脑缺血再灌注的双血管闭塞小鼠模型
Summary
建立脑缺血再灌注小鼠模型, 探讨脑卒中的病理生理。我们将右大脑中动脉和右颈总动脉进行远端结扎, 在10或40分钟缺血后恢复血液流动。
Abstract
本研究采用大脑中动脉闭塞小鼠模型对脑缺血再灌注进行研究。一种可重复、可靠的小鼠模型可用于探讨脑缺血再灌注的病理生理学, 确定脑卒中患者的潜在治疗策略。c57bl6 小鼠威利斯圆圈的变化影响其脑缺血损伤后的梗死体积。研究表明, 远端 mca 闭塞 (mcao) 可以克服这一问题, 并导致一个稳定的梗死大小。在本研究中, 我们建立了一个双血管闭塞小鼠模型的脑缺血再灌注通过中断血液流向右 mca。我们将右 mca 和右颈总动脉 (cca) 进行远端连接, 并在一定时期的缺血后恢复血液流动。这种缺血再灌注损伤会诱发大小稳定的梗塞和行为缺陷。外周免疫细胞在24小时浸润期内浸润缺血性脑。此外, 在更长的再灌注时间内, 皮质区域的神经元丢失较少。因此, 该双血管闭塞模型适用于研究脑缺血后再灌注期的免疫反应和神经元恢复情况。
Introduction
脑缺血再灌注小鼠模型是研究缺血所致脑损伤1的最常用的实验方法之一.由于脑缺血再灌注激活外周免疫系统, 外周免疫细胞浸润到缺血性脑, 并造成神经元损伤2。因此, 一个可靠的和可复制的小鼠模型, 模仿脑缺血再灌注是必要的, 以了解中风的病理生理学。
c57bl/6j (b6) 小鼠是中风实验中最常用的菌株, 因为它们很容易被基因操纵。有两种常见的模拟脑缺血再灌注状况的 mcao·再灌注模型。第一种是近端 mcao 的腔内长丝模型, 其中采用硅涂层长丝对 mca 中的血流进行血管内闭塞;随后将闭塞的长丝取出, 以恢复血液流动3。短的闭塞持续时间会导致皮质下区域的病变, 而较长的闭塞持续时间会导致皮质和皮质下区域的梗塞。第二个模型是远端 mcao 的结扎模型, 它包括 mca 和 cca 的血管外结扎, 以减少通过 mca 的血液流动, 之后通过切除缝合线和动脉瘤夹 4恢复血液流动。在这种模型中, 皮质区域发生梗死, 死亡率较低。由于 mcao\ 再灌注模型的结扎需要颅骨切除术来暴露远端 mca 的部位, 因此可以很容易地确认该部位, 并检查在手术过程中远端 mca 的血流是否中断是很简单的。
b6 小鼠在威利斯圆圈的解剖结构上表现出相当大的变化;这可能会影响脑缺血再灌注5,6, 7 后的梗死体积。目前, 这个问题可以通过远端 mca8的结扎来解决。在这项研究中, 我们建立了一种方法, 以闭塞 mca 血流和使再灌注后预定的缺血期。脑缺血再灌注模型的双血管闭塞通过右远端 mca 和右 cca 的结扎, 引起 mca 区域的短暂性缺血, 在一定时期的缺血后恢复血液流动。这种 mcao\ 再灌注模型诱导了一个大小稳定的梗塞, 在缺血性脑中的大量脑浸润免疫细胞, 并在脑缺血再灌注4后的行为缺陷。
Protocol
Representative Results
Discussion
mcao·再灌注小鼠模型是一种常用于模拟人类短暂性脑缺血的动物模型。该动物模型可应用于转基因小鼠和敲除小鼠菌株, 探讨脑卒中的病理生理学。协议中的几个步骤尤其重要。(1) 在头骨上创建孔时, 必须小心使用微钻头, 不适当的操作很容易导致 mca 出血。(2) mca 不应损坏, 在结扎手术前后必须避免出血。mca 损伤影响缺血性脑的再灌注水平7。必须在 mcao 之后检查 mca 再灌注状态?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了台湾科技部 (最大部分106-2320-b-038-024、最-ec-93-008-my3 和最十 104-2320-b-424-001) 和台北医科大学医院 (107tmuh-sp-01) 的支持。这份手稿是由华莱士学术编辑编辑。
Materials
| Bone rongeur | Diener | Friedman | |
| Buprenorphine | Sigma | B-044 | |
| Cefazolin | Sigma | 1097603 | |
| Chloral hydrate | Sigma | C8383 | |
| Dissection microscope | Nikon | SMZ-745 | |
| Electric clippers | Petpro | ||
| 10% formalin | Sigma | F5304 | |
| Germinator dry bead sterilizer | Braintree Scientific | ||
| Iris Forceps | Karl Klappenecker | 10 cm | |
| Iris Scissors | Diener | 9 cm | |
| Iris Scissors STR | Karl Klappenecker | 11 cm | |
| Microdrill | Stoelting | FOREEDOM K.1070 | |
| Micro-scissors-Vannas | HEISS | H-4240 | blade 7mm, 8 cm |
| Mouse brain matrix | World Precision Instruments | ||
| Non-invasive blood pressure system | Muromachi | MK-2000ST | |
| Operating Scissors STR | Karl Klappenecker | 14 cm | |
| Physiological Monitoring System | Harvard Apparatus | ||
| Razor blades | Ever-Ready | ||
| Stoelting Rodent Warmers | Stoelting | 53810 | Heating pad |
| Suture clip | Stoelting | ||
| Tweezers | IDEALTEK | No.3 | |
| Vetbond | 3M | 15672 | Surgical glue |
| 10-0 suture | UNIK | NT0410 | |
| 2,3,5-Triphenyltetrazolium chloride | Sigma | T8877 |
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