Name: experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia
Uploaded: 2017-07-31T12:30:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

这项工作由中国国家自然科学基金（81573406,81373393,81273506,81221003,81473186和81402907），浙江省自然科学基金（LR15H310001）和浙江省科技创新队队长计划（2011R50014）资助。

所有实验均经浙江大学动物实验委员会道德准则批准实施，完全符合国家卫生研究院"实验动物护理与使用指南"。努力减少任何疼痛或不适，并使用最少数量的动物。 皮质硬膜下切片的OGD <ol><li> 解决方法: <ol><li>制备1000毫升的r-ACSF（124毫摩尔/ L，氯化钠，5毫摩尔/ L的KCl，1.25毫摩尔/ L KH 2 PO 4，2毫摩尔/升MgSO 4，26毫摩尔/ L的NaHCO 3，2毫摩尔/ L的CaCl 2， 10 mmol / L葡萄糖，最终pH 7.4）。在整个实验中，具有5％CO 2和95％O 2的气泡。 </li><li>如上所述制备200mL无葡萄糖的ACSF（gf-ACSF），但不包括葡萄糖，以5％CO 2和95％N 2鼓泡。气泡气体30分钟，然后应用于brain切片以减少溶液中的氧含量。继续冒泡直到OGD程序完成。 </li><li>通过用20％CO 2和80％O 2平衡200mL r-ACSF来制备酸性ACSF（a-ACSF）。气泡气体30分钟，然后应用于脑切片，将溶液的pH值降至pH6.8，继续鼓泡，直到APC程序完成。 </li><li>将三个组织架分别放入r-ACSF，gf-ACSF，a-ACSF中。 </li></ol></li><li> 脑切片准备: 注意:本研究使用8周龄的雄性C57B1 / 6J小鼠。将动物饲养在受控温度（22±2℃）下，12小时光暗循环周期，并进入沉淀的食物和水。 <ol><li>在感应室中用异氟醚过量的小鼠牺牲。放弃鼠标。用小剪刀和镊子解剖大脑。用薄的铲子取出大脑，小心地放入玻璃烧杯中用5％CO 2和95％O 2平衡的冰冷r-ACSF。 注意:这些步骤应尽可能快速但精细地进行。 </li><li>将大脑置于冰冷的r-ACSF中5分钟。调整气压以避免大脑的运动。 </li><li>将低温沉淀胶放在vibratome板上两条。将一块3％的琼脂糖放在离开刀片的胶条上，以支撑大脑。 </li><li>将10厘米培养皿倒入冰中，然后将滤纸放在盘子上。用冰冷的r-ACSF滴加湿滤纸。 </li><li>用镊子将大脑转移到滤纸上。用刀片和镊子切断额极和小脑。 注意:横截面应尽可能平滑，垂直于矢状面。 </li><li>将剩余的脑组织垂直放置在另一条胶水上，并保持大脑靠在琼脂糖上。将冰冷的r-ACSF加入切割槽淹没大脑，并向冰块区域加冰。将r-ACSF置于用5％CO 2和95％O 2鼓泡的容器中。 </li><li>提高水库并调整剃须刀的位置，使剃须刀尽可能靠近大脑，并在大脑之上。 </li><li>选择"CONT"模式，连续切片脑部。将切割厚度设置为400μm，振动频率为6 - 8，速度为3 - 4.按"启动/停止"按钮开始自动切割。 </li><li>切掉3 mL巴斯德吸管的尖端，使其与大脑切片的大小相匹配。 注意:开口应足够大，以避免对脑切片造成额外的损害。 </li><li>用tip-cut巴斯德移液器逐个抽出五个脑切片，并将其放入冰冷的r-ACSF中的组织保持器中，用5％CO 2和95％O 2鼓泡。不要收集生产的第一片。调整气压到avoi脑部切片的运动。 注意:脑切片不应相互覆盖。 </li><li>将脑切片与r-ACSF在室温下放置30分钟，然后在37℃下在水浴中放置另外10分钟以恢复突触功能。 </li></ol></li><li> OGD和APC: <ol><li>将gf-ACSF和a-ACSF放入37°C水浴中，并用上述1.1.2和1.1.3中所述的相应气体将缓冲液鼓泡30分钟。 </li><li>在r-ACSF中留下一片作为对照。将其他四个脑片转移到具有预热的gf-ACSF的组织保持器中并孵育15分钟。将切片转移回r-ACSF中的组织支架以实现再灌注。 </li><li>研究APC的时间窗口，将一片直接从gf-ACSF传输到a-ACSF。首先将其他三个切片转移到r-ACSF，然后分别在再灌注后5和15分钟内将其中两个转移到a-ACSF中的组织支架。 </li><li> Incubate-ACSF中的两个切片3分钟，然后将其转移回r-ACSF。将r-ACSF中的三个切片孵育另外1小时。将r-ACSF中的剩余切片设置为OGD组。 </li><li>对于剂量反应研究，首先在OGD后将所有四个切片转移到r-ACSF中的组织保持器，并孵育5分钟。然后在r-ACSF中留下一片作为OGD组，并将其他三个切片转移到-ACFF。将a-ACSF中的切片分别孵育1,3和5分钟，然后将其转移回r-ACSF。将r-ACSF中的三个切片孵育另外1小时。 </li></ol></li><li> 切片生存力测定: <ol><li>制备0.25％2,3,5-三苯基四唑盐酸盐（TTC）生理盐水溶液。加入1.25 g TTC粉末至500 mL生理盐水溶液。 注意:粉末完全溶解后，将溶液转移到覆盖在箔中的24孔板（500μL/孔）中并将其储存在4℃。用TTC染色的TTC和组织轻sitive。 </li><li>将切片转移到含有TTC溶液的24孔板（每孔一片）中，并在溶液中拉伸切片。将TTC溶液中的切片在37℃下在浅水浴中孵育30分钟。 </li><li>将切片转移到覆盖在箔中的干净的1.5 mL离心管中，并测量切片的干重。 </li><li>将乙醇/二甲基亚砜（1:1）加入到离心管中（v = 10:1），以提取甲。。在室温下将切片在乙醇/二甲基亚砜中孵育24小时。 </li><li>将管中的所有乙醇/二甲基亚砜加入到96孔板中，并通过读板器测量490nm处的吸光度。 </li><li>将吸光度归一化到切片的干重，并将生存力表示为对照切片的百分比。 </li></ol></li></ol> MCAO <ol><li> 制备: <ol><li>消毒工作台，激光多普勒流量计的表面y（LDF）仪器及其配件，含70％乙醇。 </li><li>准备高压灭菌仪器:两把剪刀，10厘米;两镊子，10厘米;一个眼科钳，11厘米;一只微型眼科剪刀，9厘米;一个微血管夹，1.8厘米; oneneedle驱动，r 12.5厘米;几条外科缝合线（△1/2 4×10）;棉签。 </li><li>用盐水溶液准备注射器（无针头）以保持水合作业区域。 </li><li>准备麻醉气体（100％O 2 +异氟烷）。 </li></ol></li><li> 脑血流量检测: <ol><li>设置LDF仪器。 </li><li>腹腔注射小鼠镇痛药:metamizol 200 mg / kg，carprofen 4 mg / kg，丁丙诺啡0.1 mg / kg。 </li><li>将小鼠放入感应室中，用4％异氟烷在氧气中进行麻醉，直到身体自发移动，并将其停止。 </li><li>将鼠标置于俯卧位置，其鼻子装入鼻锥并维持异氟醚为1.5％，以后的程序。 </li><li>在双眼睛上涂抹松香酚眼膏。 </li><li>用70％乙醇消毒头部。 </li><li>使用手术刀在眼睛和耳朵之间做出正确的paramedian皮肤切口，以暴露胭脂红。 </li><li>用无菌棉揉颅骨。 </li><li>在头骨表面涂一滴至两滴手术胶。 </li><li>将光纤探头的尖端5 mm尾部放置到腋下，并将中线的外侧6 mm。 </li><li>开始在计算机软件上记录血流量。等待直到发生稳定的血流基线，然后在胶上涂上20μL催化剂以固定探针。 注意:理想的基线应该超过500个通量。 </li><li>如果基线小于200通量，请精细调整位置以获得合适的基线。在实验期间，将探头连接到颅骨。 </li><li>用碘伏消毒头连接光纤探头。 </li></ol></li><li> MCAO: <ol><li>将麻醉的小鼠（用LDF探针连接到其头骨）放置并固定，仰卧位置，并在整个手术过程中使用加热灯保持其体温。用70％乙醇消毒颈部。 </li><li>使用手术刀在颈部做一个paramedian皮肤切口;用镊子钝化软组织以暴露血管。向暴露的组织中加入一滴盐水以保持水分。 </li><li>使用眼科钳将周围组织和迷走神经解剖颈总动脉。小心不要损伤迷走神经。颈总动脉暴露后再次用碘伏消毒颈部皮肤。 </li><li>在颈总动脉的远端放置微血管夹，然后在近端用6-0丝线缝合死结。将夹具尽可能靠近以进行后续操作。确保长度钳夹与死结之间的血管尽可能长，以便后续操作。 </li><li>通过捆扎一个松散的结将临时缝合线靠近夹具。确保单丝插入之间有足够的空间。 </li><li>用微型眼科剪刀在两节之间进行一个小的纵向切口。确保切口尽可能靠近死结，以便后续操作。小心不要切断船只。 </li><li>通过切口插入12毫米尖端钝的单丝进入动脉内腔，并将其推进几毫米。拧紧单丝尖端的松散结，然后取下夹具。 </li><li>使用眼科钳将长丝推进颈内动脉（10mm，以阻塞大脑中动脉），直到LDF软件显示血流量急剧下降（基线血流减少80％以上）。记录遮挡的开始时间。 注意:LDF仪器设置为在2.2节中描述。 </li><li>大脑中动脉闭塞1 h。切断LDF探针，并将其放入30℃的培养箱中，以保持闭塞状态。 </li></ol></li><li> 再灌注和APC: <ol><li>在开始闭塞后55分钟，用异氟醚再次麻醉动物。将小鼠放置并固定在仰卧位。打开颈部切口并重新暴露颈总动脉。 </li><li>在闭塞期后用眼科钳子轻轻拉出长丝，以达到再灌注，并通过拧紧结而将临时缝合线变成永久缝合线。 </li><li>为酸中毒的治疗，再灌注后5，50，或100分钟改变由鼻锥吸入到含有20％CO 2，20％O 2和60％N 2 5分钟混合气体的气体。用中断的手术缝合缝合切口。 </li><li>将小鼠放在30°C的保温笼中，直到小鼠恢复意识然后将老鼠送回清洁的个别笼子里。为小鼠提供水培养和滋润的食物。手术后密切观察小鼠的过度疼痛和死亡。 </li></ol></li><li> 脑梗塞体积测量: <ol><li>再灌注24 h后采用TTC染色测量脑梗死体积。 </li><li>在指定的牺牲时间之前，按步骤1.4.1中所述准备0.25％的TTC溶液。将溶液转移到覆盖在箔中的24孔板（1mL /孔）中并将其储存在4℃。 注意:TTC和TTC染色的组织对光敏感。 </li><li>在再灌注后24小时，在诱导室中用异氟醚过量处死动物。解脱老鼠用小剪刀和镊子解剖大脑。检查大脑上的流血点，以排除在威利斯圈发生蛛网膜下腔出血的小鼠。 </li><li>将大脑放在-20°的干净的玻璃片上<html> ; C冰包。将大脑和玻片放在-20°C冰箱中5分钟，使大脑更容易切片。 </li><li>将大脑和玻璃片从-20°C取出，放回-20°C冰袋。用刀片和镊子解剖额极和小脑。 </li><li>用刀片将脑切片水平切割成1mm厚度以产生5片。将切片转移到含有TTC溶液的24孔板（每孔1片）中并用镊子拉伸，并拉伸溶液中的切片。将TTC溶液中的切片在37℃下在浅水浴中孵育30分钟。 </li><li>吸出TTC溶液。加入10％甲醛（1mL /孔），并在室温下孵育30分钟。将切片按切片顺序放置在玻璃纸上，并将片段扫描到照相板成像仪中。 </li><li>使用ImageJ分析软件分析梗塞面积占整个脑切片的百分比lass ="xref"&gt; 8基于视觉识别; 见图2 （左图）。 </li></ol></li></ol>

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Z., Jiang, S., Zeng, Y. M., Tang, Q. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+study+of+the+neuroprotective+effect+of+propofol+on+hypoxic+hippocampal+slice.">An in vitro study of the neuroprotective effect of propofol on hypoxic hippocampal slice.</a> Brain Inj. 28 (13-14), 1758-1765 (2014).</li><li>Niu, Y., 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=Chemical+profiling+with+HPLC-FTMS+of+exogenous+and+endogenous+chemicals+susceptible+to+the+administration+of+chotosan+in+an+animal+model+of+type+2+diabetes-induced+dementia.">Chemical profiling with HPLC-FTMS of exogenous and endogenous chemicals susceptible to the administration of chotosan in an animal model of type 2 diabetes-induced dementia.</a> J Pharm Biomed Anal. 104, 21-30 (2015).</li></ol>

作者没有什么可以披露的。

在这里我们提出两个实验模型来研究APC对脑缺血的神经保护作用。在脑切片中，APC通过在再灌注发生后用20％CO 2鼓泡的酸性缓冲液中孵育小鼠皮质纹状体切片来实现，而在MCAO模型中，APC是通过在再灌注后向小鼠吸入20％CO 2而实现的。这两种模型反映了APC对脑缺血的神经保护作用。保护与通过缺血后处理实现的保护相当，但是具有更宽的时间窗口。在MCAO模型，再灌注后相比10分钟对缺血后处理15的时间窗口可以是只要50分钟。此外，APC的过程更简单，更安全。 在皮质硬膜切片模型中，温和和快速的手术对于最大限度地保证脑切片的活力至关重要。对于MCAO表现，脑血流量监测为必须观察血流的急剧下降，以确保大脑中动脉闭塞。大脑切除TTC染色后，仔细检查大脑是排除蛛网膜下腔出血所必需的。 酸中毒的延长和持续时间对于实现APC的神经保护至关重要。长时间的酸中毒治疗对皮质硬膜切片模型无益（ 图 1A ）。另外，我们之前的研究表明，这两个10％和20％CO 2吸入，而不是30％的CO 2吸入赋予小鼠8神经保护。这些表明轻度酸中毒对于APC的神经保护至关重要，因此CO 2的浓度和APC的持续时间对于酸中毒神经保护是不可或缺的。 除了TTC染色，许多技术可以组合成satisfy多样化的研究需求。例如，可以添加细胞外的电记录到最后一步，观察缺血和酸中毒如何影响神经电位16 。也可以收集脑切片的灌注液，测定APC后的氨基酸神经递质浓度变化17 。可以准备某些脑区域的切片来研究特定区域对缺血和APC的反应。总的来说，可以引入许多替代方案来照亮缺血和酸中毒的影响。

中风是全球死亡和残疾的主要原因之一。在过去几十年中，为了找到有效的卒中治疗，已经做出了巨大的努力，但是成绩还不尽如人意。后处理是在缺血发作后由亚毒性应激操纵的过程。后处理，包括缺血性，缺氧，低血糖和远程缺血后处理，触发内源性适应机制，并已被证明是有前途的治疗对脑缺血1，2，3，4。然而，缺血后处理可能引起额外的损伤。肢体远程缺血后处理通常需要5的几个周期-在同侧或双侧后肢5，6，7 20分钟闭塞和再灌注。钍因此，这些后处理操作在临床实践中是危险的或不切实际的。为了克服这些缺点，我们开发了APC为小鼠8局灶性脑缺血的疗法。通过吸入20％CO 2诱导，APC以更可行和更安全的方式显着减少缺血性脑损伤。最近我们已经证明，APC延长了再灌注窗口，突出了APC对卒中治疗的重要性9 。 在这里我们提出两个实验模型来研究APC对脑缺血的神经保护作用。第一个是小鼠皮质纹状体切片中的氧 - 葡萄糖剥夺（OGD）模型。通过人造脑脊液（ASCF）将脑切片快速制备和转移到人造环境中，可以维持细胞活力和神经元电路，这样可以在体外研究脑功能10sup&gt;， 11 。在ASCF模仿脑缺血缺氧缺糖，诱导缺血性损伤12，13，14。在OGD之后，将脑切片用常规ASCF（r-ASCF）刷新以提供再灌注，然后使用用20％CO 2鼓泡的酸性ASCF处理APC。与原代培养细胞相比，皮质硬膜下切片维持完整的组织学表征。 为了研究体内脑功能，采用小鼠中脑动脉闭塞（MCAO）模型。通过颈总动脉插入火焰钝的单丝阻止大脑中动脉。作为最广泛使用的中风模型之一，MCAO模型显示临床相关性，单丝的应用使得更容易实现再灌注。简单地通过reperfusio发病后吸入含有20％CO 2含氧量正常的混合气体n，APC对脑梗死体积减少所表明的脑缺血有显着的保护作用。

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>2,3,5-triphenyltetrazolium hydrochloride</td><td>Sigma</td><td>T8877</td><td></td></tr><tr><td>Absolute Ethanol</td><td>Aladdin Industrial Corporation</td><td>E111993</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Laser Doppler Flowmetry</td><td>Moor Instruments Ltd</td><td>Model Moor VMS-LDF2</td><td></td></tr><tr><td>Diethyl ether anhydrous</td><td>Sinopharm Chemical Reagent Corporation</td><td>80059618</td><td></td></tr><tr><td>Trichloroacetaldehycle hydrate</td><td>Sinopharm Chemical Reagent Corporation</td><td>30037517</td><td></td></tr><tr><td>10% Formalin</td><td>Aladdin Industrial Corporation</td><td>F111936</td><td></td></tr><tr><td>24-well plates</td><td>Jet Biofil</td><td>TCP-010-024</td><td></td></tr></tbody></table>

experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

中风是全球死亡和残疾的主要原因之一，治疗方法有限。作为神经保护的内源性策略，后处理已被证明是有希望的针对脑缺血的治疗方法。然而，复杂的程序和潜在的安全问题限制了其临床应用。为了克服这些缺点，我们开发了酸性后处理（APC）作为实验性局灶性脑缺血的治疗方法。 APC是指温和酸中毒治疗由局部缺血再灌注期间吸入的CO 2。在这里，我们分别提出了两种模型来在体外和体内执行APC。使用小鼠的氧 - 葡萄糖剥夺（OGD）治疗和小鼠的皮质静脉闭塞和中脑动脉闭塞（MCAO）来模拟脑缺血。 APC可以通过将脑切片转移到用20％CO 2鼓泡的酸性缓冲液中来实现r通过吸入20％CO 2的小鼠。 APC显示出对脑缺血的显着保护作用，如组织存活力和脑梗死体积所反映的。

在上述皮质纹状体切片模型中，在再灌注后1小时通过TTC测定定量皮质硬膜下切片存活力。通过将490nm处的吸收归一化到对照切片来计算TTC转化。根据TTC转换，APC在起始时间和持续时间依赖的方式保护OGD诱导的再灌注损伤。详细地，1和3分钟的酸中毒治疗在OGD 15分钟后5分钟显着改善了活力，而5分钟没有（OGD:0.609±0.029,5 / 1:0.758±0.034,5 / 3:0.821±0.041， 5/5:0.672±0.053，数据报告为平均值±SEM）（ 图 1A ）。再灌注后5分钟内，酸中毒治疗的神经保护作用保持不变，15分钟（OGD:0.584±0.044,0/3:0.762±0.036,5 / 3:0.833±0.062，15/3:0.627±0.038）（ 图1 </strong&gt; B ）。 在MCAO模型中，在再灌注发生后5分钟开始5分钟的酸中毒治疗，拯救了由缺血性损伤引起的细胞死亡，反映为梗死体积较小（MCAO:33.4±4.4％，5/5:16.6±2.7％，50 / 5:19.5±2.1％，100/5:37±2.1％）。即使发作时间延迟到再灌注后的50分钟，神经保护仍然是健壮的。然而，在100分钟开始的酸中毒治疗并没有阻止缺血性损伤（ 图2 ）。 收集所有数据并以盲法分析。数据以平均值±SEM表示。在皮质组织切片模型中，每组有6-8个样品。在MCAO模型中，每组有9-10个样本。单因素方差分析差异最小，用于多重比较。 图1 。 APC保护OGD诱导的再灌注损伤皮质神经节切片。在酸中毒（pH 6.8）处理指示的持续时间（A）并在OGD后指示恢复期（B）治疗皮质纹状体切片。再灌注24 h后，通过3- [4,5-二甲基噻唑-2-基] -2,5-二苯基四唑溴化物测定法评估细胞存活率，再灌注后1 h，通过TTC测定定量皮质硬膜下切片的活力。值显示平均值±SEM。 n = 6〜8; * p &lt;0.05和** p &lt;0.01通过单向方差分析; R，再灌注<a href="http://ecsource.jove.com/files/ftp_upload/55931/55931fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2"class ="xfigimg"src ="/ files / ftp_upload / 55931 / 55931fig2.jpg"/&gt; 图2 。 APC防止MCAO引起的小鼠损伤。动物经受60分钟MCAO，并在再灌注后5,50或100分钟通过吸入20％CO 2处理5分钟。在再灌注后24小时，通过2,3,5-三苯基四氮唑盐酸盐染色定量梗塞体积（左图中以黑色虚线表示）。值显示平均值±SEM。 n = 8〜10; * p &lt;0.05和** p &lt;0.01通过单向方差分析; R，再灌注<a href="http://ecsource.jove.com/files/ftp_upload/55931/55931fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

酸性后处理可防止脑缺血。在这里我们提出两个模型来执行APC。分别通过体外氧葡萄糖剥夺后将皮质硬膜下切片转移到酸性缓冲液中，并在体内大脑中动脉闭塞后吸入20％CO 2 。

实验模型研究酸性后处理对脑缺血的神经保护作用

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

experimental-models-to-study-neuroprotection-acidic-postconditioning

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.4K 次观看.  Zhejiang University. 酸性后处理可防止脑缺血。在这里我们提出两个模型来执行APC。分别通过体外氧葡萄糖剥夺后将皮质硬膜下切片转移到酸性缓冲液中，并在体内大脑中动脉闭塞后吸入20％CO 2 。 

视频: Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

冷预处理神经保护作用的研究策略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

科研

神经科学

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

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

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

在小鼠永久性大脑中动脉结扎术中的应用

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

医学

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

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

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 ...

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

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

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 ...

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

一种中脑动脉闭塞技术在大鼠脑卒中后抑郁中的诱导

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

行为学

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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 ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

保留 ECA 的改良 MCAO/R 模型，用于增强再灌注和稳定性

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

作者聚焦：脑缺血建模的新方法 – 增强再灌注和简化手术

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

实验模型研究酸性后处理对脑缺血的神经保护作用

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