Summary

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

Published: October 23, 2020
doi:

Summary

此协议描述了在同一组啮齿动物脑样本上测量缺血性脑损伤三个最重要的参数的新技术。仅使用一个大脑样本在道德和经济成本方面非常有利。

Abstract

全世界发病率和死亡率最常见的原因之一是缺血性中风。从历史上看,用于刺激缺血性中风的动物模型涉及中脑动脉闭塞(MCAO)。梗塞区、脑水肿和血脑屏障 (BBB) 故障被测量为反映 MCAO 后脑损伤程度的参数。这种方法的一个重大限制是,这些测量通常在不同的大鼠大脑样本中获得,由于大量大鼠需要安乐死以达到适当的样本大小,因此会导致伦理和经济负担。在这里,我们提出了一种方法,通过测量同一组大鼠大脑中的梗塞区、脑水肿和BBB渗透率,准确评估MCAO之后的脑损伤。这种新技术为评估中风的病理生理学提供了更有效的方法。

Introduction

全世界发病率和死亡率最常见的原因之一是中风。在全球范围内,缺血性中风占所有中风病例的68%,而在美国缺血性中风占中风病例的87%。1,2。据估计,中风的经济负担达到340亿美元,在美国2和450亿欧元在欧盟3。动物中风模型是研究其病理生理学,开发新的评价方法,并提出新的治疗方案4。

缺血性中风发生在一个主要脑动脉的闭塞,通常是中脑动脉或其分支之一5。因此,缺血性中风的模型历来涉及中脑动脉闭塞(MCAO)6,7,8,9,10,11,12。继 MCAO 之后, 神经损伤最常通过测量梗塞区 (IZ) 来评估,使用 2,3,5 三苯甲酸酯氯化物 (TTC) 染色方法13,脑水肿 (BE) 使用 干燥或计算半球体积14,15,16,和血脑屏障(BBB)渗透性光谱技术使用埃文斯蓝色染色17,18,19。

传统的 MCAO 方法在三个大脑测量中每个都使用单独的大脑集。对于大量的样本量,这导致大量的安乐死动物,增加了伦理和财务考虑。减轻这些成本的替代方法将涉及在一组后 MCAO 啮齿动物大脑中测量所有三个参数。

先前曾尝试测量同一大脑样本中的参数组合。同时免疫荧光染色方法20 以及其他分子和生化分析21 已描述后TTC染色在同一大脑样本。我们以前计算过脑半球的体积来评估脑水肿,并进行了TTC染色,以计算同一大脑集15的梗塞区。

在本协议中,我们提出了一种经过改进的 MCAO 技术,通过确定同一组啮齿动物大脑中的 IZ、BE 和 BBB 渗透性来测量缺血性脑损伤。IZ通过TTC染色测量,BE通过计算半球体积来测量,BBB渗透性通过光谱方法19获得。在此协议中,我们使用了经过修改的 MCAO 模型,基于将单滤导管直接插入和固定到内部胡萝卜动脉 (ICA) 和进一步阻断流向中脑动脉 (MCA)22的血液。与传统的MCAO方法16、22相比这种修改后的方法表明死亡率和发病率有所下降。

这种新方法为测量 MCAO 之后的神经损伤提供了财务健全和道德模型。这种对缺血性脑损伤主要参数的评估将有助于全面研究其病理生理学。

Protocol

下列程序是根据《赫尔辛基宣言》和《欧洲共同体实验动物使用准则》的建议进行的。内盖夫本古里安大学的动物护理委员会也批准了这些实验。 1. 为实验程序准备大鼠 选择成年雄性斯普拉格-道利大鼠,没有明示病理学,每只体重在300至350克之间。 将所有大鼠在室温下保持在22°C,在实验前有12小时的光和暗周期。 确保食物和水是可用的广告利比妥姆…

Representative Results

梗塞区测量 一项独立样本T测试表明,与16只假手术大鼠相比,19只接受永久性MCAO的老鼠的大脑梗塞体积显著增加(MCAO=7.49%,±3.57对。 香=0.31%±1.9,t(28.49)=7.56,p<0.01(见图2A)。 数据表示为反半球± SD 的平均百分比。 脑水肿测量 一项独立样本T测试表明,与16只假手术大…

Discussion

本议定书的主要目标是证明对缺血损伤的三个主要参数的一致测量:IZ、BE和BBB渗透性。先前在这一领域的研究表明,有可能在同一样本中一起执行其中一个或两个参数。除了这种由三部分组成的方法提供的成本降低外,它还提供了一个更理想的生物伦理模型,限制必须操作并随后实施安乐死的动物数量。与所有病理学技术一样,该方法受到无法动态观察缺血损伤的限制。

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Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们感谢马里娜·库舍里亚瓦、马克西姆·克里沃诺索夫、达里娜·亚库门科和叶夫根尼亚·贡查里克,他们支持我们的讨论,并为我们的讨论作出了有益的贡献。获得的数据是鲁斯兰·库茨博士论文的一部分。

Materials

2 mL Syringe Braun 4606027V
2% chlorhexidine in 70% alcohol solution Sigma-Aldrich 500 cc Provides general antisepsis of the skin in the operatory field
27 G Needle with Syringe Braun 305620
3-0 Silk sutures Henry Schein 1007842
4-0 Nylon suture 4-00
Brain & Tissue Matrices Sigma-Aldrich 15013
Cannula Venflon 22 G KD-FIX 183603985447
Centrifuge Sigma 2-16P Sigma-Aldrich Sigma 2-16P
Compact Analytical Balances Sigma-Aldrich HR-AZ/HR-A
Digital weighing scale Sigma-Aldrich Rs 4,000
Dissecting scissors Sigma-Aldrich Z265969
Eppendorf pipette Sigma-Aldrich Z683884
Eppendorf tube Sigma-Aldrich EP0030119460
Fluorescence detector Tecan, Männedorf Switzerland Model: Infinite 200 PRO multimode reader Optional.
Fluorescence detector Molecular Devices LLC VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument Optional.
Gauze sponges Fisher 22-362-178
Heater with thermometer Heatingpad-1 Model: HEATINGPAD-1/2
Hemostatic microclips Sigma-Aldrich
Horizon-XL Mennen Medical Ltd
Infusion cuff ABN IC-500
Micro forceps Sigma-Aldrich
Micro scissors Sigma-Aldrich
Multiset Teva Medical 998702
Olympus BX 40 microscope Olympus
Operating forceps Sigma-Aldrich
Operating scissors Sigma-Aldrich
Optical scanner Canon Cano Scan 4200F Resolution 3200 x 6400 dpi
Petri dishes Sigma-Aldrich P5606
Purina Chow Purina 5001 Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment.
Rat cages Techniplast 2000P Conventional housing for rodents. Cages were used for housing rats throughout the experiment
Scalpel blades #11 Sigma-Aldrich S2771
Software
Adobe Photoshop CS2 for Windows Adobe
ImageJ 1.37v NIH The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA
Office 365 ProPlus Microsoft Microsoft Office Excel
Windows 10 Microsoft
Reagents
2,3,5-Triphenyltetrazolium chloride Sigma-Aldrich 298-96-4
50% trichloroacetic acid Sigma-Aldrich 76-03-9
Ethanol 96 % Romical Flammable liquid
Evans blue 2% Sigma-Aldrich 314-13-6
Isoflurane, USP 100% Piramamal Critical Care, Inc NDC 66794-017

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Frank, D., Gruenbaum, B. F., Grinshpun, J., Melamed, I., Severynovska, O., Kuts, R., Semyonov, M., Brotfain, E., Zlotnik, A., Boyko, M. Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples. J. Vis. Exp. (164), e61309, doi:10.3791/61309 (2020).

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