Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Siobhan Crilly; Alexandra Njegic; Adrian R. Parry-Jones; Stuart M. Allan; Paul R. Kasher

doi:10.3791/59716

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

斑马鱼幼虫研究出血性脑卒中的病理后果

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06:36 min

June 5, 2019

DOI: 10.3791/59716-v

Siobhan Crilly¹, Alexandra Njegic², Adrian R. Parry-Jones^2,3, Stuart M. Allan^1,3, Paul R. Kasher^1,3

¹Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Manchester Academic Health Science Centre,University of Manchester, ²Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre,University of Manchester, ³Lydia Becker Institute of Immunology and Inflammation,University of Manchester

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Overview

This study presents a protocol for quantifying brain injury, locomotor deficits, and neuroinflammation in zebrafish larvae following intracerebral hemorrhage (ICH), a critical human medical condition. Utilizing the transparent nature of zebrafish larvae allows for real-time observation of cellular responses in a live brain model post-hemorrhagic stroke.

Key Study Components

Area of Science

Neuroscience
Neuroinflammation
Stroke Recovery

Background

Intracerebral hemorrhage (ICH) is a serious medical condition lacking specific treatments.
Zebrafish larvae serve as an innovative model to study brain responses due to their transparency.
Real-time imaging provides insights into cellular dynamics following brain injury.
The study employs fluorescent microscopy to assess neuroinflammation and other cellular responses within the brain.

Purpose of Study

To develop a pre-clinical model for studying the cellular response to hemorrhagic stroke.
To investigate the impacts of ICH on locomotion and neuroinflammation.
To explore potential drug candidates for mitigating the effects of ICH.

Methods Used

The main platform used is fluorescent microscopy for imaging cellular responses.
Zebrafish larvae are the biological model, with a focus on brain injury from induced hemorrhaging.
Key timelines involve embryo collection, treatment application, and subsequent imaging at specified post-fertilization hours.
Critical phases include monitoring motility and assessing neuroinflammation responses over several days.

Main Results

Significant cellular responses were observed, including clusters of dying cells in hemorrhaged larvae.
A decrease in motility was noted post-hemorrhage, with partial recovery by 120 hours.
Activated macrophages displayed morphological changes associated with the ICH response.
This model enables assessment of potential therapeutic interventions for stroke.

Conclusions

This research establishes a zebrafish model for studying ICH and its cellular implications.
The approach may facilitate drug screening efforts to improve outcomes after brain hemorrhage.
Overall, it advances the understanding of cellular dynamics following brain injury and potential recovery mechanisms.

Frequently Asked Questions

What are the advantages of using zebrafish larvae as a model?

Zebrafish larvae offer a transparent view of brain activity, enabling real-time imaging of cellular responses to injury, which is not possible in mammalian models.

How is brain injury induced in zebrafish larvae?

Brain injury is induced by applying Atorvastatin to achieve a specific percentage of hemorrhaged larvae at defined post-fertilization intervals.

What types of data are collected during the study?

Data includes quantification of cellular responses, neuroinflammation, and behavioral assessments of locomotion over specified time points.

How can the method be adapted for drug screening?

The model can be used to test potential drug candidates aimed at mitigating the severity of ICH effects, providing an avenue for screening therapeutics.

What considerations are important when preparing zebrafish larvae?

It's crucial to thoroughly examine larvae for hemorrhage presence before conducting phenotyping assays to ensure valid results.

在这里, 我们提出了一个程序来量化脑损伤, 运动缺陷和神经炎症出血后, 脑出血的斑马鱼幼虫, 在人的脑出血 (ICH) 的情况下。

这种方法提供了一个免费的临床前方法，用于研究出血性中风后大脑血液的即时细胞反应，这是一种非常严重的疾病，我们没有为患者提供特定的药物。与啮齿动物模型不同，斑马鱼幼虫的透明度使我们能够使用荧光显微镜实时观察活体完整动物大脑中的细胞反应。首先，使用茶过滤器收集所有受精胚胎从自然产卵的繁殖箱中产生的一只雄性和一到两只雌性成年斑马鱼。

将100个胚胎转移到每个含有标准E3胚胎培养的培养皿中。在28摄氏度下孵育，并按照标准指南进行阶段孵育。在受精后6小时，使用巴斯德移液器从培养皿中取出死亡和未受精的胚胎，将盘子放回培养箱。

受精后24小时，在明亮的场立体显微镜下，使用锋利的超薄解剖钳对胚胎进行修饰，用于治疗阿托伐他汀。然后将30毫升E3胚胎培养剂加入两个干净的培养皿中，一个用于治疗，另一个用于控制。从治疗盘中去除60微升胚胎水，加入60微升0.5毫摩尔阿托伐他汀，达到80%的幼虫出血。

使用巴斯德移液器，将100个胚胎在尽可能少的水中转移到每道菜。在28摄氏度下孵育两道菜。受精50小时后，在显微镜下使用巴斯德移液器小心地将出血的鱼与非出血种群分离，将幼虫转移到含有新鲜E3介质的新菜肴中。

为了更容易分离出血阳性幼虫，您可以使用没有色素的鱼或鱼在红血球中表达荧光蛋白。第三天，在荧光显微镜下筛选幼虫，以确保荧光蛋白的表达。然后用含有 0.2%MS-222 的 E3 介质填充光片安装室，用于麻醉。

使用巴斯德移液器，将含有一到六个幼虫的单个液滴转移到干培养皿表面进行安装。使用移液器去除尽可能多的液体。将 45 摄氏度热块的低熔体气糖加入到幼虫的 1.5% 低熔体中，并使用 800 微米安装毛细管首先将幼虫拉上头部。

如果定位不准确，将幼虫从红糖中排出并再次安装。让毛细管冷却，然后插入光片室。在 ZEN 成像软件上，连续按压以定位幼虫，然后按获取获得眼镜之间头部的 z 堆栈图像。

在处理选项卡中，从每个 z 堆栈生成最大强度投影图像。要随机选择运动性测定，麻醉后将24只幼虫转移到新鲜的E3介质中，让动物从麻醉中恢复。幼虫完全从麻醉中恢复后，使用带末端切口的移液器将回收的幼虫转移到E3介质中，无甲基蓝。

在24个井板每井一毫升内板一个幼虫。将板装入摄像机室。在 EthoVision XT 跟踪软件中，调整实验设置以设置白光启动程序，以增加自发游泳和检测运动 10 分钟。

在受精素后96小时和120小时重复运动测定。使用转基因泛素分泌的附件V M静脉报告线评估脑细胞死亡，结果在出血性幼虫中，所有非出血性幼虫中不存在的明确明确的死亡细胞群，绿色荧光亮场图像表明存在脑出血。在阿托伐他汀和泡沫头模型中观察到出血幼虫的死亡细胞。

MPEG1阳性巨噬细胞的形态在脑内出血阳性幼虫中变化，因为细胞采用活性圆形类黄体形状。这些激活的圆形细胞随着时间的推移被监测，以显示泛素分泌附件V M静脉表达垂死细胞在脑内出血阳性幼虫的噬细胞增加的噬菌体反应。脑出血与受精后72小时和96小时运动能力显著下降有关，相比之下，脑内出血阴性兄弟姐妹控制。

施肥后120小时的活力恢复到接近基线水平。这个程序最重要的方面是，在进行表型检测之前，确保彻底检查幼虫是否存在脑出血。此模型还可用于药物筛选，以确定出血后表型严重性能否得到改善，这种方法可能导致我们在未来确定新的候选药物。

这种技术使我们能够探索大脑出血后立即的细胞反应，在一个时间点，已经出了名的难以研究之前。尽管本协议中描述的试剂或仪器均无特别危险，但无论何时使用化学品、利器或激光器，都必须采取标准注意事项和预防措施。

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