轻度脑外伤的幼年大鼠一种新模式

摘要

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

摘要

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

引言

虽然有许多广泛使用的方法的中度至重度创伤性脑损伤（TBI），极少数技术已被开发以诱导温和的生成，关闭头部受伤在啮齿类动物。由于这样的事实，轻度创伤性脑损伤（MTBI）比中度和重度脑损伤合并¹三次多见，MTBI的可靠模型，需要以促进关于研究生理学，神经生物学和行为结果，和治疗策略。例如，部分由于当前的动物模型²的局限性，在过去十年中已经有超过200个失败的临床药物试验用于TBI ³的治疗。当转化研究研究，产生模拟系统，对调查结果的适用性取决于实施该模型的有效性。对于MTBI /脑震荡的研究中，一个可靠的动物模型不仅模仿生物力学力量​​responsible对于损伤的病因，而且还会引起与报告的临床相关人口一致的症状。此外，由于儿童处于特别高风险MTBI，优化建模系统将适用于广大青少年啮齿动物，除了成年当量。

，其中运动员有持续mTBIs震荡或脑损伤的情况下，生物力学分析表明，最关键的预测因子的伤害是快速的头加速和高速冲击^4。大部分目前用于TBI诱导啮齿类动物模型的允许头^5（综述参见^2）很少或没有移动。这里列出的模式，提供了高速冲击物理潇洒的少年大鼠是伴随着一个180°旋转和自由落体适用加速/减速力被摄对象的头部和身体的头。牛逼这里有与MTBI的诱导本变形重量滴技术相关联的两个主要优点。首先，模型产生临床相关的震荡一样症候，而不会造成任何明显的损害到大脑（完整描述行为结果见^6）。与后震荡综合征的临床报告也一致，此修改重锤落下技术生产异构的结果。虽然从MTBI的效果是显著，存在经历了MTBI上的多个结果的措施检查当啮齿动物之间大幅变化。其次，该方法允许重复MTBI ⁷的研究。由于大多数现有的TBI模型造成如此严重的伤害，这是常常难以诱导的第二损伤，并且几乎不可能研究重复TBI而不对整个皮质广泛损害。

因此，对于使用改性重量滴法对工业家里的主要理由MTBI的ction是产生伤害更紧密地代表了青少年群体的病理生理和脑震荡的症状学和重复TBI。随着MTBI的发病率越来越高，涉及到体育，坠落，交通事故，特别是在儿童时期，MTBI这种独特的啮齿动物模型为研究人员提供了震荡式的脑损伤，可以很容易地应用到多个命中研究的宝贵工具范式。

研究方案

注:所有实验，进行了符合动物护理的加拿大议会和批准了卡尔加里大学，动物保健伦理委员会。

1.育种和动物准备

1. 按照标准养殖协议，订购孕鼠从内部标准实验动物供应商或品种的幼崽。
2. 房子所有动物随意获得食物和水，在温度受控的大鼠容纳室（21℃），即保持在12:12小时的光:暗周期。
3. 当幼崽出生后达到21天（P21），断奶同性群体的3或4，从他们的母亲和幼仔的房子。

2.建立轻度脑外伤（MTBI）的设备

1. 此前毕业典礼程序，重磨到所需的质量（ 如 150克）。牢固地附着在金属环的重量的顶端使渔线被连接固定的，以重量。
2. 得分锡箔用锋利的剃刀刀片。确保打进锡箔支撑体重的大鼠的但与鼠的加速度在与重量头部冲击不会干涉。
3. 磁带上的计分锡箔到U形平台由透明塑料（38×27×27 ^毫升 ），其中包含一个集合海绵（38×25×15cm的^3）（ 图1），以便它是绷紧。
4. 将U形的塑料阶段在导向管由透明塑料下的正确位置。
5. 持在塑料导向管（2.2厘米直径的乘以1.5米）代替用夹子支架和导向管的位置，以便它为3.5厘米以上的计分锡箔。
6. 通过金属环连接的钓线确保了重量的底部自由悬挂2.5厘米上面拿下锡箔的重量。
7. 牢固地安装鱼线的固定支架。
   注意:系链的重量的蛤P支架以2.5厘米以上的锡箔高度防止重新命中，而大鼠是从冲击到集合海绵）落下。
8. 通过塑料导管与鱼线拉起来的重量拿在地方与六角扳手销1.0米

3.感应MTBI的

1. 当老鼠到达P30招进笼子里的程序空间。
2. 放置大鼠在异氟醚的腔室并轻轻麻醉大鼠，直到它是非响应于一个爪子或尾巴捏。
3. 赶紧把老鼠的得分锡箔在落锤的路径胸部朝下直接发挥得淋漓尽致。参见图2，如果老鼠开始移动或唤醒之前可放置在打进锡箔，返回到异氟烷室，直到非响应于一个爪子或尾巴捏和再次开始。
4. 拉六角扳手引脚，允许重量垂直落下通过塑料导管和罢工老鼠的头部。日Ë老鼠会很快接受一个180°旋转和土地在仰卧位。
5. 立即删除从集合海绵大鼠和应用利多卡因于老鼠的头部用棉签。
6. 将鼠在由经市售加热垫设置它加热在干净笼子仰卧位。
7. 用秒表和数字计时器获得"时间到右"。时间到右是采取的大鼠从麻醉醒来并从仰卧位翻转到俯卧姿势或开始行走的时间。
8. 返回老鼠的家笼后，已恢复正常的行为（美容，散步，游览等 ）。
9. 每增加大鼠需要MTBI重复步骤3.2-3.8。
10. 重复在同一大鼠的程序，在重复性实验MTBI多个时间点。

4.感应深水损伤

1. 轻轻ANESThetize用异氟烷将大鼠直到它是非响应于一个爪子或尾巴捏。
2. 赶紧把老鼠的得分锡箔在落锤的路径胸部朝下直接发挥得淋漓尽致。
3. 取下锡箔的老鼠没有从塑料导管拉六角扳手引脚和应用外用利多卡因的大鼠头部用棉签。
4. 将鼠在由经市售加热垫设置它加热在干净笼子仰卧位。
5. 使用秒表或数字计时器来获取"时间到右"。
6. 返回老鼠的家笼后，已恢复正常的行为（美容，散步，游览等 ）。

5.验证MTBI与梁步行测试⁸

1. 感应的MTBI或假伤愈复出动物的过程房间后24小时。
2. 转移大鼠到一个干净的保持笼并放置老鼠的家笼在的165厘米锥形光束，使得家笼的开放端部面向所述光束的较窄端的较窄端。
   注:锥形束为165厘米长。光束的中心平台具有6厘米在广角端和1.75厘米的窄端的宽度。该中心梁具有壁架（2厘米，宽2厘米中心束以下），提供安全，当老鼠的脚打滑。放置光束下面泡沫填充以减少鼠可能在测试期间脱落光束伤害的风险。
3. 放置一个摄像机在锥形光束和位置的宽端/变焦摄像机以确保研究者观看录像可以清楚地看到的大鼠的运动为锥形束的整个长度。
4. 将大鼠在锥形束的广角端，并鼓励其跨梁步行到其家笼。一旦老鼠穿过锥形束，让大鼠在其家笼至少60秒川NFORCE目标位置。这是试验＃1和不包括在分析中。
5. 打开视频摄像头，并允许大鼠完成梁行走任务，4个额外的时间（保持在家里笼60秒加固周期）。一旦老鼠已经完成了所有的试验，返回老鼠的家笼。
6. 得分，记录后肢脚打滑发生的时间/持续时间越过横梁，为每个单独的试验的次数。使用此数据来计算的后腿英尺单和时间的平均数量越过横梁每只大鼠。

结果

上述改性重锤落下技术对于轻度创伤性脑损伤（MTBI）在幼年大鼠中诱导的可靠方法。利用150克重量的影响，该技术已成功应用于范围从50-120克幼年大鼠。此外，该程序可以很容易地反复在同一动物重复MTBI的研究。虽然醒来后的动物，经历一个单一MTBI展览增加了时间-右（ 图3）和出现愣，它们迅速地恢复正常的活动，并从假损伤动物视觉上没有区别。鉴于该损伤是温和的，局部的利多卡因这消除了与掠冲击相关联的任何疼痛，需要唯一的止痛剂。这是研究的重要的止痛药是已知的干扰典型的炎症和恢复过程。由于缺乏明显的症状学的，光束行走任务是一个可靠的，以醇可用于验证MTBI的诱导。需要注意的是，并非所有的动物，体验MTBI将展示在梁行走任务赤字是很重要的，但作为一个群体，少年只用MTBI展示显著更多的后腿脚打滑时相比，幼年大鼠假伤（ 图4）。

这个修改​​重锤落下技术的另一个重要特点是在伤害感应应用到青少年老鼠缺乏约束。通过提供一个一眼打击头部后迅速旋转加速，减速，这种模式更加紧密地代表归因于MTBI和脑震荡的生物力学的力量。在此过程中被应用到幼年大鼠或成年小鼠，死亡率极低（二百零二分之七幼年动物〜3.4％的死亡率），以及颅骨骨折和颅内出血是非常罕见^6,7。此外，该模型会产生临床相关SYmptomology。在经历一个MTBI少年老鼠表现出平衡和运动行为缺陷，随着赤字的行政职能，提高抑郁样行为，并改变了社会交往^6,9。同样，成年小鼠还显示，随着时间的⁷轻度恢复平衡和协调赤字。最后，使用感应MTBI这种模式的需要最少的麻醉，不涉及手术准备，​​或钻入头骨。结果，因此不通过由手术或麻醉触发混杂炎性或免疫效应偏置。此外，开放性伤口的快速恢复时间和缺乏使更多的测试范例开始出现老鼠体验MTBI后不久。

图1: > 的U形塑料阶段，收集海绵与所有相关方面。必须保持在收集海绵和塑料的阶段，以确保青少年老鼠顶部之间的10厘米的距离C artoon代表有足够的时间来完成180 °旋转。

图2:伤害感应平台（A）摄影代表性。少年大鼠放置在打进锡箔胸部向下，使得头部直接落锤下方。损伤感应平台（B）的侧视图。（C）的 MTBI的诱导中使用的重量的照相示范。

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图3:那经历了一个MTBI并经历了假伤害幼鼠（* P <0.01），老鼠收到了MTBI展览在一个显著增加时间到右幼年大鼠之间的平均差异的图形表示的持续时间需要从仰卧位纠正自己。

图4:后腿足单由幼年大鼠表现出的光束行走的任务，经历了一个MTBI并经历了假伤害幼鼠（* P <0.05）的平均数量的图形表示。

讨论

Reliable modelling systems are needed to effectively cultivate basic science research that has significant translational validity. In response to rising occurrences and popular media, the investigation of mTBI and concussion has become a priority in many disciplines. However, despite increased research, there have been only incremental improvements in therapeutic strategies and treatment options ³. This lack of progress may be partially due to a discrepancy between the modeling systems employed and actual injury etiology. The majority of studies utilized rodent models that failed to reproduce the important biomechanical forces and appropriate post-injury symptomology. The current human definition of mTBI specifies that the injury results from acceleration and deceleration forces associated with a blunt trauma ¹⁰. The modified weight drop technique described here is therefore an ideal model for the study of mTBI and concussion because it uses a glancing impact to cause rapid rotational acceleration and deceleration to the head of an unrestrained animal, mimicking the biomechanical forces identified in sports-related injuries and automobile accidents. In addition, this model is easily adapted to examine repetitive mTBI, a phenomena that is emerging as a serious medical and socioeconomic issue. Studies indicate that rodents may be exposed to a series of 10 distinct mTBIs with minimal mortality ⁷. Finally, the method is inexpensive and can be carried out rapidly, allowing for high-throughput examination of a many therapeutic compounds and treatment regiments.

Just as with any procedural technique, certain aspects of the protocol are particularly important to the generation of reliable results. First, the tin foil needs to be scored effectively. If the tin foil is not properly scored, the force imparted by the weight during the glancing impact will not be enough to propel the juvenile rat through the tin foil onto the collection sponge. In these situations, the rat will remain in the starting position (chest down on the tin foil) and the mTBI will result from the blunt trauma from the weight impacting the stationary head, not the rotational acceleration and deceleration desired. Second, during the induction of the mTBI and the sham injury, the level of anesthetic applied to each rat should be consistent. Owing to the fact that time-to-right is used as marker of mTBI, the researcher should try to ensure that animals receiving a mTBI and animals receiving a sham injury are exposed to similar levels of anesthetic. A major advantage to this technique over many other TBI procedures is the low level and duration of anesthesiology. However, the juvenile rat needs to be non-responsive to a toe or tail pinch to ensure they do not wake-up on the stage before the injury is induced. Finally, in order to maintain a consistent injury etiology, the positioning of the rat’s head is particularly important. Ideally the weight should impact the center of the dorsal surface of the head. Caution should be taken to avoid positioning the path of the weight too near the caudal/posterior portion of the head, as impacting the brainstem and cerebellum is associated with increased mortality and seizure activity.

Based upon the biomechanical pathophysiology of injury induction and the behavioural outcomes examined, the modified weight-drop technique appears to be a reliable model for the investigation of paediatric mTBI and concussion. Although preliminary studies of this novel model have assessed some basic molecular and structural changes ⁷ future studies will be needed to ascertain how the brain responds to a mTBI with this injury etiology. An in-depth analysis of the neuroanatomical and biological changes that occur at the cellular and epigenetic level would increase model validity and translational applicability. In addition to stimulating the generation of targeted pharmacological therapies, understanding the pathophysiological changes that occur in the brain in response to mTBI and concussion would also direct the research related to clinical biomarkers that have the ability to predict outcomes following injury.

材料

Name	Company	Catalog Number	Comments
Brass Weights	Ginsberg Scientific	7-2500-2	Need to have metal loop attached to base
Alluminum Foil	Alcan		Available at most grocery stores
Masking Tape			Commercially available
U-Shaped Plastic Stand			Constructed by Laboratory
Clamp Stand	Sigma-Aldrich	Z190357
Plastic Guide Tube			Could be constructed or purchased at a hardware store
Fishing Line	Angler 10lb		Purchased from a sporting goods retailer
Isoflurane	Pharmaceutical Partners of Canada	DIN 02237518	Inhalation Anesthetic
Topical Lidocaine (30ml)	Astra Zeneca	DIN 0001694	Xylocaine Jelly 2%
Cotton Swabs			Commercially available
Heating Pad - 3 heat setting			Commercially available
Stop Watch	Sportline	L303	Purchased from a sporting goods retailer
Video Camera	Sony	HDR-CX260V
Sprague Dawley Rats	Charles River Laboratories	SAS SD 40	Male and females ordered from Charles River Laboratories and pups bred in-house
Balance Beam			Constructed by Laboratory