这项工作是由卫生部资助，R01 AG033620（BG）全国学院和罗伯特 - 德雷珀技术创新资金（DAW）的支持。

1.构造的HIT设备的<ol><li>装上弹簧用两个夹子和四个螺丝（ 图1A）董事会。相对于中心到电路板的宽度夹具和对接起来反对另一个与电路板的边缘外夹紧齐平。前附接夹具，用钳子为紧密地装配在弹簧弯曲它们。 注:请参阅表1的要求构建HIT装置的材料的说明。弹簧的固定端应该从板的边缘1/8英寸（3.2毫米），以及自由端应延伸到板由3/4英寸（19毫米）。调整弹簧使得其位于与董事会的长度平行。 </li><li>用魔术贴循环的胶粘带一圈包裹弹簧的自由端。魔术贴的外边缘应与弹簧的端部齐平。维可牢是重要的，因为它是用来固定小瓶到弹簧通过CRE阿婷紧压缩接头。魔术贴还允许方便地连接和拆卸小瓶，让许多小瓶在短时间内进行处理。 </li><li>放置冰桶盖倒挂，中心，紧贴木板。东方冰桶盖的凸起区域，使得长边平行于板的宽度。注意，冰桶的凸起区域是1/2英寸（13mm）的比木板更高，这样，当一个小瓶附着在弹簧的弹簧不会平躺在板上。 </li><li>对一个固定的物体，整个滑动装置如墙，使冰桶盖楔形板和物体之间并没有移动。 </li><li>胶带纸量角器纸板底部飞瓶托盘，站在它的边缘上对板的长度，使得90°标记对准弹簧，当它被拉回一个完美的垂直位置。 </li></ol> 2。哈工大设备的操作<ol><li> 1和60的CO 2 -anesthetized苍蝇在一个空的管形瓶中，并用紧身棉球塞住小瓶之间的地方。 </li><li>通过推棉球到小瓶直到它为1英寸（2.5厘米）从底部限制在果蝇到小瓶的底部1英寸（2.5厘米）。它有利于再接上瓶在1英寸（2.5厘米）的标志。需要注意的是限制苍蝇更大或更小的管形瓶中的区域可影响表型的严重程度。 </li><li>等待5分钟的苍蝇从CO 2回收流动性。需要注意的是它不知道5分钟是否足以完全除去CO 2的影响。 </li><li>插入弹簧到小瓶的端部，直到粘扣带的内边缘与所述小瓶（ 图1B）的顶部齐平。当弹簧被平放，小瓶1英寸（2.5厘米）应该重叠冰桶盖的被升高的区域。小瓶可重复使用米任何时代。 </li><li>而坐，抱用左手的拇指和食指小瓶在魔术贴区域。握住板紧用右手的台式。另外，使用C-夹持有板紧到台式。 </li><li>弹簧拉笔直回所需角度。释放弹簧。让春天的到来吧完全停止。 </li><li>拆下弹簧小瓶，让苍蝇恢复为≥5分钟。受到苍蝇到另一个罢工或与苍蝇飞食品转移到小瓶中。 注:多种分析方法可以用于评估来自HIT装置撞击的表型效应。例如，在寿命的效果可以通过分析苍蝇，在损伤后时期生存的百分比来确定对脑形态学的影响可通过头部的组织学分析来确定，并在基因表达的效果可以通过mRNA水平的定量分析来确定10。 </li><li> Determin程序电子效果，是不通过相同的处理未受到打击控制苍蝇由于打击。戴听力保护，因为对冰桶盖小瓶的影响产生巨响。 </li></ol>

<ol>
	<li>Harrison-Felix, C. L., Whiteneck, G. G., Jha, A., DeVivo, M. J., Hammond, F. M., Hart, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+over+four+decades+after+traumatic+brain+injury+rehabilitation:+A+retrospective+cohort+study.">Mortality over four decades after traumatic brain injury rehabilitation: A retrospective cohort study.</a> Arch Phys Med Rehabil. 90, 1506-1513 (2009).</li><li>Coronado, V. G., 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=Surveillance+for+traumatic+brain+injury-related+deaths+&#8211;+United+States.">Surveillance for traumatic brain injury-related deaths &#8211; United States.</a> MMWR Surveill Summ. 60, 1-32 (1997).</li><li>Masel, B., DeWitt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury:+A+disease+process,+not+an+event.">Traumatic brain injury: A disease process, not an event.</a> J. Neurotrauma. 27, 1529-1540 (2010).</li><li>Blennow, K., Hardy, J., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropathology+and+neurobiology+of+traumatic+brain+injury.">The neuropathology and neurobiology of traumatic brain injury.</a> Neuron. 76, 886-899 (2012).</li><li>Prins, M., Greco, T., Alexander, D., Giza, C. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+challenges+in+clinical+trails+in+traumatic+brain+injury.">Unique challenges in clinical trails in traumatic brain injury.</a> Crit Care Med. 37, S129-S135 (2009).</li><li>Xiong, Y., Mahmood, A., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injury.">Animal models of traumatic brain injury.</a> Nature Rev Neurosci. 14, 128-142 (2013).</li><li>Balsiger, Z., Leudkte, J., Mawer, S., Willey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> HIT device high speed analysis. , (2014).</li><li>Davceva, N., Janevska, V., Illevski, B., Petrushevska, G., Popeska, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+of+acute+subdural+haematoma+and+diffuse+axonal+injury+as+two+typical+acceleration+injuries.">The occurrence of acute subdural haematoma and diffuse axonal injury as two typical acceleration injuries.</a> J Forensic Leg Med. 19, 480-484 (2012).</li><li>Katzenberger, R. J., Loewen, C. A., Wassarman, D. R., Petersen, A. J., Ganetzky, B., Wassarman , D. A. <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, E4152-E4159 (2013).</li><li>Fang, Y., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+degeneration+and+regeneration:+insights+from+Drosophila+.models+of+nerve+injury.">Axon degeneration and regeneration: insights from Drosophila .models of nerve injury.</a> Annu Rev Cell Biol. 28, 575-597 (2012).</li><li>Babcock, D. T., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+accurate+and+rapid+measurement+of+flight+performance+in+Drosophila.">An improved method for accurate and rapid measurement of flight performance in Drosophila.</a> J Vis Exp. (84), e51223 (2014).</li><li>Tully, T., Preat, T., Boynton, S. C., Vecchio, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+consolidated+memory+in+Drosophila.">Genetic dissection of consolidated memory in Drosophila.</a> Cell. 79, 35-47 (1994).</li><li>Andretic, R., Shaw, P. 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我们有没有利益冲突披露。

所述HIT器件的方法是从由它引起闭头而不是穿透TBI 11的事实造成外伤性损伤的苍蝇等方法区分。此外，HIT器件的方法花费较少的时间，精力和技能造成的TBI许多苍蝇，所以该方法比其它方法来大规模遗传筛选更适合。最后，一​​个事实，即对HIT设备造成主伤害不限于大脑既是限制和优点。它是一种限制，因为附加的研究来评估表型是否是由于外伤性损伤的大脑或其他身体部位。另一方面，多发伤（外伤性损伤到多个身体部分）通常伴随TBI并且被认为调节TBI的表型，所以HIT装置的方法可用于研究多发伤到TBI表型的贡献。飞TBI模式也有过啮齿动物和非人类灵长类动物模型的TBI优势<s达&gt; 10。大量动物可以检查因为果蝇繁殖迅速（女性躺在〜100粒/天），具有短的生命周期（从蛋10天至成熟成年在25℃），并保持在小药瓶和饲料便宜的介质上（〜20美分/月/ 100苍蝇）。此外，脑外伤的结果可以容易地在整个生命周期评估，因为成蝇具有相对短的寿命（50-100天，在25℃）。最后，从100多年以前的研究研制的苍蝇实验工具和技术产生巨大的军火库提供了实验控制基因表达的精确时空调控无与伦比的程度;的行为，发育和电生理学分析; 在体内成像等 。 本协议描述的HIT装置，其目的是造成闭头TBI在果蝇的建设和运营。我们已经使用了HIT设备结合对MI 24 &lt;/ SUB&gt;分析研究有机体死亡以下TBI 7。然而，飞TBI模型可以以许多其它方式来了解死亡以及TBI的其他后果可以使用。例如，创伤性脑损伤物理后果可使用测定法的参数，如爬山或飞行，和TBI的行为后果可使用测定法的参数，如睡眠以及学习和记忆10,12-14进行测量来测量。可以使用的成像技术如免疫荧光显微镜和透射电子显微镜来确定对大脑结构效应。可以使用基因组分析等的RNA以次或蛋白质组分析，如质谱法来确定对大脑分子的效果。可使用的方法，如条件性基因敲除和过表达来确定对大脑的遗传效应。关于TBI的后果的环境影响可以通过改变前或后损伤的参数，如温育温度和饮食或参数来确定所述HIT装置如弹簧偏转的角度或重复撞击之间的时间。最后，HIT设备可用于大规模遗传筛选，以解决根本问题，例如为何TBI的后果是在老年个体比年轻个体更坏，以及对可以用于鉴定治疗大规模药物屏幕TBI的后果。 当以一致的方式使用的，我们已经发现，对HIT装置产生间独立的实验和用户再现的表型。可以影响的再现性的参数是设置了HIT装置和操作。表型可以显著受冰桶盖相对于主板，棉花球在飞瓶的位置，位置的微小变化，以及小瓶相对于尼龙带的位置。此外，如示于图2，表型可以显著受的微小变化弹簧偏转的角度。因此，HIT设备的新用户应进行监测，以确保设置和操作是相同的，其他用户。我们经常通过让他们确定0-7日龄W¯¯1118苍蝇的MI 24校准新用户。最后，HIT装置不需要是相同的，在协议中所描述的。我们预计，HIT设备的大部分地区，同时保持对敌人造成TBI在果蝇的能力来改变。

An author's name was corrected in the publication of <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52905">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila</a>. The second to last author's was spelled incorrectly. It has been updated from:
Barry Ganetky
to:
Barry Ganetzky

An author's name was corrected in the publication of <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52905">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila</a>.

Erratum: A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

创伤性脑损伤（TBI），从外部机械力定义为损伤到大脑。最常见的是，自闭头的力量TBI的结果，如钝力和惯性加速和减速的力量导致大脑罢工颅骨里面。在美国，据估计，50,000人每年死于脑外伤和2.5-6.5亿人患有TBI的后果，包括衰弱的身体，认知和行为问题1,2。 TBI的后果不仅由于一次侧机械损伤大脑而且还发生在时间3-5次级细胞和分子的伤害到大脑以及其他组织。的方法来诊断和治疗TBI的发展已被证明是困难的，因为TBI是一种复杂的疾病过程。主要受伤，人体生理学和环境因素的变化性质导致异构辅助Injuries和病理。底层可变因素包括原发性损伤的个体的严重程度，重复初级损伤之间的时间，以及患者的年龄和基因型。如何理解每个变量因素有助于TBI的后果是可能的方法来诊断和治疗TBI 6,7的发展提供帮助。 在这里，我们描述了用于造成闭头TBI 果蝇 （果蝇），可用于描绘的可变因素的TBI的后果的贡献的方法。该方法是基于强烈反对击球手的手掌一飞文化小瓶的侧面造成野生型的初步观察苍蝇成为暂时丧失劳动能力，TBI的可能后果。因此，我们构建了高撞击伤（HIT）设备从手击球动作概括的加速和减速的力量。一个高速电影显示，从单一的罢工HIT设备导致飞往多次与他们的头部和身体接触8小瓶墙。从某种程度上说，所有接触都可能造成飞大脑弹和对变形头壳，类似于发生在人类的跌倒和车祸9什么。因此，苍蝇与脑损伤，包括暂时失能跟着共济失调，流动性逐步恢复，在头部基因表达的变化，并逐步在神经退行性大脑10相一致的HIT装置显示的表型治疗。因此，HIT设备使得有可能使用的实验工具和苍蝇开发的技术的巨大军火库研究TBI。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Zinc plated compression spring</td>
			<td>The Hillman&#160; Group</td>
			<td>540189</td>
			<td>9 7/8 in (length), 15/16 in (outer diameter), 0.12 in (wire size)</td>
		</tr>
		<tr>
			<td>Wooden board</td>
			<td></td>
			<td></td>
			<td>9 in (length), 6.5 in (width), 0.75 in (height)</td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Sigma Electrical Manufacturing Corporation</td>
			<td>49822</td>
			<td>3.10 in (length), 0.68 in (width), 1.11 in (height), EMT Two Hole Straps, click on type for 1 inch steel EMT conduit</td>
		</tr>
		<tr>
			<td>Loop half of self-adhesive velcro</td>
			<td></td>
			<td></td>
			<td>3 in (length), (3/4 in width)</td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket cover</td>
			<td>Fisher Scientific</td>
			<td>02-591-45</td>
			<td>9 1/8 in (length), 9 1/8 in (width), 1 1/4 in (height)</td>
		</tr>
		<tr>
			<td>Plastic fly vials</td>
			<td>Applied Scientific</td>
			<td>AS-510</td>
			<td>3 11/16 in (height), 1 1/16 in (inner diameter), 1 1/8 in (outer diameter)</td>
		</tr>
		<tr>
			<td>Large cotton balls</td>
			<td>Fisher Scientific</td>
			<td>22-456-883</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper protractor</td>
			<td></td>
			<td></td>
			<td>10 in (diameter)</td>
		</tr>
	</tbody>
</table>

a method to inflict closed head traumatic brain injury in drosophila

创伤性脑损伤（TBI）影响数以百万计的人，每年造成的身体，认知和行为功能和死亡的损害。使用果蝇研究在理解神经过程贡献重要突破。因此，随着理解人类TBI病理的细胞和分子基础的目标，​​我们制定了高撞击伤（HIT）设备造成闭头TBI的苍蝇。苍蝇受到人类TBI一致的HIT装置显示的表型，如暂时失能和渐进神经退行性疾病。所述HIT设备使用基于弹簧的机构，以推动苍蝇对小瓶的壁上，造成机械损伤对苍蝇大脑。该设备是便宜的，并易于构造，其操作简便，快速，和它产生可重复的结果。因此，HIT设备可与现有的实验工具和技术，苍蝇处理基本组合有关TBI问题，这可能会导致诊断和治疗TBI的发展。特别是，HIT装置可用于执行大规模遗传筛选以了解TBI病理的遗传基础。

我们感兴趣的是理解为什么原发性损伤苍蝇后不久死亡。量化死亡，我们确定在24小时的死亡率指数（MI 24），这是苍蝇该内的原发性损伤的24小时死亡的百分比。蝇从HIT设备进行打击孵育在25℃下与飞食品小瓶，和死蝇数24小时后进行计数。我们使用这种方法来确定影响的MI 24因素，发现MI 24不受苍蝇在小瓶中（10〜60苍蝇被测试）的数量，重复的撞击之间的时间（1到60分钟进行试验），或者飞10的性别。与此相反，我们发现，年龄在原发性损伤的时间和基因型确实影响了MI 24。年长的果蝇有较高的MI 24比年轻的苍蝇，苍蝇不同基因型有显著不同的MI 24秒。 在图2中，我们测试了原发性损伤的严重程度是否影响对MI 24。以改变的原发性损伤的严重程度，我们偏转的弹簧，以不同的角度。对于每一个角度，无论是0-7或20-27日龄W¯¯1118苍蝇（标准实验室品系）的小瓶受到四个罢工以罢工为5分钟。三小瓶60苍蝇进行了检查每个角度。蝇转移至小瓶糖蜜食物，保温在25℃，和死蝇数24小时后进行计数。算出平均值的平均MI 24和 ​​标准误差为每个角度。这些数据表明，较大的偏转，角度即 ，更严重的初级损伤，导致较高的MI 24比偏转小角度， 即 ，不太严重的伤害。这是观察到两个0-7和20-27日龄苍蝇。此外，符合Katzenberger 等 10，20-27日龄FLIES有显著高于MI 24比0-7日龄苍蝇角度≥50°。因此，这些数据表明，在多种年龄的MI 24相关的原发性损伤的严重性。 <img alt="图1" src="/files/ftp_upload/52905/52905fig1.jpg" /> 图1:对HIT装置的图（A）的示意图对HIT装置在静止位置的顶视图。 （B）的示意图HIT装置的侧视图。所述弹簧被示出处于静止位置（暗红色）和偏转到90°（淡红色）。 <img alt="图2" src="/files/ftp_upload/52905/52905fig2.jpg" /> 图2:原发性损伤严重程度与心梗24我们确定的MI为24天0-7岁W¯¯ <eM&gt; 1118苍蝇（浅灰色柱）和20-27日龄W¯¯1118苍蝇（深灰色条）从与春天HIT设备受到四个罢工的偏向度的角度表示的 0度数据对于苍蝇不进行对HIT设备。示出的是平均的MI 24和 ​​平均值的标准误差。 0-7和20-27天龄的苍蝇的MI 24是显著不同为50°（P &lt;0.05，一是尾t检验）和角度&gt; 50°（P &lt;0.001，一是尾t检验），但不用于角&lt; 50°（P&gt; 0.1，1尾t检验）。

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A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

在这里，我们描述了一种方法，在果蝇造成闭合性颅脑创伤性脑损伤（TBI）。此方法提供了一种网关，调查所依据使用的实验工具和技术可用于苍蝇繁多TBI病症的细胞和分子机制。

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 ...

a-method-to-inflict-closed-head-traumatic-brain-injury-in-drosophila

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

11.4K 次观看.  University of Wisconsin-Madison. 在这里，我们描述了一种方法，在果蝇造成闭合性颅脑创伤性脑损伤（TBI）。此方法提供了一种网关，调查所依据使用的实验工具和技术可用于苍蝇繁多TBI病症的细胞和分子机制。 

视频: A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

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 ...

科研

神经科学

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

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 ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

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 ...

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

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

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 ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

研究小鼠创伤性脑损伤后凯库姆微生物群的改动

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

低强度冲击波模型用于啮齿动物闭头轻度创伤性脑损伤的临床前评估

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

创伤性脑损伤的神经炎症和血流动力学反应的系统分析

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

小鼠轻度创伤性脑损伤的电磁控制闭头模型

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

方法对敌人造成闭合性颅脑创伤性脑损伤

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