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Summary
Abstract
Introduction
Protocol
Results
Discussion
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Materials
References
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神经科学

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

Published: May 09, 2020
doi:
10.3791/61198
, , , , , , , , ,
1Division of Anesthesia and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences,Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences,Ben-Gurion University of the Negev, 3Department of Anesthesiology,Yale University School of Medicine, 4Recanati School for Community Health Professions, Faculty of Health Sciences,Ben-Gurion University of the Negev, 5Department of microbiology and immunology, Faculty of Health Sciences,Ben-Gurion University of the Negev

Summary

该协议验证了大脑扩散性连体损伤 （DAI） 的可靠、易于执行和可重复的啮齿动物模型，这种模型可引起大范围的白质损伤，而不会造成颅骨骨折或挫伤。

Abstract

创伤性脑损伤 （TBI） 是导致死亡和残疾的主要原因。扩散性斧伤 （DAI） 是需要住院的很大一部分 TBI 患者的主要伤害机制。DAI 涉及因晃动、旋转或爆炸伤害而造成的广泛斧头损伤，导致快速的斧头拉伸损伤和与功能恢复的长期影响相关的二次斧头变化。从历史上看，没有焦伤的DAI实验模型一直难以设计。在这里，我们验证了DAI的简单、可重复和可靠的啮齿动物模型，该模型不会造成广泛的白质损伤，而不会造成颅骨骨折或挫伤。

Introduction

创伤性脑损伤 （TBI） 是美国死亡和残疾的主要原因。结核病占所有与伤害有关的死亡1，22的约30%。TBI的主要原因因年龄组而异，包括跌倒、运动期间高速碰撞、故意自残、机动车碰撞和袭击11、2、3。2,3

脑扩散性斧头损伤（DAI）是一种特殊类型的TBI，由旋转加速、晃动或爆炸损伤引起的大脑在受伤后瞬间不受限制的头部运动44，5，6，7，8。5,6,7,8DAI涉及广泛的斧子损伤，导致长期神经损伤，与不良的结果，沉重的医疗费用，和33-64%的死亡率1,1，2，4，5，9，10，11。2,4,5,9,10,11尽管最近对DAI的发病机制进行了大量研究，但关于最佳治疗方案11、12、13、14,12,13,14仍没有达成共识。

在过去的几十年里，许多实验模型试图准确地复制DAI11、12、15、1612,15,16的不同方面。11然而，与其他焦伤相比，DAI 具有独特的表现方式，这些模型存在局限性。这些以前的模型不仅在白质区域造成斧伤，而且还会导致焦点脑损伤。临床上，DAI伴有微出血，这可能是白质损伤的主要原因。

只有两种动物模型被证明复制DAI的主要临床特征。Gennarelli和他的同事在1982年制造了第一个侧头旋转装置，使用非冲击头旋转加速诱导昏迷与DAI在非人类灵长类动物模型15。这种灵长类动物模型采用控制的单次旋转来加速和减速，在10-20毫秒内将头部通过60°取代。 这项技术能够模拟意识受损和广泛的斧头损伤，类似于在人类大脑中观察到的严重TBI的影响。然而，灵长类动物模型是非常昂贵的44，11，16。11,16部分基于以前的模型，猪模型旋转加速脑损伤是在1994年（罗斯等人）设计，类似的结果14。

这两种动物模型虽然产生了不同的典型病理学的介绍，但大大增加了DAI发病机制的概念。快速头部旋转被公认为诱导DAI的最好方法，啮齿动物为快速头部旋转研究11、16,16提供了一个更便宜的模型。在这里，我们验证了DAI的简单、可复制和可靠的啮齿动物模型，该模型在不造成颅骨骨折或挫伤的情况下造成广泛的白质损伤。目前的模型将使人们更好地了解DAI的病理生理学和更有效的治疗方法的发展。

Protocol

这些实验是在《赫尔辛基和东京宣言》和欧洲共同体《使用实验动物准则》的建议下进行的。这些实验得到了内盖夫本-古里安大学动物护理委员会的批准。 1. 为实验程序准备大鼠 注：选择成年雄性斯普拉格-达利大鼠体重300-350克。 获得机构动物护理和使用委员会的批准，以进行这些实验。 将大鼠保持在 22 ± 1 °C 的室温下，12 小时照明和 …

Representative Results

表 1说明了协议时间线。DAI模型的死亡率为0%。Mann-Whitney的一项测试表明，15只DAI大鼠的神经缺陷明显大于干预后48小时15只假鼠（Mdn = 1 vs. 0），U = 22.5，p < 0.001，r = 0.78（见表2）。数据以计数为单位进行测量，并呈现为中位数和 25-75 个百分位数范围。 图1显示了脑组织下部具有代表性的显微图。与对照组（67.46 × 30 vs. 0） 相比，?…

Discussion

该协议描述了DAI的啮齿动物模型。在DAI中，大脑旋转加速导致剪切效应，触发斧和生化变化，导致在渐进过程中失去六角函数。二次斧子变化是由快速的斧子拉伸损伤产生的，其范围和严重程度44、5、105,10是可变的。在主损伤后几到数天内，生化变化将导致斧功能44、5、105,<…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢内森·克利奥林博士（内盖夫本-古里安大学机械工程系）在生物力学测量方面的协助。此外，我们还感谢乌克兰德尼普罗大学生理学系的奥莱娜·斯潘诺夫斯卡教授、玛丽安娜·库舍里亚瓦教授、马克西姆·克里沃诺索夫教授、达里娜·亚库缅科教授和叶夫根尼亚·贡恰雷克教授对我们的讨论给予的支持和有益贡献。

Materials

0.01 M sodium citrate SIGMA – ALDRICH
2.5% normal horse serum SIGMA – ALDRICH H0146 Liquid
4 % buffered formaldehyde solution
Anti-Amyloid Precursor Protein, C – terminal antibodyproduced in rabbit SIGMA – ALDRICH Lot 056M4867V
biotinylated secondary antibody Vector BA-1000-1.5 10 mM sodium phosphate, pH 7.8, 0.15 M NaCl, 0.08% sodium azide, 3 mg/ml bovine serum albumin
bone-cutting forceps
DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3’-diaminobenzidine vector laboratory
embedding cassettes
ethanol 99.9 % ROMICAL Flammable Liquid
guillotine
Hematoxylin SIGMA – ALDRICH H3136-25G
Hydrogen peroxide solution Millipore 88597-100ML-F
Isofluran, USP 100% Piramamal Critical Care, Inc
Olympus BX 40 microscope Olympus
paraffine paraplast plus leica biosystem Tissue embedding medium
phosphate-buffered saline (PBS) SIGMA – ALDRICH P5368-10PAK Contents of one pouch, when dissolved in one liter of distilled or deionized water, will yield 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl – 0.0027 M); pH 7.4, at 25 °C.
Streptavidin HRP ABCAM ab64269 Streptavidin-HRP for use with biotinylated secondary antibodies during IHC / immunohistochemistry.
xylene
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Tags

Diffuse Axonal Brain InjuryTraumatic Brain InjuryRotational AccelerationSprague-Dawley RatsNeurological Severity ScoreBeta-amyloid Precursor ProteinImmunochemical StainingBrain Injury Model

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Frank, D., Melamed, I., Gruenbaum, B. F., Grinshpun, J., Kuts, R., Shvartsur, R., Azab, A. N., Assadi, M. H., Vinokur, M., Boyko, M. Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration. J. Vis. Exp. (159), e61198, doi:10.3791/61198 (2020).
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