Name: a novel method to model chronic traumatic encephalopathy in drosophila
Uploaded: 2017-07-04T13:00:00
Description: Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

作者没有什么可以披露的。

打击装置装配（ 图1 ） <ol><li>从1 mL结核菌素注射器中取出柱塞。切成1毫升标记的桶。 </li><li>从200μL移液器吸头上取下气溶胶屏障（直径3mm，直径4 mm），并将其用作冲击器。将冲击器放在注射器筒内。轻轻敲击枪管将冲击器移动到前端，平面覆盖喷嘴开口。 </li><li>将枪管末端连接到连接到果蝇麻醉站的二氧化碳（CO 2 ）流量调节器的塑料管上。 </li><li>将桶?...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+traumatic+encephalopathy:+where+are+we+and+where+are+we+going?.">Chronic traumatic encephalopathy: where are we and where are we going?.</a> Curr Neurol Neurosci Rep. 13 (12), 407 (2013).</li><li>McKee, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+disease+in+chronic+traumatic+encephalopathy.">The spectrum of disease in chronic traumatic encephalopathy.</a> Brain. 136 (Pt 1), 43-64 (2013).</li><li>Petraglia, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+neurobehavioral+sequelae+after+repetitive+mild+traumatic+brain+injury:+a+novel+mouse+model+of+chronic+traumatic+encephalopathy.">The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.</a> J Neurotrauma. 31 (13), 1211-1224 (2014).</li><li>Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+in+the+study+of+human+neurodegeneration.">Drosophila melanogaster in the study of human neurodegeneration.</a> CNS Neurol Disord Drug Targets. 9 (4), 504-523 (2010).</li><li>Littleton, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter+hemolymph+sampling+and+analysis+of+individual+adult+Drosophila+melanogaster.">Nanoliter hemolymph sampling and analysis of individual adult Drosophila melanogaster.</a> Anal Chem. 84 (10), 4460-4466 (2012).</li><li>Katzenberger, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+closed+head+traumatic+brain+injury.">A Drosophila model of closed head traumatic brain injury.</a> Proc Natl Acad Sci USA. 110 (44), E4152-E4159 (2013).</li><li>Barekat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Drosophila+as+an+integrated+model+to+study+mild+repetitive+traumatic+brain+injury.">Using Drosophila as an integrated model to study mild repetitive traumatic brain injury.</a> Sci Rep. 6, 25252 (2016).</li><li>Katzenberger, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+to+Inflict+Closed+Head+Traumatic+Brain+Injury+in+Drosophila.">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.</a> J Vis Exp. (e52905), (2015).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat Methods. 6 (6), 451-457 (2009).</li><li>Straw, A. D., Dickinson, M. 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忠实地建模CTE特征的动物模型，包括神经生理学改变，神经病理学特征和神经行为缺陷，对于揭示疾病机制和开发诊断和治疗目标至关重要。可以理解，人类疾病的动物模型在模拟所有临床相关终点方面是完美的。然而，我们认为，强大的CTE模型应该满足以下三个要求：（1）影响必须直接适用于具有完整的头皮和颅骨保护的头部; （2）在冲击暴露期间头部不应固定，以便允许快速加速 - 减速和旋...

慢性创伤性脑病（CTE）最近被认为是一种独特的神经变性疾病，与其他tau病，如阿尔茨海默病1分离 。与阿尔茨海默病和其他常见的tau蛋白病（其最重要的危险因素是年龄增长和痴呆家族史）不同，CTE如其名称所示，意味着与脑外伤历史密切相关，很可能与体育运动员接触，例如拳击手和足球运动员，以及2,3,4,5的退伍军人。它被认为是通过反复的震荡和冲击打击脑袋。患者可能会出现症状和体征，如认知缺陷，情绪和行为变化以及运动功能障碍，其与阿尔茨海默病，额颞叶痴呆，路易体痴呆和帕金森病6 。相比之下，脑组织的死后检查显示在皮层sulci深度周围的小血管周围的超磷酸化tau积累的不同模式，在其他退化病症中未观察到的病理特征7 。然而，到目前为止，关于导致疾病表现的发病机制知之甚少。这在很大程度上是由于缺乏一个忠实的动物模型 - 只是最近才有了5,8的鼠类模型。这些模型生物体具有成本密集型护理和相对较长使用寿命的缺点，不太适用于神经退行性疾病研究。 与哺乳动物相比，无脊椎动物如果蝇是一种很好的替代方案，具有成本效益的维护，广泛的解剖遗传决定因素的工具，寿命相对较短9 。值得注意的是，飞行和人类大脑分享进化上保守的分子和细胞通路，以及解剖学相似性10,11,12 。已经报道了两个巧妙的果蝇模型来研究创伤性脑损伤。由Katzenberger及其同事设计的第一个“高冲击创伤”（HIT）装置将塑料瓶中的自由移动的苍蝇与金属弹簧13,15的自由端相连。当塑料小瓶倾斜直立并释放时，它会撞到聚氨酯垫，并在苍蝇弹到小瓶壁并反弹时给野生动物施加创伤。相比之下，Barekat和同事们设计了一种不同的送货方式使用Omni Bead Ruptor-24匀浆机平台14 。苍蝇与CO 2失活，并置于2mL螺旋管中，将其固定在均化器上并进行预编程的摇动条件。使用组织匀浆器系统的一个好处是，实验者可以调节受伤的强度，受伤持续时间和伤害发作次数。然而，这两个制度都有同样的缺点：头部的主要伤害在冲击位置和强度方面是随机的。此外，这两种方法导致相当大的死亡率，这是由身体和内脏其他部位的不可避免的附带损害造成的。在这里，我们描述了一种在果蝇中诱导mTBI的新方法。我们的装置由气体推进的弹道冲击器组成。与现有的果蝇模型14,15相比，我们的方法具有独特的优势可引起影响，仅针对自由移动的飞头，从而允许准确控制各种因素，如影响严重性，影响之间的时间间隔以及持续影响的总数。

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Aerosol Barrier</td>
			<td style="width:151px;">USA Scientific</td>
			<td style="width:119px;">1120-8810</td>
			<td style="width:207px;">Used as an impactor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">200 ul Pipette Tip</td>
			<td style="width:151px;">USA Scientific</td>
			<td style="width:119px;">1111-0706</td>
			<td>Used as a fly head holder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">1000 ul Pipette Tip</td>
			<td style="width:151px;">USA Scientific</td>
			<td style="width:119px;">1122-1830</td>
			<td>Used as a connector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">1 ml Tuberculin Syringe</td>
			<td style="width:151px;">Becton Dickinson</td>
			<td style="width:119px;">309625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">60 mm Petri Dishes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td>FB0875713A</td>
			<td>Used as a tracking arenas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Flow Regulator</td>
			<td>Genesee Scientific</td>
			<td style="width:119px;">59-122WC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Standard Clamp Holder/stand</td>
			<td style="width:151px;">EISCO Scientific</td>
			<td style="width:119px;">CH0688</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Fine Brush</td>
			<td style="width:151px;">Genesee Scientific</td>
			<td style="width:119px;">59-204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Flypad</td>
			<td style="width:151px;">Genesee Scientific</td>
			<td style="width:119px;">59-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sylgard Silicone Elastomer</td>
			<td style="width:151px;">Dow Corning</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD Camera</td>
			<td style="width:151px;">Microsoft&#160;</td>
			<td style="width:119px;">HD-5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ctrax Walking Fly Tracker</td>
			<td style="width:151px;">Caltech</td>
			<td style="width:119px;">Ctrax 0.2.11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MATLAB Image Processing Toolbox</td>
			<td style="width:151px;">MATLAB</td>
			<td style="width:119px;">R2015b</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel method to model chronic traumatic encephalopathy in drosophila

慢性创伤性脑病（CTE）是一种既定的神经退行性疾病，与暴露于重度轻度创伤性脑损伤（mTBI）密切相关。尽管最近有一个界定神经病理学标准的共识，但其复杂的病理变化的机制仍然很难捉摸。在这里，我们描述了一种在黑腹果蝇 （ 果蝇 ）中开发CTE模型的新方法，旨在鉴定导致大脑中特征性过度磷酸化tau积累和神经元死亡的关键基因和途径。对可能受到轻微封闭伤害的强度影响直接传递给飞头，使头部快速加速和减速。我们的方法消除了其他果蝇 mTBI模型固有的潜在问题（ 例如，动物死亡可能是由于损伤引起的身体其他部位或内脏）。较少的劳动力和成本密集的动物护理，短寿命和广泛的遗传工具使果蝇成为研究CTE发病机理的理想选择，并且可以进行大规模，全基因组的前期遗传和药理筛选。我们预计，模型的持续表征将为疾病预防和治疗方法产生重要的机械观点。

为了建立使用成年果蝇的CTE模型，我们确定了我们的装置在单次闭合性头部受伤时的有效性。为了消除与基因型，性别或年龄有关的变化，我们在实验中使用了2日龄的Canton-S WT雌性苍蝇。通过在100 kPa的恒定气压下调节CO 2的流量，可以很容易地控制冲击器的强度。以最高流速（15L / min）暴露于单次冲击的苍蝇显示出最小的外部缺陷（ 图2</stron...

Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

在这里，我们描述了一种在黑腹果蝇中引起封闭头部创伤性脑损伤的新方法。我们的方法的优点是可以直接将可重复的冲击强度提供给头部。无脊椎动物系统的进一步探索将有助于阐明慢性创伤性脑病的发病机制。

慢性创伤性脑病模型的一种新方法<em&gt;果蝇</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

The overall goal of this novel procedure is to inflict closed traumatic brain injuries in the fruit fly, Drosophila, to establish a new chronic traumatic encephalopathy model. This method can help answer key questions in the chronic traumatic encephalopathy field, such as how the repetitive trauma induces neuron degeneration. The main advantage of this technique is that it delivers the repetitive impacts of adjustable strengths to the fly head directly without causing damage to other parts of the animal body.Real world demonstration of this method is critical as assembly of the device and fly loading steps are difficult to learn, because the Drosophila head is so small and it is important to apply the impact only to the head. To make the apparatus used to concuss or strike the fruit flies, first, make the impactor portion of the apparatus. Remove the plunger from a one milliliter polycarbonate tuberculin syringe and then cut the barrel at the one milliliter mark.For an impactor, use the aerosol barrier found on a 200 microliter pipette tip. This impactor should be three millimeters long and four millimeters in diameter. Then, pack the impactor into the syringe barrel with the flat side towards the nozzle.To provide force, attach the syringe barrel to a carbon dioxide flow regulator. The regulator must have an On Off toggle switch and good control of the gas flow rate. Then, clamp the barrel vertically to an iron stand, such that the impactor will fall to the bottom of the barrel.Next, make the fly holder. Start with a 200 microliter pipette tip. Cut the pipette's tip five millimeters from the small end to make a 8 millimeter diameter opening.A fly head will fit through this hole, but the thorax will not. Lastly, make a for the fly holder and impactor device. First, cut a one milliliter pipette tip 44 millimeters from the small opening.Second, cut a six millimeter length from the needle cover for a one milliliter syringe. Then, push the two together. Anesthetize a single, two-day old adult female fly using carbon dioxide.From the carbon dioxide pad, use a fine brush to gently transfer the fly into the holder. Then, gently tap the holder until the head protrudes from the tip. Of the proboscis is exposed, use a blunted, one milliliter syringe needle to gently tuck it back inside the tip.It is critical to keep the fly body, especially the mouth part, inside the holder. Otherwise, the fly may die from damage to the internal organs or from not being able to ingest food. Next, set the gas pressure to 100 kilopascals and adjust the flow as needed.Once the fly is loaded, tighten the holder to the syringe barrel using the connector, such that the fly head faces downward. Then, using the toggle switch, send a burst of gas that moves the impactor to strike the fly once and only once. Now, detach the fly holder and dump the fly back onto the carbon dioxide pad.Brush the fly into an empty vial until it recovers. Keep one fly per vial. The recovery takes only a few minutes.Repeat the process to test four flies per experimental group. Processing two groups, a test and a sham, should only take 20 to 30 minutes. Treat shams just like test animals, except do not include an aerosol barrier in the impactor tube.To track fly movements in a Petri dish, first, fill a six centimeter dish with transparent silicon elastomer to make the tracking arena. Leave a three millimeter gap between the silicon and the lid so that flies can walk freely in the gap, but not take flight. Beneath the tracking arena, have a white background.Next, anesthetize four flies from the same treatment group and place them in the arena. Let the flies acclimate for an hour to the arena, which should be fully lit and at about 22 degrees Celsius. Not hotter than room temperature.After an hour, use a charge-coupled device camera positioned above the arena to record the fly's activity for five minutes. After the five minute recording, anesthetize the flies in the arena and return them to a new home vial. Then, dispose of the arena.Later, analyze the recordings using the freely available Ctrax software. This software generates tracking data, which can be exported in a programmable language compatible format, such as the MATLAB format. From the data, calculate the distance traveled per frame, the mean walking distance for each fly, and the average distance traveled per fly.For statistical significance, expect to test about 100 flies per treatment group. To establish a chronic traumatic encephalopathy model, the effectiveness of a single, closed-head injury made by the impacting apparatus at different speeds was analyzed using two-day old Canton-S females. The gas pressure was held at 100 kilopascals.Flies exposed to a single strike at the highest tested flow rate, exhibited minimal external defects. Despite no clear evidence for external damage, injuries at a flow rate of 15 liters per minute were acutely lethal, resulting in less than a 10%survival within 24 hours. Survival increased with lower flow rates.One hundred percent survival was achieved at five liter per minute or less, so five liters per minute was selected as the standard for the model. Just after being struck, flies gradually recovered their mobility within four minutes. Recovery for the shams was about a minute quicker.Post-impact, video-assisted movement tracking was used to measure locomotive function. During the two days after the impact, locomotion slowly returned to normal. This evidence for gradual recovery is in line with CTE recovery in humans.To evaluate long term effects, a repetitive traumatic brain injury protocol of five strikes over five days was applied. Twenty days post-treatment, the locomotion of surviving members in each group was compared and the treated group clearly walked less. The lifespan of the treated group was also substantially reduced, with a median at 26 days compared to 37 days for controls.After its development, this technique paves the way for researchers in the field of mild repetitive traumatic brain injury to explore pathogenic mechanisms in the classic animal model Drosophila melanogaster. While attempting this procedure, it is important to remember to shield the proboscis, thorax and abdomen from direct impact by the aerosol barrier.

Research

JoVE Journal

Neuroscience

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Novel Apparatus and Method for Drug Reinforcement

新型仪器和药物加固方法

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

科研

神经科学

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

在分析癫痫样活动和运动的一种低成本方法<em&gt;果蝇</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

药品监督管理局多个斑马鱼的新方法（<em&gt;斑马鱼</em&gt;）和退出的量化

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...