一种体内评价大鼠缺血性中风模型中血脑屏障的破坏
Summary
本程序的总体目标是为在体内评估缺血性中风模型大鼠血脑屏障中断提供一种高度可重复的技术。
Abstract
缺血性中风导致血管源性脑水肿和随后的原发性脑损伤, 这是通过破坏血脑屏障 (BBB) 介导的。建立了诱导缺血性中风大鼠, 并作为体内模型对脑屏障功能完整性进行了研究。分光光度法检测脑标本中的埃文斯蓝 (EB), 可以为新的治疗方式的研究和开发提供可靠的依据。这种方法产生可重现的结果, 并适用于任何实验室, 不需要特殊设备。在此, 我们提出了一个可视化的技术指南, 以检测的 EB 渗出后, 诱导的缺血性中风的大鼠。
Introduction
血管源性脑水肿由于血脑屏障 (BBB) 中断仍然是缺血性中风的重要并发症, 也是中风患者存活率的主要决定因素1, 2.血脑屏障 (BBB) 由脑毛细血管内皮细胞 (BCECs) 形成, 由不同的神经血管成分组成 (例如, BCECs、毛细血管、星形胶质和神经细胞之间的紧密连接3), 提供了一种中枢神经系统 (CNS) 与外周血循环之间的专门和动态接口4,5。诸如缺血再灌注损伤等侮辱可能扰乱脑屏障功能完整性, 导致循环白细胞随后渗入大脑实质, 最终引发脑部炎症和原发性脑损伤。6,7. 需要动物模型来精确检测中风发生后的脑屏障功能障碍。这些模型对于研究潜在的病理生理学机制和引入新的神经保护策略具有重要意义。体外基于细胞培养的脑屏障模型已得到高度发展, 并用于生理病理的分子研究(bbb) 8, 9, 10.然而, 在这方面,在体内动物模型, 它产生的脑屏障的缺血性损伤类似于人类的临床条件, 也是非常值得的。定量检测埃文斯蓝 (EB) 的渗出是一种公认和敏感的技术, 已用于评估脑屏障完整性和功能的神经退行性疾病, 包括缺血性中风11,12,13,14. 该方法成本效益高, 可行, 重现性好, 完全适用于任何实验实验室。它的实现不需要先进的设备, 如放射性示踪剂15或磁共振成像 (MRI)16, 这是其他方法的先决条件。本文对缺血性脑卒中大鼠模型中 EB 渗出进行了脑屏障的基本技术过程的综合演示。
Protocol
所有的程序都按照阿尔达比勒大学医学研究委员会的指导方针进行动物研究 (道德 ID 号: IR。ARUMS。1394.08). 在这一可视化研究中, 我们使用了由牧场研究所 (伊朗德黑兰) 获得的成年雄性大大鼠 (300-350g)。 1. 麻醉和血流仪 麻醉使用4% 异氟醚, 并保持它与异氟醚 (1-1. 5%) 在一氧化氮 (70% 伏/五) 和氧 (30% v/v) 的混合物在手术期间。 放置一个麻醉动物在一个反馈控制的加?…
Representative Results
右半球 EB 水平与假手术大鼠左半球无显著性差异 (分别为 1.06 0.1 µg/克和 1.1 @ 0.09 µg/克)。如图 2 a-2 b所示, 在缺血性大鼠的左半球, 诱导短暂缺血 (90 分钟缺血/24 小时再灌注) 导致 EB 水平显著差异 (10.41 @ 0.84 µg/g, p < 0.001), 与各自半球在假操作的老鼠。总的来说, 这些发现表明, 在正常情况下, eb 不能轻易地越过脑屏障进入大脑实质和脑缺血性侮辱诱发 EB ?…
Discussion
迄今为止, 各种方法, 如显影和检测放射性示踪剂24,25, 免疫荧光显微镜26,27, EB 渗出技术20, 23已被用来评估血脑屏障损伤。EB 染料强烈能够绑定到血清白蛋白, 并作为追踪器, 以检测血管渗漏和量化的 BBB 击穿11,28,<su…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者感谢阿尔达比勒医学大学 (阿尔达比勒, 伊朗) 研究的副校长为财政支持 (授予 No: 9607)。
Materials
| Isoflurane | Piramal | AWN 34041100 | 20 – 25 °C |
| 2,3,5-Triphenyltetrazolium chloride (TTC) | Molekula | 31216368 | 4 years |
| Sprague–Dawley rats | Pasture Institute (Tehran, Iran) | 300-350g | |
| Evans Blue | Sigma-Aldrich | 314-13-6 | |
| Trichloroacetic acid | Sigma-Aldrich | 76-03-9 | 2 years |
| Bupivacaine HCl (0.5%) | Delpharm Tours | below 25 °C | |
| Bupernorphine | Exir (Iran) | ||
| Sodium Carbonate | Sigma-Aldrich | 497-19-8 | |
| Sodium chloride | Sigma-Aldrich | 7647-14-5 | |
| Di- Sodium hydrogen phosphate | EMD Millipore | 231-448-7 | |
| Potassium chloride | Sigma-Aldrich | 7447-40-7 | |
| Ethanol | Sigma-Aldrich | 64-17-5 | |
| silicone(Xantopren) | Heraeus | EN ISO 4823 | |
| Activator universal plus | Heraeus | 66037445 | |
| Micro-Dissecting forceps | Stoelting | 52100-41 | |
| Spring Scisors | Stoelting | 52130-00 | |
| Operating Scissors | Roboz | 52140-70 | |
| Brain matrix | Stoelting | 51390 | |
| Anesthesia Machine for Small Animals | | Kent Scientific | SS-01 | |
| Power Lab system | AD Instruments | ML880 | |
| Laser Doppler flowmeter | AD Instruments | ML191 | |
| Heating feed back system | Harvard Appratus | 72-7560 | |
| Vascular micro clamp | FineScience Tools | 18055-03 | |
| Silk 5-0 suture thread | Ethicon | 682G | |
| Ethilon 4-0 suture thread | Ethicon | EH6740G |
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Diesen Artikel zitieren
Panahpour, H., Farhoudi, M., Omidi, Y., Mahmoudi, J. An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke. J. Vis. Exp. (133), e57156, doi:10.3791/57156 (2018).