我们要感谢中南大学实验动物系的所有研究员。本研究得到了国家自然科学基金（第81971791号）的支持;法医学上海市重点实验室， 法医学部重点实验室（法医学研究院）KF202104）。

动物实验由中南大学动物护理和使用委员会批准。所有研究均按照实验动物的福利和伦理原则进行。
1.动物喂养和麻醉程序
<ol><li>群居280-320克Sprague-Dawley雄性大鼠，并将它们维持在12小时/ 12小时的光/暗循环中， 并随意获得食物和水。在大鼠适应环境6天后进行研究。</li>
	<li>在诱导箱中以0.6L / min的气流用3%异氟醚麻醉大鼠，直到它对爪子或尾巴捏没有反应。保持流速30秒。 注意：不使用止痛药，因为它们会干扰大鼠在神经行为评估中的反应。</li>
</ol>2. 术前设置
<ol><li>将硬度值为 35D（海绵重量为 35 kg/m3）的海绵放置在没有顶盖的亚克力盒 （15 cm x 22 cm x 43 cm） 中，长度和宽度相同，但厚度为 12 cm。</li>
	<li>修剪锡箔（厚度为20μm）并使用胶带将其固定在亚克力盒上，以形成能够支撑大鼠重量的可破坏表面。此外，标记一条大约 10 厘米的切割线作为定位大鼠头部的指定位置。</li>
	<li>借助铁架，将 PVC 管牢固地固定到位。准备一个穿孔砝码，重 550 克，直径为 18 毫米。将重物连接到聚氯乙烯或 PVC 管内 1 米高的钓鱼线上。并将导管的位置调整到锡箔上方 3 厘米处。</li>
	<li>准备头盔和枕头。使用直径为 10 毫米、厚度为 3 毫米的不锈钢圆盘制作头盔。准备一个楔形海绵枕头放在老鼠的头部下方，确保它垂直于重力方向。 注： 冲击装置的示意图如 图 1 所示。头盔的作用是识别撞击位置并增强外力的分布。枕头用于保证均匀稳定的损坏。</li>
</ol>3. mTBI诱导
<ol><li>迅速将麻醉的大鼠放在锡箔纸上的胸部。 注意：mTBI 诱导需要两名操作员 - 一名用于准备，另一名用于验证。</li>
	<li>准备：将枕头放在老鼠下面，确保其头部与铝箔纸平行。将头盔与老鼠的耳朵对齐并将其固定到位。</li>
	<li>验证：验证 PVC 管是否位于头盔的正上方。两个操作员确认设置正确后，继续下一步。</li>
	<li>诱导头部旋转：释放重物，让它下落并撞击大鼠的头部，导致跌落到海绵上并旋转 180°。</li>
	<li>将老鼠放在干净的笼子里。</li>
</ol>4. 假诱导
<ol><li>以与先前mTBI诱导描述相同的方式对待大鼠，但不要使其受到头部撞击。</li>
</ol>5. 验证程序：急性神经行为评估
注意：以下评估是根据 Flierl 等人 10 的神经严重程度评分9 和协议修改的。所有这些评估都是在大鼠恢复矫正反射后 10 分钟进行的。
<ol><li>意识丧失时间：记录从大鼠被麻醉到恢复矫正反射的持续时间。 注意：矫正反射是大鼠仰卧时翻身的过程。矫正反射的丧失应被视为人道的终点，并且必须按照机构指南对动物实施安乐死。</li>
	<li>第一次寻找行为时间：记录从大鼠被麻醉到第一次出现寻找行为的持续时间。 注意：寻求行为是对环境感兴趣的标志，是一种生理反应。</li>
	<li>逃生能力<ol><li>将大鼠放在带有出口（长12.5厘米，宽9厘米）的圆形装置（直径0.5米，高0.3米）的中间。</li>
		<li>记录老鼠离开圆圈所需的时间。 注意：如果大鼠在180秒内没有离开圆圈，则将时间记录为180秒。</li>
	</ol></li>
	<li>光束平衡能力测试<ol><li>将大鼠放在3cm，2cm和1.5cm宽的光束上1分钟。</li>
		<li>如果大鼠在横梁上保持平衡，保持稳定姿势，则将其打为 0。</li>
		<li>如果老鼠抓住横梁的一侧，则给 1 分。如果老鼠抱住横梁，一只肢体从横梁上掉下来，则将其打成 2。</li>
		<li>如果老鼠拥抱横梁，两肢从横梁上掉下来或在上面旋转（>60 秒），则将其打成 3。</li>
		<li>如果大鼠试图在横梁上保持平衡但掉下来（> 40 秒），则将其打成 4。</li>
		<li>如果大鼠试图在横梁上保持平衡但掉下来（>20 秒），则将其打成 5。</li>
		<li>如果老鼠没有试图平衡或挂在横梁上并在 20 秒内掉下来，则将其打为 6。 注意：光束平衡测试不需要预试验。</li>
	</ol></li>
</ol>6. 验证程序：神经行为评估
注意：在行为实验之前，连续3天每天处理大鼠2分钟，以尽量减少压力和新奇性干扰。所有行为实验都是通过在实验开始前将动物置于测试环境中 60 分钟来进行的。
<ol><li>运动协调能力（光束任务）<ol><li>实验装置<ol><li>将大鼠放在平衡木的一端（长1.5 m，离地面75厘米）。在另一端放置一个逃生箱（倾斜的床上用品家庭笼子）。</li>
			<li>在横梁下方放置泡沫衬垫，以减轻在测试过程中跌倒时对大鼠造成伤害的潜在风险。</li>
			<li>打开摄像机。</li>
			<li>将检测日安排在受伤后或假治疗后的特定时间点（例如，第 1 天、第 3 天和第 7 天）。</li>
		</ol></li>
		<li>培训阶段（2天）<ol><li>训练大鼠连续 3 次穿过 4 厘米宽的光束，然后在 2 厘米宽的光束上进行两次试验。</li>
			<li>在训练过程中，轻轻地引导老鼠穿过光束，直到它们可以不受干扰地轻松穿过光束。</li>
		</ol></li>
		<li>平衡木实验<ol><li>将大鼠放在2厘米宽的光束上连续5次试验。</li>
			<li>当大鼠的鼻子分别越过起点和终点线时，记录每次试验的开始和结束。</li>
			<li>在实验结束时将大鼠放回笼子。</li>
		</ol></li>
		<li>基线测试<ol><li>在受伤或治疗前进行平衡木实验。</li>
			<li>计算这 5 次连续试验的平均值，以建立每只大鼠的基线。</li>
		</ol></li>
		<li>数据分析<ol><li>使用对实验条件不知情的研究人员的视频分析，分析穿过横梁的时间和后足滑倒的总数。</li>
		</ol></li>
	</ol></li>
	<li>焦虑（开放场测试）<ol><li>实验装置<ol><li>准备空旷的场地，确保它干净，没有任何以前的气味提示。将竞技场分为三个区域：中央内区（33 cm x 33 cm）、中间区（66 cm x 66 cm）和外区。</li>
		</ol></li>
		<li>测试阶段<ol><li>将一只老鼠放在空旷场地的中央并启动计时器。让老鼠自由探索竞技场 5 分钟。5分钟后，小心轻轻地将老鼠放回笼子里。</li>
		</ol></li>
		<li>数据采集<ol><li>测量大鼠在 5 分钟的探索期间行进的总距离。确定大鼠在中央内区、中区和外区停留的时间。</li>
		</ol></li>
		<li>数据分析<ol><li>使用行进的总距离作为整体探索行为和运动能力的衡量标准。计算在中央内区花费的时间，作为焦虑样反应的指标。</li>
		</ol></li>
	</ol></li>
	<li>学习和记忆能力（莫里斯水迷宫测试）<ol><li>确保水迷宫设备处于正常状态。将水染成黑色，并在四个基本方向上放置提示。将平台放置在水面以下 2.5 厘米处。</li>
		<li>设置监控系统来记录和观察老鼠的行为。</li>
		<li>步道日<ol><li>迅速将老鼠放入水迷宫中。如果老鼠未能在2分钟内到达平台，请用木棍轻轻引导它。</li>
			<li>让老鼠在平台上站立 20 秒时熟悉迷宫环境，然后将其移除。一旦老鼠在平台上，让它停留 20 秒，然后将其移除。</li>
		</ol></li>
		<li>每日重复<ol><li>重复训练日程序，将大鼠放入不同象限的水中。重复步骤 6.3.3。连续 5 天继续训练。</li>
		</ol></li>
		<li>探针测试日：在第 6天，取下平台并将大鼠置于同一象限2分钟。</li>
		<li>观察和记录：利用监测系统监测大鼠在试验和探测测试日的行为。</li>
		<li>清洁：将老鼠从水迷宫中取出后，用毛巾彻底擦干。</li>
	</ol></li>
</ol>

在 mTBI 大鼠模型中验证损伤反应和行为评估

<ol>
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作者没有经济利益需要披露。

该模型成功地模拟了闭合的头部 mTBI，而无需头皮切口或颅骨开口，从而更准确地表示了在人类病例中观察到的撞击场景。避免头皮切口有助于防止可能与实际情况不符的炎症反应。与 Richelle Mychasiuk 的儿科模型12 相比，本研究中使用的模型专为体重在 280-320 g 之间的成年大鼠量身定制，使我们能够获得对 mTBI 对成年个体影响的宝贵见解。此外，结合枕头和头盔等关键部件有助于?...

轻度创伤性脑损伤 （mTBI） 或脑震荡是最普遍的损伤类型，可导致各种短期和慢性症状1。这些症状可能包括头晕、头痛、抑郁和快感缺乏等，导致受 mTBI2 影响的个体遭受重大痛苦。由于大多数 mTBI 是由钝器外伤引起的 3，因此必须开发准确模拟此类损伤的动物模型。这些模型对于更好地了解损伤及其潜在机制至关重要，与人类研究相比，提供了一个可控的环境，减少了变异性和异质性。
已经开发了许多成熟的啮齿动物模型来治疗创伤性脑损伤 （TBI），包括液体叩击损伤 （FPI）4、受控皮质冲击 （CCI）5、体重下降损伤6、爆炸性创伤性脑损伤7 等。然而，这些模型主要侧重于复制中度至重度 TBI 情景。相比之下，专门设计用于模拟 mTBI 的实验模型受到的关注相对较少，并且仍未得到充分探索8。因此，迫切需要建立一个稳定且可重复的动物模型，以准确表示mTBI。这样的模型将显着增强我们对与 mTBI 相关的神经生物学和行为后果的理解。
在麻醉效果消失后，人们无法通过随意观察来区分mTBI大鼠与正常大鼠的功能缺陷。因此，有必要进行特定的测试。在人类中，广泛的临床评估用于评估患者 9,10,11。同样，在大鼠模型中建立一个成功的模型也需要使用快速评估工具来确定其有效性。
在这项研究中，我们提出了一个闭头mTBI大鼠模型，能够以与人类状况非常相似的方式研究mTBI。对模型及其验证程序的详细描述提供了对研究 mTBI 所用实验方法的全面理解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylic box</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>15 cm x 22 cm x 43 cm</td>
		</tr>
		<tr>
			<td>Anesthesia Machine</td>
			<td>RWD Life Science Co.</td>
			<td>R540 Mice &#38; Rat Animal Anesthesia Machine</td>
			<td></td>
		</tr>
		<tr>
			<td>Helmet</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>Stainless-steel disk measuring 10 mm in diameter and 3 mm in thickness</td>
		</tr>
		<tr>
			<td>Morris water maze</td>
			<td>RWD Life Science Co.</td>
			<td></td>
			<td>Diameter 150 cm, height 50 cm,platform diameter 35 cm</td>
		</tr>
		<tr>
			<td>Open field</td>
			<td>RWD Life Science Co.</td>
			<td>63007</td>
			<td>Width100 cm, height 40 cm</td>
		</tr>
		<tr>
			<td>Panlab SMART V3.0</td>
			<td>RWD Life Science Co.</td>
			<td>SMART v3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Perforated weight</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>Weight of 550 g and diameter of 18 mm</td>
		</tr>
		<tr>
			<td>Pillow</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>Wedge-shaped sponge to place beneath the rat&#39;s head</td>
		</tr>
	</tbody>
</table>

rat model of closed-head mild traumatic injury and its validation

动物模型对于促进我们对轻度创伤性脑损伤 （mTBI） 的理解和指导临床研究至关重要。为了获得有意义的见解，开发稳定且可重复的动物模型至关重要。在这项研究中，我们报告了闭头 mTBI 模型的详细描述以及使用 Sprague-Dawley 大鼠验证建模效果的代表性验证方法。该模型涉及将 550 克的质量砝码从 100 厘米的高度直接落到可破坏表面上的大鼠头部，然后进行 180 度转弯。为了评估损伤，大鼠在受伤后 10 分钟接受了一系列神经行为评估，包括意识丧失时间、首次寻找行为时间、逃生能力和光束平衡能力测试。在受伤后的急性和亚急性阶段，进行行为测试以评估运动协调能力（光束任务）、焦虑（开放场地测试）以及学习和记忆能力（莫里斯水迷宫测试）。闭头 mTBI 模型以最低的死亡率产生了一致的损伤反应，并复制了现实生活中的情况。验证方法有效地验证了模型的开发，保证了模型的稳定性和一致性。

Mild traumatic brain injury (mTBI), or concussion, is the most prevalent type of injury and can lead to various short-term and chronic symptoms1. These symptoms may include dizziness, headaches, depression, and anhedonia, among others, leading to significant suffering for individuals affected by mTBI2. Since most mTBIs are caused by blunt force trauma3, it becomes imperative to develop animal models that accurately mimic such injuries. These models are essential for gaining a better understanding of the injury and its underlying mechanisms, offering a controlled environment with reduced variability and heterogeneity compared to human studies.
Numerous well-established rodent models have been developed for traumatic brain injury (TBI), including fluid percussion injury (FPI)4, controlled cortical impact (CCI)5, weight-drop injury6, blast traumatic brain injury7, and others. However, these models primarily focus on replicating moderate to severe TBI scenarios. In contrast, the experimental models specifically designed to simulate mTBI have received relatively less attention and remain underexplored8. Therefore, there is a critical need to establish a stable and reproducible animal model that accurately represents mTBI. Such a model would significantly enhance our understanding of the neurobiological and behavioral consequences associated with mTBI.
One cannot distinguish the functional deficits in mTBI rats compared to normal rats via casual observation after the effects of anesthesia have worn off. Therefore, it is necessary to administer specific tests. In humans, a wide range of clinical assessments are used to evaluate patients9,10,11. Similarly, establishing a successful model in the rat model also requires using rapid assessment tools to determine its validity.
In this study, we present a closed-head mTBI rat model, enabling the investigation of mTBI in a manner that closely resembles the human condition. The detailed description of the model and its validation procedure provides a comprehensive understanding of the experimental approach utilized in studying mTBI.

作者聚焦：推进创伤性脑损伤研究 - 用于准确复制和快速评估的闭头模型 

闭合头轻度创伤大鼠模型及其验证

In this study, we introduced a closed-head, mild traumatic brain injury rat model, and provided a comprehensive overview of its validation. Our goal was to offer a straightforward approach for replicating this type of brain injury and a rapid assessment method for confirming the success of the model. This model successfully simulates a closed-head, mild traumatic brain injury without the need for scalp incision or skull opening, provide a more accurate representation of the impact scenario observed in human cases.The avoidance of scalp incision helps to prevent inflammatory responses that may not align with the active situation. Mild traumatic brain injury lacks subjective and accurate diagnostic methods. In the future, our laboratory will continue to explore possible biomolecular markers and their deep molecular mechanisms, and help to transform the obtained molecular markers into clinical and forensic identification practices.

这项工作中使用的设备是 Kane 模型和 Richelle Mychasiuk 的儿科模型11,12 的改进版本。在这项研究中，SD大鼠被分配到假手术组和mTBI组。为了证明该模型的可重复性，我们对该模型进行了三次独立重复以及急性神经行为评估，每个实验涉及 8-12 只大鼠。在这项研究中，我们使用了 30 多只 mTBI 大鼠，其中 2 只大鼠因麻醉而死亡。然而?...

Watch this Scientific Journal Video about Advancing Traumatic Brain Injury Research — A Closed-Head Model for Accurate Replication and Rapid Assessment at JoVE.com

在这里，我们提出了一个闭合式轻度创伤性脑损伤 （mTBI） 大鼠模型及其验证，在急性和亚急性阶段的行为表现方面与人类 mTBI 表现出显着的相似性。

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...

在这项研究中，我们介绍了一个闭头、轻度创伤性脑损伤大鼠模型，并对其验证进行了全面概述。我们的目标是提供一种简单的方法来复制这种类型的脑损伤，并提供一种快速评估方法来确认模型的成功。该模型成功地模拟了闭合的头部、轻度创伤性脑损伤，无需头皮切口或颅骨开口，更准确地表示了在人类病例中观察到的撞击场景。避免头皮切口有助于防止可能与活动情况不一致的炎症反应。轻度创伤性脑损伤缺乏主观和准确的诊断方法。未来，我们的实验室将继续探索可能的生物分子标志物及其深层分子机制，并帮助将获得的分子标记物转化为临床和法医鉴定实践。

rat-model-of-closed-head-mild-traumatic-injury-and-its-validation

Research

JoVE Journal

Neuroscience

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

717 次观看.  School of Basic Medical Science, Central South University. 在这项研究中，我们介绍了一个闭头、轻度创伤性脑损伤大鼠模型，并对其验证进行了全面概述。我们的目标是提供一种简单的方法来复制这种类型的脑损伤，并提供一种快速评估方法来确认模型的成功。该模型成功地模拟了闭合的头部、轻度创伤性脑损伤，无需头皮切口或颅骨开口，更准确地表示了在人类病例中观察到的撞击场景。避免头皮切口有助于防止可能与活?...

视频: 作者聚焦：推进创伤性脑损伤研究 - 用于准确复制和快速评估的闭头模型 

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

科研

神经科学

Experimental Set Up to Create a Rat Model of Mild Traumatic Brain Injury (mTBI)

Experimental Set Up to Create a Rat Model of Mild Traumatic Brain Injury (MTBI)  

To begin, place a 12-centimeter thick sponge with a 35D hardness in an acrylic box, lacking a top cover. Then, cut a sheet of tinfoil and adhere it to the acrylic box with adhesive tape to create a fragile surface capable of supporting the weight of a rat. Also, mark a 10-centimeter long line as the designated location for positioning the rat's head.Using an iron stand, firmly secure a polyvinyl chloride or PVC tube in place. Prepare a perforated weight weighing 550 grams with an 18-millimeter diameter. Attach the weight to a fishing line at a height of one meter inside the PVC tube and adjust the position of the guide tube three centimeters above the tin foil.Next, make a helmet from a stainless steel disc. Prepare a wedge-shaped sponge pillow to place under the rat's head. Swiftly position the anesthetized rat on its chest in the tinfoil.Slide the pillow beneath the rat, ensuring its head remains parallel to the foil surface. Align the helmet with the rat's ears, and securely fasten it in place. Confirm that the PVC pipe is directly above the helmet.Then, release the weight and allow it to impact the rat's head resulting in a fall onto the sponge and a 180 degree rotation. Afterwards, position the rat on its back inside a clean cage.

创建轻度创伤性脑损伤 （mTBI） 大鼠模型的实验装置

Acute Neurobehavioral Assessments of the Closed-Head Mild Traumatic Brain Injury (mTBI) Rat Model

Acute Neurobehavioral Assessments of the Closed-Head Mild Traumatic Brain Injury (MTBI) Rat Model  

After inducing a mild traumatic brain injury in an anesthetized rat, position it on its back inside a clean cage. Record the duration from when the rat is anesthetized to when it regains the righting reflex. Also record the time it takes for the rat to exhibit its first seeking behavior from the moment of anesthesia.Next place the rat in the center of a circular apparatus with an exit. Measure and record the time it takes for the rat to exit the circle. Then place the rat successively on three beams of varying length each for a duration of one minute.Score the rat on a scale of zero to six, depending on their performance on the beam. mTBI rats exhibited a significantly prolonged recovery from unconsciousness. The seeking behavior increased significantly in the mTBI group during the recovery period.Also, the mTBI rats took longer to exit the circle compared to sham rats. The beam balance test showed a significant injury effect in wide beam tasks indicating balance impairment in mTBI rats 10 minutes post impact. The 2 and 1.5 centimeter wide beams effectively differentiated between sham and mTBI groups in three independent experiments.

闭合式头部轻度创伤性脑损伤 （mTBI） 大鼠模型的急性神经行为评估

Neurobehavior Assessment of the Closed-Head Mild Traumatic Brain Injury (mTBI) Rat Model

Neurobehavior Assessment of the Closed-Head Mild Traumatic Brain Injury (MTBI) Rat Model  

After inducing a mild traumatic brain injury in an anesthetized rat, position it on its back inside a clean cage. Place foam padding beneath the balance beam to reduce the risk of injury to the rats in case of falls during the test. Place an escape box at one end of the beam.Activate the video camera to record the test. Once the animal recovers, position the rats at the other end of the balance beam. Position the rats on the two centimeter wide beam for a series of five consecutive trials.Record the initiation and conclusion of each trial when the rat's nose crosses the starting and finishing lines respectively. Prepare the open field arena, ensuring it is clean and devoid of any prior odor cues. Divide the arena into three distinct zones:a central inner zone, a middle zone, and an outer zone.Place a rat in the center of the open field arena and initiate the timer. Allow the rat to freely explore the arena for a duration of five minutes. After this period, carefully and gently return the rat to its home cage.Quantify the total distance covered by the rat during the five-minute exploration period. Additionally, determine the amount of time the rat spends in the three zones. Ensure that the water maze apparatus is in proper working condition.Place the platform at a depth of 2.5 centimeters below the water surface. Dye the water black and position cues in the four cardinal directions. Establish a monitoring system to record and observe the behavior of the rats.Then introduce the rat into the water maze. If the rat is unable to reach the platform within a two-minute timeframe, gently guide it using a wooden stick. Allow the rat to become accustomed to the maze environment while standing on the platform for a duration of 20 seconds, then remove it.On the sixth day, which is the probe test day, remove the platform from the water maze and position the rat in the same quadrant for a duration of two minutes. Employ the monitoring system to continuously observe and record the rat's behavior. Motor coordination was assessed pre and post-anesthesia or injury using the beam task.At day one post-injury, mTBI rats showed significantly more hindlimb slips compared to shams. However, after two days, hindlimb slips returned to sham levels. All six mTBI rats had more post-impact hindlimb slips with no differences in beam traversal times between mTBI and sham rats.Distance traveled showed no significant differences between sham and mTBI groups. Anxiety-like behavior was evident in mTBI rats at three and seven days post-injury, spending less time in the center zone during the open field test. Morris water maze results indicated impaired spatial learning and memory in mTBI rats.They took longer to find the hidden platform and spent less time searching for the removed platform during the probe trial without differences in swimming speed suggesting no impact on spontaneous locomotor function.