Name: a scalable model to study the effects of blunt-force injury in adult zebrafish
Uploaded: 2021-05-31T15:00:00
Description: Watch this Scientific Journal Video about A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish at JoVE.com

作者要感谢海德实验室成员的深思熟虑的讨论，弗雷曼生命科学中心负责斑马鱼护理和饲养的技术人员，以及圣母大学光学显微镜核心/ NDIIF使用仪器及其服务。这项工作得到了圣母大学斑马鱼研究中心，圣母大学干细胞和再生医学中心的支持，以及NIH R01-EY018417国家眼科研究所（DRH），国家科学基金会研究生研究奖学金计划（JTH），LTC Neil Hyland圣母院奖学金（JTH）的资助， 自由哨兵奖学金（JTH）和Pat Tillman奖学金（JTH）。

斑马鱼在弗雷曼生命科学中心的圣母院斑马鱼设施中饲养和维护。本手稿中描述的方法得到了圣母大学动物护理和使用委员会的批准。
1. 创伤性脑损伤范式
<ol><li>将1 mL 2-苯氧乙醇加入1 L系统水中（60mg Instant Ocean在1L去离子RO水中）。</li>
	<li>在室温下准备一个装有2L系统水的曝气回收罐。</li>
	<li>选择滚珠轴承的所需重量以及钢/塑料管的所需长度和直径，并确定能量和冲击力...

<ol>
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长期以来，神经创伤和相关后遗症的研究一直集中在传统的非再生啮齿动物模型20上。直到最近，研究才将各种形式的CNS损伤应用于再生模型9，11，13，14，21。虽然这些模型具有洞察力，但它们受到使用在人群中罕见的损伤方法（穿透性创伤，化学消融，爆...

创伤性脑损伤（TBIs）是一场全球性健康危机，也是死亡和残疾的主要原因。在美国，每年约有 290 万人患有 TBI，在 2006-2014 年期间，TBI 或 TBI 后遗症导致的死亡率增加了 50% 以上1。然而，TBI 的病因、病理学和临床表现各不相同，这在很大程度上部分归因于损伤机制 （MOI），这也会影响治疗策略和预测预后2。虽然TBI可以由各种MOI引起，但它们主要是穿透性或钝力创伤的结果。穿透性创伤占TBI的一小部分，并产生严重的局灶性损伤，局限于直接和周围的刺穿脑区域3。相比之下，钝力TBI在一般人群中更常见，涵盖一系列严重程度（轻度、中度和重度），并产生累及多个脑区域的弥漫性、异质性和整体性损伤1，4，5。
斑马鱼 （Danio rerio） 已被用于检查跨越中枢神经系统（CNS）的各种神经损伤6，7，8，9。与哺乳动物不同，斑马鱼还具有先天而强大的再生反应，以修复中枢神经系统损伤10。目前的斑马鱼创伤模型使用各种伤害方法，包括穿透，切除，化学侮辱或压力波11，12，13，14，15，16。然而，这些方法中的每一种都利用了人群很少经历的MOI，不能在一系列损伤严重程度上扩展，并且不能解决钝性TBI后报告的异质性或严重程度依赖性TBI后遗症。这些因素限制了斑马鱼模型的使用，以了解与人群中最常见的TBI形式（轻度钝力损伤）相关的病理的潜在机制。
我们的目标是开发一种快速且可扩展的钝力TBI斑马鱼模型，为研究损伤病理学，TBI后遗症的进展以及先天再生反应提供途径。我们修改了常用的啮齿动物Marmarou17 重量下降，并将其应用于成年斑马鱼。该模型产生可重复的严重程度范围，从轻度，中度到重度。该模型还以严重程度依赖性的方式概括了人类TBI病理学的多个方面，包括癫痫发作，水肿，硬膜下和脑内血肿，神经元细胞死亡和认知缺陷，如学习和记忆障碍。受伤后几天，病理和缺陷消散，恢复到类似于未受损对照的水平。此外，该斑马鱼模型在损伤严重程度方面显示出整个神经轴的强健增殖和神经元再生反应。
在这里，我们详细介绍了钝力创伤的设置和诱导，创伤后癫痫发作的评分，血管损伤的评估，准备脑切片的说明，量化水肿的方法以及对损伤后增殖反应的见解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Sigma Alderich</td>
			<td>77699</td>
			<td></td>
		</tr>
		<tr>
			<td>#00 buckshot</td>
			<td>Remington</td>
			<td>RMS23770</td>
			<td>3.3g weight for sTBI</td>
		</tr>
		<tr>
			<td>#3 buckshot</td>
			<td>Remington</td>
			<td>RMS23776</td>
			<td>1.5g weight for miTBI/moTBI</td>
		</tr>
		<tr>
			<td>#5 Dumont forceps</td>
			<td>WPI</td>
			<td>14098</td>
			<td></td>
		</tr>
		<tr>
			<td>5-ethynyl-2&#8217;-deoxyuridine</td>
			<td>Life Technologies</td>
			<td>A10044</td>
			<td>EdU</td>
		</tr>
		<tr>
			<td>5ml glass vial</td>
			<td>VWR</td>
			<td>66011-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit</td>
			<td>Life Technologies</td>
			<td>C10340</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoOne 12-well plate</td>
			<td>USA Scientific</td>
			<td>CC7682-7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Super frost postiviely charged slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP Pen Liquid Blocker</td>
			<td>Ted Pella</td>
			<td>22309</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium</td>
			<td>VWR</td>
			<td>15148-031</td>
			<td></td>
		</tr>
	</tbody>
</table>

a scalable model to study the effects of blunt-force injury in adult zebrafish

钝力创伤性脑损伤（TBI）是最常见的头部创伤形式，其严重程度范围广泛，并导致复杂和异质的继发效应。虽然没有机制来替换或再生人类TBI后丢失的神经元，但斑马鱼具有在整个身体（包括大脑）中再生神经元的能力。为了检查斑马鱼在钝力TBI后表现出的病理学的广度，并研究随后的神经元再生反应背后的机制，我们修改了常用的啮齿动物Marmarou体重下降，以用于成年斑马鱼。我们的简单钝力TBI模型是可扩展的，可诱导轻度，中度或重度TBI，并概括了在人类TBI之后观察到的许多表型，例如接触性和创伤后癫痫发作，水肿，硬膜下和脑内血肿以及认知障碍，每种表型都以损伤严重程度依赖性的方式显示。TBI后遗症在受伤后几分钟内开始出现，在受伤后7天内消退并恢复到接近未受损的控制水平。再生过程早在损伤后48小时（hpi）就开始了，细胞增殖峰值在60 hpi处观察到。因此，我们的斑马鱼钝力TBI模型产生类似于人类TBI的特征性原发性和继发性损伤TBI病理，这允许研究疾病的发作和进展，以及斑马鱼独有的神经元再生机制。

准备损伤诱导装置可以快速而简单地向成年斑马鱼提供可扩展的钝力TBI。损伤模型的分级严重程度提供了几个易于识别的成功损伤指标，尽管血管损伤是最简单和最突出的病理之一（图3）。受伤期间使用的鱼品系可以使该指标更容易或更难识别。当使用野生型AB鱼（WTAB，图3A-D）时，很难区分miTBI或moTBI的?...

Watch this Scientific Journal Video about A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish at JoVE.com

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

我们修改了成年斑马鱼的Marmarou体重下降模型，以检查钝力创伤性脑损伤（TBI）后的病理范围以及随后神经元再生的机制。这种钝力 TBI 模型具有可扩展性，可诱发轻度、中度或重度 TBI，并概括在人类 TBI 中观察到的损伤异质性。

研究钝力损伤对成年斑马鱼影响的可扩展模型

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

该协议提供了一种简单，快速且可重复的方法来诱导成年斑马鱼的可扩展钝力创伤性脑损伤。经历这种钝力创伤性脑损伤的成年斑马鱼表现出许多在患有钝力TBI的人类中观察到的特征。演示该程序的将是James Hentig先生和Kaylee Cloghessy女士，他们是我实验室的两名博士生。首先用造型粘土填充培养皿。然后用手指或一对镊子的背面用额外的建模粘土创建一个凸起的平台。使用剃须刀片将凸起的平台纵向分成两个大致相等的两半。将两半形成一个通道，使其容纳成年鱼的长度。使用额外的粘土来建造墙壁，以确保2/3的鱼体头部暴露在外。在垂直于墙壁的暴露头部区域模制一个小支撑，以避免头部受伤时旋转或反冲。确保通道的侧面不会阻碍掉落的重量。使用迷你打孔机从 22 号钢闪光灯中创建 3 毫米钢盘。将一条对尾巴捏合无反应的麻醉鱼放在通道内的粘土模具上，其背侧朝上，使其身体固定在侧面。将3毫米22号钢盘放在头部，以所需冲击点为中心。垂直对齐鱼，以避免其头部向一侧倾斜，这可能会导致不均匀的撞击。使用标准的环形支架和臂夹来固定钢管或塑料管，确保它是直的，因此管的底部在斑马鱼头部上方1.5厘米处。向下看管道，确保其与钢板上方对齐。根据所需的损伤严重程度选择合适的滚珠轴承。滚珠轴承从预定高度沿着卡套管下降到钢板上。然后将受伤的鱼放入恢复池中进行监测。用建模粘土填充培养皿，并创建一个小腔以在解剖过程中支撑身体。将鱼放入粘土模具中，背部朝上。放置两个解剖销，一个穿过身体中间的中线，另一个在头部底部后方约5毫米处。使用一对5个Dumont镊子钝地切断视神经并去除眼睛。将5个镊子的一端放在右顶板下。故意做一个剪刀动作，向喙端移动，并取出右额板。然后将鱼顺时针旋转90度。将5个镊子的一端放在左顶板下方，并使用相同的剪刀运动去除左顶板和额板，暴露大脑的整个背侧。使用5个镊子钝切上颌骨，使嗅球保留不被损坏。使用相同的镊子，卸下右吊钳、前吊钳、互操作器和子吊钳。然后钝地切除牙龈尾端的肌肉组织，以暴露脊髓。使用镊子，钝性地切除脊髓。将镊子小心地放在大脑下方，轻轻地将大脑从髑髅地取出。使用文本手稿中的说明解剖整个大脑或感兴趣的区域，并使用精细的镊子立即将大脑放在小称重船上，注意不要刺伤或刮伤大脑。将大脑转移到焦油干燥称重船上，并记录大脑的湿重。调整大脑的方向，将它们平放在称重船上，背侧朝上。然后将大脑和干燥的称重船放入设置为60摄氏度的杂交烤箱中八小时。要将大脑转移到一个新的焦油小秤上，一旦它干燥，就把细镊子捏在一起，从大脑的腹侧开始，向上舀。在湿海绵上做一个部分切口。一次将一条鱼放在开口处，腹侧朝上，并使用30号针将大约40微升的10毫摩尔EdU注入鱼体内。然后将鱼放回装满系统水的储水箱。如前所述收集大脑，并将它们作为一组放置在一个9毫升的玻璃小瓶中，该小瓶含有2毫升的9份100%乙醇到一份37%甲醛。将大脑固定在4摄氏度的摇臂平台上。使用低温恒温器卡盘将大脑嵌入TFM中，在干冰上以所需的方向嵌入。血管损伤被发现是通过该模型识别成功损伤的最简单和最突出的病理之一。识别指标的能力随着受伤期间使用的鱼品系而变化。野生AB型的血管损伤的鉴定很难区分轻度或中度TBI和未受损的对照鱼，因为色素沉着。受伤后，轻度TBI鱼的表面擦伤很小，而中度TBI鱼表现出有限的脑出血。损伤程度在严重的TBI鱼中很明显。相比之下，使用白化病或Casper鱼时可以很容易地识别血管损伤。使用水肿评估由于损伤引起的脑肿胀。相比之下，中度TBI和重度TBI都有明显的水肿，1 dpi和3 dpi，但中度TBI和重度TBI的液体含量恢复到类似于5 dpi的未受损对照的水平。这种钝力损伤导致横跨神经轴的强烈细胞增殖反应。与未受损的对照组相比，在前脑的心室和脑室下区域观察到EdU标记增加。与未受损的鱼脑相比，受伤的大脑在脑室周围灰色区域，光学直肠叶和下丘脑前部方面显示出增加的EdU标记。在严重TBI之后，与未受损的大脑相比，后脑中的神经源性区域表现出增加的细胞增殖。为了正确诱导伤害，请确保鱼在模具中固定和稳定，并且管子是直的，以避免偏离中心冲击或改变重量下降轨迹。该程序提供了一种快速且具有成本效益的钝力损伤诱导方法，应用于再生模型，特别适用于重复的头部创伤或损伤诱导的再生研究。

Research

JoVE Journal

Neuroscience

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

科研

神经科学

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

斑马鱼成人端脑的一刀，受伤：一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

大鼠模型中的面部神经外科研究同氧抑制与再生

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

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

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

结合行为和脑电图研究正念冥想对情景记忆的影响

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

创伤性脑损伤后评估成年大鼠空间工作记忆的指标测试

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...