应用短暂的中脑动脉闭塞小鼠模型评价甘草提取物的神经保护作用
Summary
在本研究中, 我们通过建立大脑中动脉闭塞 (mcao) 小鼠模型, 修改了现有的实验方法, 以获得更多的重现性结果。口服甘草和根状茎 (gr) 甲醇提取物 (grex), 脑卒中诱导后, 与未治疗对照组相比, 总梗死量显著降低。
Abstract
脑卒中后脑血流再灌注后的缺血导致神经细胞死亡和脑组织丢失。脑卒中最常用的动物模型是大脑中动脉闭塞 (mcao) 模型。以前的研究报告了不同的梗死大小, 即使在类似的 mcao 条件下使用相同的实验动物物种。因此, 我们开发了一种改进的实验方法来解决这种差异。小鼠接受 mcao 使用长丝作为遮挡材料, 以模拟人类中风条件和灯丝厚度进行优化, 以建立更可重现的梗死体积。脑卒中诱导后用甘草提取物处理的小鼠总梗死体积明显下降, 存活细胞数量相对于未治疗的对照组有所增加。该改进的实验方案成功地和可重现地证明了 grex 对缺血性脑卒中的有益作用。
Introduction
脑血流缺血再灌注引起的脑损伤导致神经细胞死亡和脑组织丢失。随着肥胖、高血压和糖尿病等代谢疾病的传播, 脑血管疾病的发病率不断上升, 这种类型的脑损伤继续增加.全世界老年中风患者的绝对人数急剧增加, 这些患者的医疗费用是一个主要的社会负担, 这些患者往往患有长期残疾。因此, 应尽可能减轻继发性残疾, 以减轻经济负担1,2。
脑梗死最常用的啮齿类动物模型是大脑中动脉闭塞 (mcao) 模型, 其中 mca 被硅涂层手术缝合长丝封闭, 以阻断血液流动, 导致缺血性中风3,4. 使用灯丝作为闭塞材料, 可以通过控制腔内灯丝插入的持续时间来控制闭塞时间和持久性。
以往的研究表明, 即使使用相同的啮齿类动物 mcao 模型, 脑梗死的总量也因实验而异, 导致研究的重现性较低。为了提高重现性, 我们优化了实验中使用的细丝薄荷的厚度。脑缺血期和诱发梗死的初步研究结果表明, 缺血期超过 60分钟, 可以观察和量化受损脑组织的体积区域。
甘草, 又名甘草, 由甘草和葛根的干根和根状茎组成. 它已被用于中国和韩国传统医学的各种用途, 包括作为食品添加剂和药用5,6,7。
在先前的一项研究8中, gr 甲醇提取物 (grex) 预处理显示出 mcao 小鼠的抗凋亡作用, 包括显著防止 b 细胞淋巴瘤 2 (bcl-2) 和 bcl 超大 (bcl-xl) 蛋白表达的降低。本研究通过评价传统 mcao 小鼠模型在确定 grex 在梗死后处理中的效率, 有效地降低了 mcao 所致脑损伤的梗死体积, 从而提高了该模型的重现性。
Protocol
所有涉及动物的程序都得到了釜山国立大学道德委员会的批准 (批准号码, pu-2016-1087)。图 1显示了这项研究的图形概述。 1. grex 的准备和管理 注: 本研究中使用的 gr 是从一家商业制药公司购买的。 将200克 gr 放入 2, 000 毫升甲醇中, 在室温 (25°c) 下孵育5天。 使用厚度为 0.26 mm、孔径为5μm 的滤纸对混合物进行过滤, 然后?…
Representative Results
在假操作的正常组, 没有观察到脑梗塞, 而在对照组, 观察到相对广泛的损伤区域。在 mcao 模型组给小鼠的 300 mg/kg grex 中, 损伤面积显著减少 (图 2)。 用 h & amp; e 或 cresyl 紫染色缺血性脑切片, 研究其组织学改变。h & amp; e 染色提供有关细胞10的结构信息和特定功能信息, 而 cresyl 紫色染色用…
Discussion
随着慢性高血压、糖尿病、高脂血症等代谢疾病的发病率不断上升, 脑卒中的主要危险因素, 脑卒中的预防和治疗已成为医学研究的一个重要领域12.13. 中风后语言和运动方面的缺陷与脑组织14的损伤程度密切相关, 导致患者及其家属的生活质量差.重要的是要使用一个适当的中风动物模型, 其中涉及相同的病理变化发生?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
不适用。
Materials
| Glycyrrhizae Radix et Rhizoma | Gwangmyoung Pharmaceuticals Co., Korea | Glycyrrhizae Radix et Rhizoma | |
| Qualitative filter paper | Advantec | Filter paper No. 2 | Qualitative filter paper |
| Dimethyl sulfoxide (DMSO) | Sigma | D8418-250ML | Dimethyl sulfoxide (DMSO) |
| Syringe filter (0.45 µm) | Sigma | CLS431220 | Syringe filter (0.45 µm) |
| Stereo Microscope | Leica | M50 | Stereo Microscope |
| Stereo Microscope | Nikon | SMZ745 | Stereo Microscope |
| Laser Doppler | Moor Instrument | moorVMS-LDF | Laser Doppler |
| Anesthesia Tabletop Bracket with N2O&O2 Flowmeter System | Harvard Appratus | 34-1352 | Anesthesia Tabletop Bracket with N2O&O2 Flowmeter System |
| Homeothermic Monitoring System | Harvard Appratus | 55-7020 | Homeothermic Monitoring System |
| Digital Camera | Canon | Eos-M2 | Digital Camera |
| Cryostat | Leica | CM3050S | Cryostat |
| Microscope | Carl Zeiss | Zeiss Axio | Microscope |
| Data Analysis | Systat Software Inc. | SigmaPlot version 12 | Data Analysis |
| Data Analysis | NIH Image | ImageJ | Data Analysis |
| Mouse diet | Doo Yeol Biotech | Standard rodent chow | Mouse diet |
| Isoflurane | JOONGWAE | A02104781 | Isoflurane |
| Isoflurane | TROIKAA | ISOTROY 100 | Isoflurane |
| Silk suture (4-0 Black silk) | AILEE | SK47510 | Silk suture (4-0 Black silk) |
| Silk suture (3-0 White silk) | Baekjae | 57 | Silk suture (3-0 White silk) |
| Nylon suture (8-0 monofilament) | AILEE | NB825 | Nylon suture (8-0 monofilament) |
| 2,3,5-triphenyltetrazolium chloride (TTC) | Sigma | T8877-25G | 2,3,5-triphenyltetrazolium chloride (TTC) |
| Formalin (Formaldehyde solution) | JUNSEI | 69360-1263 20KG | Formalin (Formaldehyde solution) |
| Hematoxylin (Harris Hematoxylin) | YD Diagnostics | EasyStain | Hematoxylin (Harris Hematoxylin) |
| Eosin (1% Eosin Y Solution) | MUTO PURE CHEMICALS | 3200-2 | Eosin (1% Eosin Y Solution) |
| Cresyl violet (acetate) | Sigma | C5042-10G | Cresyl violet (acetate) |
| Paraformaldehyde | Sigma-Aldrich | P6148-1KG | Paraformaldehyde |
| Sucrose | JUNSEI | 31365-0350 1KG | Sucrose |
| Optimum cutting temperature (OCT) compound | Scigen | 4583 | Optimum cutting temperature (OCT) compound |
| Disecting Knife | Fine Science Tools | 10055-12 | Disecting Knife |
| #4 Forcep | Fine Science Tools | 11241-30 | #4 Forcep |
| #5 Forcep | Fine Science Tools | 11254-20 | #5 Forcep |
| #6 Forcep | Fine Science Tools | 11260-20 | #6 Forcep |
| #7 Fine Forcep | Fine Science Tools | 11274-20 | #7 Fine Forcep |
| Surgical Scissors | Fine Science Tools | 14001-12 | Surgical Scissors |
| Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | Extra Fine Bonn Scissors |
| Moria Pascheff-Wolff Spring Scissors | Fine Science Tools | 15371-92 | Moria Pascheff-Wolff Spring Scissors |
| Vessel Dilating Forcep | Fine Science Tools | 18153-11 | Vessel Dilating Forcep |
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Tags
NeuroprotectiveGlycyrrhizae Radix Et RhizomaTransient Middle Cerebral Artery OcclusionMouse ModelNatural CompoundsIschemic TimingMiddle Cerebellar ArteryGlycyrrhizae Radix Et Rhizome ExtractMethanol ExtractionFreeze-dryingDimethylsulfoxideC57BL/6 MouseCommon Carotid ArteryExternal Carotid ArteryInternal Carotid ArterySilicone-coated Monofilament
Citazione di questo articolo
Lee, S., Lim, C., Lee, M., Kim, C., Kim, H., Lee, B., Cho, S. Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model. J. Vis. Exp. (142), e58454, doi:10.3791/58454 (2018).