚的过麻醉牺牲鼠标。 </li><li>使用手术刀，使过沿中线颅骨切开皮肤，并缩回皮以暴露在大脑中的整个颅骨。在枕骨大孔开始，切开颅骨沿两侧大脑的周边横向，然后小心地抬起而不触及它下面的大脑。切断从脊柱脑和附着在颅骨的基础上的神经。 </li><li>取出大脑，它从嗅球和小脑分开，并解剖沿纵裂大脑进入缺血和非缺血大脑半球，如前所述。9 </li><li>通过如前面描述的4,9比较湿到干比（WDR）评估脑水肿。权衡组织之前和在烘箱中在100℃下干燥3天。计算脑含水量（BWC）的％H 2 O =（1-干重/湿重）×100％。 </li></ol>

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这项研究有临床前研究中风重要的价值。这项研究表明，小鼠实验中风后考尼伐坦（0.2毫克/天）的持续静脉注射，有效降低治疗后的48小时脑水肿。 IP注射相同剂量对脑水肿考尼伐坦的效果也进行了研究。考尼伐坦治疗由双方IV和IP路由产生aquaresis由所示小鼠:1）血浆渗透压略高于生理水平的提高;和2）在尿渗透压降低由于在肾水再吸收的堵塞。因此，基于对治疗结果，我们报告中指出，四交付方法，对中风引起的水肿形成另外一个重要的有益作用。 我们的主要发现是，IV处理产生的脑水肿减轻比考尼伐坦IP路由效果更佳。考尼伐坦是组合V1a和V2受体拮抗剂，可用于临床使用至COR矩形低钠血症，血容量和渗透压。16考尼伐坦已经显示纠正患者低钠血症各种条件17考尼伐坦的临床情况表明，它在脑水肿的上下文勘探可导致快速长凳到床头的翻译。脑室内递送实验选择性V1A受体阻断剂的使用已经被证明可以减少由其他研究者描述局部缺血后的脑水肿的形成。3,18然而，无论是在这些研究中使用的药物，也没有治疗路径的药物建议在可能的临床应用不远的将来。 机制，使大脑缺血引起AVP脑水肿是复杂的。然而，一些重要的分子的玩家是设置在血脑屏障界面定位于血管壁钠和氯共转运19,20和V1A受体。 V2的阻挡考尼伐坦的作用是负责exces排泄由肾和血液渗透压，这也可能有助于防止脑水肿的发展的高程。21这些机制的水资源可能仍然在播放时考尼伐坦被直接输送到循环，以避免可能降低考尼伐坦可用性组织障碍。然而，剂量和治疗持续时间的理由是基于以前出版的观察结果。4它仍有 ​​待确定局部血流的损伤诱导的下降后考尼伐坦是否到达脑的缺血部位。它也需要进行调查大脑是否接收足够量的考尼伐坦的由在脑动脉阻塞V1A受体以产生期望的局部作用，如预防血管收缩。 实验方案的目的是要解决的选择的问题为考尼伐坦的治疗途径。根据研究结果，该研究为考尼伐坦广告的IV途径大力支持服侍。然而，需要建立和为了检测小鼠卒中后在治疗结果显著差别微调协议的最重要的步骤。在这些步骤是:1）在MCAO（60分钟）的持续时间; 2）日剂量（0.2毫克），和输注速度（1.5毫升/ kg /小时）;和3）的治疗（48小时）的长度。 MCAO的持续时间决定的局部缺血损伤的程度这对于协议，因为它不可逆地损害脑组织极为重要。太严重受伤将难以为动物以48小时存活，或动物可能成为对脑水肿考尼伐坦治疗不太敏感。另一方面，MCAO的持续时间小于60分钟，可能不足以诱导缺血性损伤的小鼠，并且，因此，不会产生脑水肿。剂量和治疗的长度被选择的基础上对考尼伐坦使用美国FDA建议。16然而，已经报道了对考尼伐坦人用剂量是效率不高的小鼠中风后减轻脑水肿。因此，剂量的小鼠，它诱导了针对局部缺血后的脑水肿显著保护要增加10倍。4选择正确的滴注速度的重要性在于，它可以影响总血量，血压和间质液的积累，如果率大大超过建议的速率。然而，总血容量和血压测量是非常丰富。 在缺血的手术感应是一种侵入性的过程，需要精确的重视协议的所有步骤，并重新认识该技术的故障排除技巧是很重要的。一些意想不到的手术并发症，如过度动脉出血由于一个意外撕裂动脉减少用于实验的成功率，提高死亡率。然而，静脉出血可以通过施加轻微的压力瓦特停止第i个无菌海绵。 本协议描述的脑缺血，这意味着该MCA闭塞生产无论是在向右或向左MCA单方面的诱导。然而，该协议可以根据外科医生的优选MCA闭塞和IV导管安装侧被修改。 图1图解描述了MCAO程序， 图1A和B中 ，IV导管安装协议的最关键部分的图， 图如图1C所示 ，并且在实验（48小时）的终点MCAO引起的脑梗塞的代表性脑切片，如先前出版，10 图1D。虽然图和代表脑切片暗示MCAO于大脑的左侧诱导，如果MCAO于大脑的右侧只要整个烯诱导作为一致性我们不认为在结果中的巨大差异轮胎的研究得以保持。 在手术过程中和麻醉后的恢复期间，动物可能会遇到的苦恼，由于手术后的疼痛和严重的神经功能缺损。在许多情况下，术后疼痛管理可以被限制为仅使用局部麻醉剂，如0.5％布比卡因。由于在人类中脑血管意外，以及在小鼠中实验性卒中中风涉及病理生理因素，如炎症，脑梗塞，和脑水肿。9，这些病理生理事件由关键分子玩家从事诸如环加氧酶（COX）13和G蛋白相关受体。22干扰涉及COX分子途径可能由NSAID药物引起的，13，如果他们在这些设置中使用。阿片类药物作用于它们的受体哪些链接到G-蛋白与第二信使12，并且可以改变行程，11和结果的病理生理结果s此研究。这些因素限制了小鼠术后止痛药的使用。 总之，我们的研究探讨再利用考尼伐坦作为一个深远的目标的可能性。我们的实验中风诱导后考尼伐坦的连续静脉输注的动物模型已经显示出产生一致的结果这表明其在中风患者的潜在用途。更具体地说，我们已经表明，持续静脉治疗中风后考尼伐坦，以防止继发性脑损伤的模型可以用于在小鼠中的临床前研究。这项研究的目的是提供治疗考尼伐坦的另一种应用程序作为一个额外的工具来研究小鼠脑水肿中风后，因为快译到临床设置可能是救命的ICU患者。

Stroke continues to be an enormous burden for patients and clinicians. Animal stroke models have been used in the laboratory setting for nearly two decades.1 Nevertheless, experimental treatments that work in animals often fail in humans.2 This discrepancy in treatment outcomes may be due to various factors, such as administration routes for drugs used in animal research, drug metabolism and elimination rate, and many other aspects. One of the major complications of stroke, brain edema, is a focus of current research in neuroscience. Mechanisms of brain edema formation involve disturbances in water and electrolyte balance triggered by the arginine-vasopressin (AVP) response to ischemic brain injury.3 Therefore, AVP and its receptors (V1a and V2) are a major focus of research studies of post-ischemic brain edema formation.3
We have developed a methodology to study the effects of mixed arginine-vasopressin (V1a and V2) receptor blocker conivaptan on post-ischemic brain edema in mice.4 Molecular targets of conivaptan5 make the drug a suitable candidate for exploration of its properties in alleviation of brain edema. Furthermore, conivaptan needs to be studied in the context of pathophysiological events of stroke.6 In designing this study, we considered comparing treatment outcomes using two different routes of administration for conivaptan: intravenous (IV)4 and intraperitoneal (IP).7 Effects of the treatments on stroke-induced brain edema were evaluated. Here detailed protocols are described for surgical induction of experimental stroke by middle cerebral artery occlusion (MCAO), and followed by continuous conivaptan treatment using the installation of a jugular IV catheter. After induction of stroke, animals were randomly allocated into the following groups: vehicle or conivaptan (0.2 mg/day) treated IV or IP.

<table><tbody><tr><td>Heated Pad</td><td>K&amp;amp;H Manufacturing Inc</td><td>1060</td><td></td></tr><tr><td>Temperature Monitor with Rectal Probe</td><td>Physitemp</td><td>7029</td><td></td></tr><tr><td>Silk Suture Spool, 6-0</td><td>Surgical Specialties Corporation</td><td>SP114</td><td></td></tr><tr><td>Silk Suture on a Needle, 3-0</td><td>Ethicon</td><td>1684G</td><td></td></tr><tr><td>Nylon Suture, 7-0</td><td>Ethicon</td><td>1696G</td><td></td></tr><tr><td>Dental Resin Polysiloxane with Hardener</td><td>Heraeus Kulzer</td><td>65817930</td><td></td></tr><tr><td>Microinfusion IV Pump</td><td>Kent Scietific</td><td>GT0897</td><td></td></tr><tr><td>Swivel 22GA</td><td>Instech</td><td>375/22PS</td><td></td></tr><tr><td>Laboratory Tubing, 0.94 mm x 0.51 mm</td><td>Dow Corning</td><td>508-002</td><td></td></tr><tr><td>Laboratory Tubing, 3.18 mm x 1.98 mm</td><td>Dow Corning</td><td>508-009</td><td></td></tr></tbody></table>

continuous iv infusion is the choice treatment route for arginine-vasopressin receptor blocker conivaptan in mice to study stroke-evoked brain edema

中风是在世界发病和死亡的主要原因之一。中风是脑水肿等病理生理事件复杂化。间在中风诱发的脑水肿的发展和演变的最重要的球员是激素精氨酸加压素及其受体，V1a和V2。最近，V1a和V2受体阻滞剂考尼伐坦受到关注作为潜在的药物，以减少中风后的脑水肿。然而，这在中风研究涉及考尼伐坦应用动物模型需要根据给药可行途径进行修改。在这里，48小时连续静脉注射（IV）的结果与小鼠实验中风后腹腔内（IP）考尼伐坦治疗相比。我们开发了在大脑中动脉闭塞与导管安装组合为IV治疗考尼伐坦（0.2毫克）或车辆的颈静脉的协议。动物的不同队列，用0.2治疗考尼伐坦或车辆的IP毫克，每天丸。实验中风诱发脑水肿连续IV和IP处理后的小鼠进行了评估。的结果比较，可知考尼伐坦的连续IV施用减轻小鼠局部缺血后的脑水肿，与考尼伐坦的IP施用。我们得出结论，我们的模型，可用于在中风和脑水肿的上下文考尼伐坦应用今后的研究。

动物的体温是在生理范围内，并且在整个冲程感应的手术过程稳定。两个小鼠表现出NDS低于2 MCAO从该研究中排除之后。 在缺血小鼠48小时生产同侧半球梗死体积。的TTC染色切片的评价表明，半球的约50％是由缺血（ 图1D）后梗塞的影响，如通过Zeynalov，E。和多尔，S。，2009年10脑缺血性中风的发病引起严重的出版在氧和营养物质的脑组织的剥夺。作为该局部脑组织缺氧的结果，神经元和胶质细胞的死亡，从而形成大脑的梗塞部分。 图1D示出了受MCAO引起梗塞代表小鼠脑切片，并给出了一个损伤的大小的估计。 <suP&gt; 10 在MCAO诱导48小时60分钟后脑含水量（BWC）的评价显示考尼伐坦IV施用（0.2毫克/天）显著减少同侧和对侧半球脑水肿，（ 图2A）。与此相反，由0.2毫克丸药考尼伐坦的IP的治疗每日未能产生对脑卒中诱发的脑水肿（ 图2B）相似的积极影响。 对血浆和尿渗透压考尼伐坦效果的证实在所有实验动物在实验的终点来实现的。考尼伐坦处理的第IV或IP增加血浆和小鼠中所有小鼠降低尿渗透压4血浆渗透压值如下:313±4.4毫渗量/公斤（车辆，IV）与355±7.5 *毫渗量/公斤（考尼伐坦，Ⅳ）如以前出版，第4和340±7.7毫渗量/公斤（车辆，IP）与383±8.8 *毫渗量/公斤（考尼伐坦，IP）。在小鼠尿渗透压值如下:1210.6±13.4毫渗量/公斤（车辆，IV）与595.0±57.4 *毫渗量/公斤（考尼伐坦，IV）中，如先前所发表的，图4和1535.0±80.9毫渗量/公斤（车辆IP）与242.0±23.2 *毫渗量/公斤（考尼伐坦，IP）。表现为所有的数据平均值±SEM，* P≤0.05与相应的控制。 <img alt="图1" src="/files/ftp_upload/54170/54170fig1.jpg" /> 图 1: 在实验性卒中模型，IV导管的安装，并且在灯丝的冲程位置后48小时代表梗塞体积的图 （以蓝色示出）预涂覆有在左侧颈内动脉树脂（绿色）（ICA）闭塞（A）和再灌注时间（B）的过程中。在左颈静脉（C）的所述柔性管子末端的正确位置。 Experimenta小鼠升行程在48小时生产同侧半球梗死体积。小鼠在48小时MCAO后处死，并取出大脑，在协议中所描述（步骤3.1-3.3）。大脑切成2毫米厚的切片，并用TTC（D）的染色。图1D已经从原来的文章Zeynalov，E和多尔，S.，2009年修改10 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54170/54170fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54170/54170fig2.jpg" /> 图 2: 连续静脉内（IV）和腹膜内（IP）的考尼伐坦（0.2毫克/天）对脑卒中诱发的脑水肿的治疗考尼伐坦静脉治疗48小时给药的影响 。考尼伐坦治疗静脉应用在同侧和对侧小时的比例减少局部缺血后的脑水肿Emisphere公司（A）。图2A已经从原来的文章由Zeynalov 等改性，2015年4考尼伐坦的IP处理48小时各组中未能减少因实验中风的脑水肿，每组10。小鼠进行缺血再灌注如上述（步骤1.1.1-1.15.3）协议中所述。然后考尼伐坦（0.2毫克）的IP注射立即给药，在22小时再灌注46小时。小鼠处死;大脑取出并干燥在烤箱，如在该协议中描述（步骤3.1-3.4）。 （二）所有的数据都以平均值±SEM，* P &lt;0.05与相应的控制。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54170/54170fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema at JoVE.c

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

持续静脉注射是精氨酸加压素受体拮抗剂考尼伐坦的选择治疗途径小鼠来研究中风诱发脑水肿

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

continuous-iv-infusion-is-choice-treatment-route-for-arginine

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.8K 次观看.  Swedish Medical Center. Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice. 

视频: Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

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 ...

科研

医学

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

神经科学

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

创伤性脑损伤的神经炎症和血流动力学反应的系统分析

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Intracranial Pressure Monitoring In Nontraumatic Intraventricular Hemorrhage Rodent Model

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage animal models is essential. In this study, we sought out ways to measure intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during nontraumatic intraventricular hemorrhage in rats. The experimental design included three Sprague Dawley groups: sham, standard 200 &#181;l intraventricular hemorrhage, and vehicle control groups. By introducing an intraparenchymal fiberoptic pressure sensor, precise intracranial pressure measurements were obtained in all groups. Cerebral perfusion pressures were calculated with the knowledge of intracranial pressure and mean arterial pressure values. As expected, the intraventricular hemorrhage and vehicle control groups both experienced a rise in the intracranial pressure and subsequent decline in cerebral perfusion pressure during intraventricular injection of autologous blood and artificial cerebrospinal fluid, respectively. The addition of an intraparenchymal fiberoptic pressure sensor is beneficial in monitoring precise intracranial pressure changes.

Monitoring intracranial pressure in rodent&#160;models of nontraumatic intraventricular hemorrhage is not common in the current literature. Herein, we demonstrate a technique for measuring intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during intraventricular hemorrhage in a rat&#160;animal model.

非创伤性脑室内出血啮齿动物模型中的颅内压监测

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

小鼠脑中动脉闭塞诱导脑卒中后脑肌粘连血管病治疗模型

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

<meta charset="utf8"></head><body>基于荧光染料的小鼠血脑屏障分解可视化

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

<meta charset="utf8"></head><body>轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

持续静脉注射是精氨酸加压素受体拮抗剂考尼伐坦的选择治疗途径小鼠来研究中风诱发脑水肿

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此视频中的章节

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

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

创伤性脑损伤的神经炎症和血流动力学反应的系统分析

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

非创伤性脑室内出血啮齿动物模型中的颅内压监测

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

小鼠脑中动脉闭塞诱导脑卒中后脑肌粘连血管病治疗模型

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价