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
Summary
我们在这里提出了啮齿动物冲击波模型的方案，以研究轻度至中度创伤性脑损伤的神经生物学和病理生理学影响。我们建立了一个气体驱动的台式设置，配备了压力传感器，允许可靠和可重复地产生爆炸引起的轻度至中度创伤性脑损伤。
Abstract
创伤性脑损伤（TBI）是一个大规模的公共卫生问题。轻度TBI是最常见的神经创伤形式，在美国占了大量的医疗就诊。目前没有FDA批准的治疗TBI的治疗方法。与军事相关的爆炸诱发的TBI的发病率增加，进一步突出了对有效TBI治疗的迫切需要。因此，新的临床前TBI动物模型概括了人类爆炸相关TBI的各个方面，将大大推进对轻度至中度TBI的神经生物学和病理生理过程的研究工作，以及开发TBI的新型治疗策略。
在这里，我们提出了一个可靠的，可重复的模型，用于研究轻度至中度原始细胞诱导的TBI的分子，细胞和行为影响。我们描述了一种分步方案，用于啮齿动物中闭合头部，爆炸引起的轻度TBI，使用由配备压电压力传感器的气体驱动减震管组成的台式设置，以确保一致的测试条件。我们建立的设置的好处是其相对低成本，易于安装，易于使用和高吞吐量容量。这种非侵入性TBI模型的其他优点包括爆炸峰超压的可扩展性和可控可重复结果的产生。该TBI模型的可重复性和相关性已在许多下游应用中进行了评估，包括神经生物学，神经病理学，神经生理学和行为分析，支持使用该模型来表征轻度至中度TBI病因背后的过程。
Introduction
仅在美国，创伤性脑损伤（TBI）每年就诊超过200万次。通常由车祸、体育赛事或跌倒引起的轻度 TBI 约占所有 TBI 病例的 80%1。轻度 TBI 被认为是“无声的疾病”，因为患者在初始损伤后的几天和几个月内通常不会出现明显的症状，但在以后的生活中可能会出现严重的 TBI 相关并发症2。此外，爆炸诱发的轻度TBI在服兵中普遍存在，并且与慢性中枢神经系统功能障碍有关3，4，5，6。由于与原始细胞相关的轻度 TBI7，8 发病率上升，与轻度 TBI 相关的神经生物学和病理生理过程的临床前建模因此成为开发 TBI 新型治疗干预措施的重点。
从历史上看，TBI研究主要集中在严重形式的神经创伤上，尽管严重的人类TBI病例数量相对较少。已经开发了用于重度人类TBI的临床前啮齿动物模型，包括受控皮质冲击（CCI）9，10 和液体叩诊损伤（FPI）11 模型，这两种模型都已得到充分验证，可产生可靠的病理生理学效应12，13。这些模型为今天已知的TBI中的神经炎症，神经变性和神经元修复奠定了基础。虽然已经对TBI的病理生理学有了相当多的了解，但目前还没有有效的，FDA批准的治疗TBI的治疗方法。
最近，TBI研究的重点已经扩大到包括更广泛的TBI相关病理学，最终目标是开发有效的治疗干预措施。然而，很少有针对轻度 TBI 的临床前模型被建立出来，显示出可测量的效果，只有少数研究调查了轻度 TBI 谱2，14，15。由于轻度TBI占所有TBI病例的绝大多数，因此迫切需要可靠的轻度TBI模型来促进对人类病因和神经病理生理学的研究，以开发新的治疗策略。
我们与生物医学工程师和航空航天物理学家一起，为轻度至中度TBI建立了可扩展的闭头冲击波模型。这种临床前啮齿动物模型专门用于研究力动力学的影响，包括爆炸波和加速/减速运动，这些影响与在军事战斗，体育赛事，车祸和跌倒中获得的人类轻度TBI有关。由于爆炸波与导致人类轻度TBI的力动力学相关，因此该模型旨在产生具有脉冲的一致弗里德兰德波形，其测量单位为磅/平方英寸（psi）*毫秒（ms）。将脉冲水平缩放至低于小鼠和大鼠定义的肺致死率曲线，以便进行临床前检查16，17，18。此外，该模型允许调查由于动物头部的快速旋转力引起的政变和撞击损伤。这种损伤是几种类型的临床TBI表现所固有的，包括在军事和平民人群中观察到的那些。因此，这种多功能模型符合包含TBI的多种临床表现的需求。
这里介绍的临床前模型产生了与临床轻度TBI相关的可靠且可重复的病理生理学变化，如先前的许多研究所证明的那样17，19，20，21，22，23。使用该模型的研究表明，遭受低强度冲击波的大鼠表现出神经炎症，轴突损伤，微血管损伤，与神经元损伤相关的生化变化以及短期可塑性和突触兴奋性缺陷19。然而，这种轻度TBI模型没有诱发任何宏观神经病理学变化，包括组织损伤，出血，血肿和挫伤19 ，这在使用中度至重度侵袭性TBI模型的研究中很常见10，24。先前的研究19，21，22，23 表明，该临床前模型可用于表征轻度和中度TBI病因的神经生物学和病理生理过程17，19，20，21，22，23。 该模型还允许测试新的治疗化合物和策略，以及确定用于开发有效TBI干预措施的新型，合适的靶标19，21，22，23。
该模型的开发是为了研究爆炸波以及快速旋转力对啮齿动物分子，细胞和行为结果的影响。与此处介绍的冲击波模型类似，已经开发了许多临床前模型，这些模型试图使用气体驱动的超压波来概括轻度至中度TBI2，14，17，25，26，27，28。其他模型的一些局限性包括：动物固定在金属丝网上，头部在撞击时固定;除了大脑之外，外围器官还暴露于波中，这产生了多发性创伤的混杂变量;并且模型很大且静止，这限制了关键参数的变化和调整，以更好地模拟人类TBI的条件。
这种台式气体驱动减震管设置的优点是其相对较低的购置和运行费用成本，以及易于安装和使用。此外，该设置允许在小鼠和大鼠中进行高通量操作并产生受控的可重复冲击波和 体内 结果。为了控制一致的测试条件（即恒定的冲击波和超压），该设置配备了压力传感器。该模型对TBI的优点包括损伤严重程度的可扩展性以及使用非侵入性闭合头手术诱导轻度TBI。峰值超压和随后的脑损伤随着聚酯膜的增厚以一致的可扩展方式增加17。通过膜厚度调整TBI严重程度的能力是确定特定结局指标（例如，神经炎症）变得明显的水平的有用工具。为外周器官提供保护性屏蔽，还可以通过避免或减少全身损伤（如肺或胸损伤）的混杂变量，重点检查轻度TBI机制。此外，这种设置允许选择方向，通过该方向，爆炸波撞击/穿透头部（即正面，侧面，顶部或下方），因此可以调查不同类型的TBI诱导侮辱。这里描述的诱导轻度至中度TBI的标准程序采用侧面暴露来评估冲击波损伤与快速旋转力引起的政变和痉挛损伤的影响。此外，为了专门研究爆炸引起的伤害，可以在该模型中采用自上而下的冲击波暴露。
Protocol
Representative Results
Discussion
我们在这里提出了一种临床前轻度TBI模型，该模型具有成本效益，易于设置和执行，并允许高通量，可靠和可重复的实验结果。该模型为外周器官提供保护性屏蔽，以便对轻度TBI机制进行重点研究，同时限制全身损伤的混杂变量。相比之下，已知其他爆炸模型会对外围器官造成损害2，39，40。与其他抛丸型号的固定位置相比，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢R. Gettens，N. St. Johns，P. Bennet和J. Robson对TBI模型的发展所做的贡献。来自大脑与行为研究基金会（F.P.和M.J.R.）的NARSAD青年研究员资助，来自Darrell K. Royal阿尔茨海默病研究基金会（F.P.）的研究资助和PhRMA基金会奖（M.J.R.）支持这项研究。这项工作得到了美国药物教育基金会（A.F.L和B.P.L.）的博士前奖学金的支持。
Materials
| 3/8 SAE High Pressure Hydraulic Hose | Eaton Aeroquip | R2-6-6-36M | Available from Grainger |
| 3/8'' Quick Connect Female Plugs | Karcher | KAR 86410440 | |
| 3/8'' Quick Connect Male Plugs | Karcher | KAR 86410440 | |
| ANY-maze video tracking software | Stoelting Co. | ANY-maze software | |
| Clear Mylar membrane | ePlastics.com | POLYCLR0.003 | http://www.eplastics.com/Plastic/Clear_Polyester_Film/POLYCLR0-003; Clear Mylar membrane is sold in various thicknesses. All are sold by vendor listed above. |
| Compound Slide Table (X2) | Grizzly Industrial | G5757 | |
| Deadman Gas Control Ball Valve | Coneraco Inc. | 71-502-01 | "Apollo", Available from Grainger |
| Driver and driven section (murine) | own design/production | n/a | For further information please contact the authors |
| Driver and driven section (rat) | own design/production | n/a | For further information please contact the authors |
| Ear Muffs | 3M | 37274 | Available from Grainger |
| Gas Regulator – Hi Flow 3500-600-580 | Harris | 3003539 | |
| Helium Gas | AirGas | HE 300 | Tanks are available in various sizes |
| Inhalation Anesthesia System | VetEquip | 901806 | |
| Input Module | National Instruments | NI 9223 | |
| Isoflurane | Baxter | NDC 10019-360-40 | Ordered by veterinarian |
| Laboratory Timer/Stopwatch | Fisher Scientific | 50-550-352 | |
| Labview version 12.0 | National Instruments | Data Acquistion Software | |
| Magnetic Dial Indicator/Micrometer | Grizzly Industrial | G9849 | |
| MATLAB | MathWorks | Software for pressure recording analysis | |
| Oxygen Regulator | Medline | HCS8725M | |
| PC for Data Processing | Dell | ||
| Polyvinylchloride Tubing – 25.4 mm | FORMUFIT | P001FGP-WH-40×3 | |
| Pressure sensors | PCB Piezotronics | 102A05 | |
| Receiver USB Chassis | National Instruments | DAQ-9171 | |
| Sensor Signal Conditioner | PCB Piezotronics | 482C series | |
| Stainless NSF-Rated Mounting Table | Gridmann | GR06-WT2448 | |
| T Handle Allen Wrench – 3/16'' | S&K | 73310 |
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