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大鼠运动皮层中的激光诱发脑损伤
Chapters
Summary September 26th, 2020
Please note that all translations are automatically generated.
Click here for the English version.
此处介绍的协议显示了创建脑损伤啮齿动物模型的技术。此处描述的方法使用激光照射和目标运动皮层。
Transcript
我们的协议提供了一种新方法，用于利用激光照射在大鼠中制造创伤性脑损伤，从而在运动皮层中产生焦点冲击。该技术的主要优点是梗塞面积变化小，死亡率低，程序相对简单，不需要专家处理。演示这个程序的将是德米特里·纳塔内尔，一个来自我们实验室的研究人员。
为了进行激光引起的脑损伤，首先将20，300至350克Sprague Dawley大鼠分配到激光组，将20只分配给控制Sham操作组。在确认对戒断反射缺乏反应后，将一只老鼠放在直肠温度调节加热垫上，并使用剃须刀从损伤部位去除足够的头发，切口周围有大约两厘米的无毛边缘。用 70% 乙醇对裸露的皮肤进行消毒，将大鼠放在立体型头架的易发位置。
做一个三厘米的切口，让头皮的横向反射暴露之间的区域， 布雷格玛和羔羊。手持峰值波长为1064纳米的Nd：YAG激光器，距离头骨两毫米的距离，以1秒脉冲持续时间对右半球上方头骨的暴露区域施用50焦耳倍10点。激光损伤后，将老鼠从设备中取出，用3-0丝丝手术缝合线关闭头皮。
然后，将大鼠放在笼子里，进行监测，直到完全卧发。手术24小时后，使用43分评分系统评估神经严重性评分。测试动物的神经缺陷、行为障碍、光束平衡任务和反射，并为更严重的残疾分配更高的分数。
评估后，根据标准规程从每个实验和控制动物中采集大脑。为了评估大脑的梗塞体积，将收获的大脑切成六个两毫米厚的日冕片，并在0.05%TTC中孵育每片，在37摄氏度下孵育30分钟。染色后，使用光学扫描仪扫描此切片，每英寸分辨率为 1600 分 1600 点。
固定大脑切片的未污染区域被定义为梗死。为了评估血脑屏障破损的发生率，激光诱导损伤后24小时，将2%的埃文斯蓝色染料用2%的Evas蓝色染料用稀释在每公斤盐水溶液中，然后通过可抽的尾静脉将溶液静脉注射给受伤并控制大鼠。在使用手术针和剪刀打开第一只动物的胸部之前，让溶液循环一小时。
通过左心室在110毫米压力下用冷却0.9%盐水将暴露的心脏灌注，直到从右中庭观察到无色填充液。接下来，收获大脑，获得两毫米的足体果子片。将左脑切片与右脑切片分开，以便分别评估受伤和非受伤的半球，并称重样本。
使用砂浆和害虫使样品均质，并在 50%三氯乙酸中孵育组织样品 24 小时。第二天，在10，000倍的G和室温下将均质脑组织样本离心20分钟，将1毫升上半液与1.5毫升96%乙醇混合，比例为1比3。然后，使用荧光探测器在620纳米的激发和680纳米发射波长，以评估血脑屏障断裂。
组织学分析指出，埃文斯蓝色染色可用于揭示激光和MCAO模型动物脑损伤的发生率。在这个代表性的分析中，对照组或实验组都没有死亡或亚布拉奇内出血，MCAO组的死亡率和亚布拉奇出血率均达到20%。尽管两组大鼠的变异性不同，但两组大鼠的相对体温变化也相似。
与Sham操作对照组相比，激光和MCAO模型的神经严重性得分都明显差。与Sham操作对照组相比，激光诱发脑损伤也导致目标半球的梗塞体积显著增加。然而，与受MCAO技术伤害的动物相比，激光模型的法化体积更小。
激光诱发脑损伤模型与沙姆操作对照组之间未观察到脑水肿差异。然而，激光模型和MCAO受伤组在脑水肿方面存在显著差异。与沙姆操作对照组相比，激光引起的脑损伤和MCAO技术都导致非损伤半球和目标半球的血脑屏障断裂显著增加。
在尝试此模型时，请记住精确定位运动皮层区域，并标准化能量、辐照点数和总展示时间。按照此过程，可以执行大量的行为测试，如光束行走、Froedert 和其他评估。
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