药学学报

Model name	Injury mechanism	Simulated clinical type	Key pathological feature	Advantage	Disadvantage
Fluid percussion injury (FPI)	Fluid pulse pressure transmission	Diffuse injury, elevated intracranial pressure (ICP), post-traumatic epilepsy	Diffuse axonal injury, cerebral edema, neuroinflammation	High reproducibility, controllable severity, suitable for diffuse injury	Requires craniotomy, non-physiological mechanism, surgical variability
Controlled cortical impact (CCI)	Mechanical impactor	Focal contusion/laceration	Cortical necrosis, hematoma, blood-brain barrier (BBB) disruption	Precise parameter control, excellent reproducibility, ideal for focal studies	Requires craniotomy, injury is localized, high surgical skill required
High-velocity projectile	High-speed penetration	Penetrating injury, simulates gunshot wounds	Diffuse injury, BBB disruption, edema	Often uses large animals, clinically relevant for ballistic trauma	High equipment cost, low survival rate
Controlled concussion	Head inertial motion	Mild TBI/concussion, chronic traumatic encephalopathy (CTE)	Microstructural damage, transient functional impairment	Reproducible mild injury	Injury is mild/transient, difficult to detect
Weight-drop injury (WDI)	Gravitational impact	Severe TBI/concussion, simulates open-head injury from falling objects	Diffuse injury, BBB disruption, edema	Simple equipment	Poor parameter control, low reproducibility, risk of cervical spine injury
Blast overpressure	High-pressure shockwave	Blast-related TBI	Diffuse vascular injury, BBB disruption	Simulates unique biomechanics, high military relevance	Complex equipment, high cost, complex mechanisms difficult to parse
Non-impact acceleration	Acceleration-deceleration forces	Diffuse axonal injury (DAI)	Axonal breakage/swelling, APP positive, pronounced injury in white matter tracts (corpus callosum, brainstem, internal capsule)	Studies pure inertial/acceleration mechanics, high controllability	Rarely causes severe contusion or major hemorrhage, not suitable for studying mixed TBI

Model name	Injury mechanism	Simulated clinical type	Key pathological feature	Advantage	Disadvantage
Fluid percussion injury (FPI)	Fluid pulse pressure transmission	Diffuse injury, elevated intracranial pressure (ICP), post-traumatic epilepsy	Diffuse axonal injury, cerebral edema, neuroinflammation	High reproducibility, controllable severity, suitable for diffuse injury	Requires craniotomy, non-physiological mechanism, surgical variability
Controlled cortical impact (CCI)	Mechanical impactor	Focal contusion/laceration	Cortical necrosis, hematoma, blood-brain barrier (BBB) disruption	Precise parameter control, excellent reproducibility, ideal for focal studies	Requires craniotomy, injury is localized, high surgical skill required
High-velocity projectile	High-speed penetration	Penetrating injury, simulates gunshot wounds	Diffuse injury, BBB disruption, edema	Often uses large animals, clinically relevant for ballistic trauma	High equipment cost, low survival rate
Controlled concussion	Head inertial motion	Mild TBI/concussion, chronic traumatic encephalopathy (CTE)	Microstructural damage, transient functional impairment	Reproducible mild injury	Injury is mild/transient, difficult to detect
Weight-drop injury (WDI)	Gravitational impact	Severe TBI/concussion, simulates open-head injury from falling objects	Diffuse injury, BBB disruption, edema	Simple equipment	Poor parameter control, low reproducibility, risk of cervical spine injury
Blast overpressure	High-pressure shockwave	Blast-related TBI	Diffuse vascular injury, BBB disruption	Simulates unique biomechanics, high military relevance	Complex equipment, high cost, complex mechanisms difficult to parse
Non-impact acceleration	Acceleration-deceleration forces	Diffuse axonal injury (DAI)	Axonal breakage/swelling, APP positive, pronounced injury in white matter tracts (corpus callosum, brainstem, internal capsule)	Studies pure inertial/acceleration mechanics, high controllability	Rarely causes severe contusion or major hemorrhage, not suitable for studying mixed TBI

Modeling agent	Typical concentration	Exposure duration	Primary target cell	Simulated TBI pathology phase/mechanism
Hydrogen peroxide (H₂O₂)^[51]	100-300 µmol·L^-1	2-6 h	Neurons, astrocytes, oligodendrocytes	Early secondary injury: Oxidative stress, ROS burst, mitochondrial dysfunction, lipid peroxidation, DNA damage
Glutamate^[52]	10-100 µmol·L^-1 (chronic); 100 µmol·L^-1-1 mmol·L^-1 (acute)	30 min-6 h	Neurons (especially cortical and hippocampal neurons)	Early secondary injury: Excitotoxicity, NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor overactivation, Ca²⁺ influx, mitochondrial damage
NMDA^[52]	10-50 µmol·L^-1	30 min-4 h	Neurons	Specific simulation of NMDA receptor-mediated excitotoxicity
TNF-α^[53]	10-20 μg·L^-1	6–48 h	Neurons, glial cells	Mid secondary injury: Inflammation amplification, apoptotic signaling tumor necrosis factor receptor 1 (TNFR1)-Caspase pathway
IL-1β^[53]	5-10 ng·mL^-1	6-48 h	Neurons, astrocytes	Mid secondary injury: Neuroinflammation, glial activation, synaptic dysfunction
LPS^[54]	10-1 000 μg·L^-1 (commonly 100 μg·L^-1)	12-72 h	Microglia (BV2 cell line, primary microglia)	Immune-inflammatory phase: TLR4-NF-κB activation, simulating the post-TBI inflammatory cascade
Cytokine cocktail (TNF-α + IL-1β + IFN-γ) ^[53]	5-20 μg·L^-1 each	12-48 h	Neuron-glia co-culture	Closer to in vivo secondary injury environment: Synergistic inflammation, apoptosis, and synaptic damage

Modeling agent	Typical concentration	Exposure duration	Primary target cell	Simulated TBI pathology phase/mechanism
Hydrogen peroxide (H₂O₂)^[51]	100-300 µmol·L^-1	2-6 h	Neurons, astrocytes, oligodendrocytes	Early secondary injury: Oxidative stress, ROS burst, mitochondrial dysfunction, lipid peroxidation, DNA damage
Glutamate^[52]	10-100 µmol·L^-1 (chronic); 100 µmol·L^-1-1 mmol·L^-1 (acute)	30 min-6 h	Neurons (especially cortical and hippocampal neurons)	Early secondary injury: Excitotoxicity, NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor overactivation, Ca²⁺ influx, mitochondrial damage
NMDA^[52]	10-50 µmol·L^-1	30 min-4 h	Neurons	Specific simulation of NMDA receptor-mediated excitotoxicity
TNF-α^[53]	10-20 μg·L^-1	6–48 h	Neurons, glial cells	Mid secondary injury: Inflammation amplification, apoptotic signaling tumor necrosis factor receptor 1 (TNFR1)-Caspase pathway
IL-1β^[53]	5-10 ng·mL^-1	6-48 h	Neurons, astrocytes	Mid secondary injury: Neuroinflammation, glial activation, synaptic dysfunction
LPS^[54]	10-1 000 μg·L^-1 (commonly 100 μg·L^-1)	12-72 h	Microglia (BV2 cell line, primary microglia)	Immune-inflammatory phase: TLR4-NF-κB activation, simulating the post-TBI inflammatory cascade
Cytokine cocktail (TNF-α + IL-1β + IFN-γ) ^[53]	5-20 μg·L^-1 each	12-48 h	Neuron-glia co-culture	Closer to in vivo secondary injury environment: Synergistic inflammation, apoptosis, and synaptic damage

Model name	Method	Simulated pathological process	Advantage	Disadvantage
Mechanical Injury	Mechanical stretch	Application of strain force to cells	Primary mechanical injury, diffuse axonal injury	High-throughput, good controllability, commercial instruments available	Difficult to simulate complex in vivo mechanical environment
	Compression Injury	Compression of cell layers/3D cultures	Focal contusion	Applicable to 3D culture, relatively simple setup	Strain rate control is imprecise
	Scratch injury	Creating a "scratch" on a cell monolayer	Wound healing, partially simulates secondary inflammation	Simple, economical, well-established, suitable for cell migration studies	Injury level cannot be precisely controlled
Chemical injury	Application of excitotoxins/ROS/inflammatory factors	Excitotoxicity, oxidative stress, neuroinflammation	Simple operation, high-throughput, clear mechanisms	Lacks primary mechanical injury, concentration difficult to control

Model name	Method	Simulated pathological process	Advantage	Disadvantage
Mechanical Injury	Mechanical stretch	Application of strain force to cells	Primary mechanical injury, diffuse axonal injury	High-throughput, good controllability, commercial instruments available	Difficult to simulate complex in vivo mechanical environment
	Compression Injury	Compression of cell layers/3D cultures	Focal contusion	Applicable to 3D culture, relatively simple setup	Strain rate control is imprecise
	Scratch injury	Creating a "scratch" on a cell monolayer	Wound healing, partially simulates secondary inflammation	Simple, economical, well-established, suitable for cell migration studies	Injury level cannot be precisely controlled
Chemical injury	Application of excitotoxins/ROS/inflammatory factors	Excitotoxicity, oxidative stress, neuroinflammation	Simple operation, high-throughput, clear mechanisms	Lacks primary mechanical injury, concentration difficult to control

Compound	Mechanism	Research stage	Company	Clinical trial information
Ronopterin	Nitric oxide synthase inhibitors	STAGE-3	veriNOS operations GmbH	NCT02794168
Biperiden hydrochloride	Muscarinic acetylcholine receptor antagonists	STAGE-3	Instituto Doutor José Frota	NCT04945213
Bezisterim	Extracellular-signal-regulated kinase 1 inhibitors	STAGE-3	Biovie	NCT05970575
Izaflortaucipir F-18	Positron-emission tomography enhancers	STAGE-3	Ac Immune	NCT05337774
Angiontensin (1-7)	Angiotensin Ⅱ receptor agonists	STAGE-3	Constant Therapeutics LLC	NCT06282965
Adezunap	Cannabinoid CB1 receptor agonists	STAGE-3	CannaXan GmbH	NCT06071949
Buntanetap	Microtubule-associated protein tau inhibitors	STAGE-3	Annovis Bio Inc.	NCT07284784
PRV-002	Neuroprotectants	STAGE-2	Avance Clinical Pty Ltd.	NCT06870240
Amantadine hydrochloride	Glutamate [NMDA] receptor antagonists	STAGE-2	SHINKEI Therapeutics Inc.	NCT02566720
AST-004	Adenosine A1 receptor agonists	STAGE-2	Astrocyte Pharmaceuticals Inc.	Astrocyte Pharmaceuticals Presents Update on AST-004 Clinical Program at 2022 Military Health System Research Symposium
ABX-101	Calmodulin inhibitors	STAGE-2	Abalonex LLC	NCT06096415
Synthetic ghrelin	Ghrelin/growth hormone secretagogue receptor agonists	STAGE-2	Oxeia Biopharmaceuticals Inc.	NCT04558346
Ibogaine	Mu opioid receptor agonists	STAGE-2	University of Sao Paulo	NCT05029401
Mebufotenin	Serotonin 1a (5-HT1a) receptor agonists	STAGE-2	GH Research Ireland Ltd.	NCT05800860
Pittsburgh compound B	Beta amyloid A4 protein inhibitors	STAGE-2	University of Pittsburgh	NCT01723553

Compound	Mechanism	Research stage	Company	Clinical trial information
Ronopterin	Nitric oxide synthase inhibitors	STAGE-3	veriNOS operations GmbH	NCT02794168
Biperiden hydrochloride	Muscarinic acetylcholine receptor antagonists	STAGE-3	Instituto Doutor José Frota	NCT04945213
Bezisterim	Extracellular-signal-regulated kinase 1 inhibitors	STAGE-3	Biovie	NCT05970575
Izaflortaucipir F-18	Positron-emission tomography enhancers	STAGE-3	Ac Immune	NCT05337774
Angiontensin (1-7)	Angiotensin Ⅱ receptor agonists	STAGE-3	Constant Therapeutics LLC	NCT06282965
Adezunap	Cannabinoid CB1 receptor agonists	STAGE-3	CannaXan GmbH	NCT06071949
Buntanetap	Microtubule-associated protein tau inhibitors	STAGE-3	Annovis Bio Inc.	NCT07284784
PRV-002	Neuroprotectants	STAGE-2	Avance Clinical Pty Ltd.	NCT06870240
Amantadine hydrochloride	Glutamate [NMDA] receptor antagonists	STAGE-2	SHINKEI Therapeutics Inc.	NCT02566720
AST-004	Adenosine A1 receptor agonists	STAGE-2	Astrocyte Pharmaceuticals Inc.	Astrocyte Pharmaceuticals Presents Update on AST-004 Clinical Program at 2022 Military Health System Research Symposium
ABX-101	Calmodulin inhibitors	STAGE-2	Abalonex LLC	NCT06096415
Synthetic ghrelin	Ghrelin/growth hormone secretagogue receptor agonists	STAGE-2	Oxeia Biopharmaceuticals Inc.	NCT04558346
Ibogaine	Mu opioid receptor agonists	STAGE-2	University of Sao Paulo	NCT05029401
Mebufotenin	Serotonin 1a (5-HT1a) receptor agonists	STAGE-2	GH Research Ireland Ltd.	NCT05800860
Pittsburgh compound B	Beta amyloid A4 protein inhibitors	STAGE-2	University of Pittsburgh	NCT01723553

抗创伤性颅脑损伤非临床药效学评价体系探索

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胡海洋 ¹ , 赵春阳 ² , 康钰莹 ¹ , 吴镭 ¹ , 唐婧姝 ¹^,^* , 王庆利 ³^,^* , 彭英 ¹^,^*

药学学报 | 综述 2026,61(6): 1995-2007

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药学学报 |综述 2026 , 61 (6) : 1995 -2007

抗创伤性颅脑损伤非临床药效学评价体系探索

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胡海洋¹, 赵春阳², 康钰莹¹, 吴镭¹, 唐婧姝¹^,^*, 王庆利³^,^*, 彭英¹^,^*

作者信息

¹中国医学科学院北京协和医学院, 药物研究所, 北京 100050

²国家药品监督管理局药品审评中心, 北京 100076

³中国食品药品检定研究院, 北京 102629

通讯作者:

唐婧姝，E-mail: tangjingshu@imm.ac.cn

王庆利，E-mail: wangql@nifdc.org.cn

彭英，E-mail: ypeng@imm.ac.cn

作者简介:

作者贡献:

胡海洋完成了文献收集和初稿的撰写; 赵春阳协助完成了本论文的文献收集和论文修改; 康钰莹完成了文献收集; 唐婧姝完成了论文的撰写和修改; 吴镭、王庆利提供了论文写作思路的建议并对论文进行了校对; 彭英为本论文进行了修改和指导。

Exploration of non-clinical pharmacodynamics evaluation system of traumatic brain injury

Hai-yang HU¹, Chun-yang ZHAO², Yu-ying KANG¹, Lei WU¹, Jing-shu TANG¹^,^*, Qing-li WANG³^,^*, Ying PENG¹^,^*

Affiliations

¹Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

²Center for Drug Evaluation, National Medical Products Administration, Beijing 100076, China

³National Institutes for Food and Drug Control, Beijing 102629, China

出版时间: 2026-06-12 doi: 10.16438/j.0513-4870.2025-1492

文章导航

摘要

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创伤性颅脑损伤(traumatic brain injury, TBI)是一种常见的神经系统损伤性疾病, 具有高发病率、高致残率和高死亡率的特点, 在全球范围内造成了沉重的健康负担和社会经济压力。其复杂的病理机制和有限的临床治疗手段使得药物研发面临重大挑战。研究人员已建立多种动物模型和体外模型用于模拟TBI及其后续病理过程, 为药物开发和机制研究提供了重要工具。本文系统综述了TBI的非临床研究评价体系, 包括常用动物模型和体外模型的建立方法、行为学与分子药效学评价指标, 并对当前治疗TBI药物的非临床评价策略提出若干规范化建议, 以期为该领域的研究提供系统性参考。

关键词

创伤性颅脑损伤 / 动物模型 / 体外模型 / 非临床药效学评价 / 新药研发

Abstract

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Traumatic brain injury (TBI) is a common neurological disorder characterized by high incidence, disability, and mortality rates, posing a substantial global health burden and socioeconomic burden. The complexity of its pathology and the scarcity of effective clinical treatments present significant challenges for drug development. Various animal and in vitro models have been established to simulate TBI and its pathological sequelae, serving as essential tools for mechanistic exploration and drug discovery. This article provides a systematic review of the non-clinical evaluation framework for TBI, including the establishment of common models and key behavioral and molecular pharmacodynamic endpoints. Furthermore, it proposes several recommendations for standardizing the evaluation strategies of anti-TBI drugs, aiming to offer a structured reference for future research in this field.

Key words

traumatic brain injury / animal model / in vitro model / non-clinical pharmacodynamics / drug discovery

引用本文

胡海洋, 赵春阳, 康钰莹, 吴镭, 唐婧姝, 王庆利, 彭英. 抗创伤性颅脑损伤非临床药效学评价体系探索. 药学学报, 2026 , 61 (6) : 1995 -2007 . DOI: 10.16438/j.0513-4870.2025-1492

Hai-yang HU, Chun-yang ZHAO, Yu-ying KANG, Lei WU, Jing-shu TANG, Qing-li WANG, Ying PENG. Exploration of non-clinical pharmacodynamics evaluation system of traumatic brain injury[J]. Acta Pharmaceutica Sinica, 2026 , 61 (6) : 1995 -2007 . DOI: 10.16438/j.0513-4870.2025-1492

正文

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创伤性颅脑损伤(traumatic brain injury, TBI)是由外部机械力引起的脑组织损伤, 导致脑功能发生暂时性或永久性障碍, 并可能伴有意识状态的改变。TBI已成球范围内致残与致死的主要病因之一, 死亡率高达20%~30%^[1], 降低TBI的发病率和死亡率仍然是一项重大挑战。近年来, 我国TBI的发病率和患病率总体上呈持续上升的趋势。2000~2019年由152.5/10万升至207.8/10万, 患病例数由298.4万增至654.6万^[2]。TBI年发病率在不同地区之间存在显著差异, 全球每年约导致50万人死亡及超过1 000万人致残。尤其在中低收入国家, 由于医疗资源有限与防护意识不足, TBI带来的社会与经济负担尤为沉重^[3]。

TBI的病理过程包括原发性损伤和继发性损伤两个阶段。原发性损伤指外力直接机械性导致的脑组织结构和功能损害, 而继发性损伤由原发性损伤出发, 涉及一系列复杂的分子与细胞事件, 如神经炎症、氧化应激、兴奋性毒性、血脑屏障破坏等, 这些过程可导致坏死、凋亡等多种形式的细胞死亡。继发性损伤在损伤后数小时至数天内持续发展, 是影响神经功能恢复与临床预后的关键决定因素^[4]。目前临床上针对中重度TBI的诊疗方案仍以对症支持为主(如气道保护、控制颅内压、补液维持血压、外科手术清除血肿等), 缺乏能够有效阻断继发性损伤并促进神经修复的特定药物。因此, 深入研究TBI的病理机制、开发有效的治疗策略, 已成为神经科学领域的重要课题。

非临床研究在TBI药物开发中起着桥梁作用, 其核心在于借助一套科学合理的药效学、药代动力学和安全性评价体系, 为候选药物进入临床试验提供坚实的科学依据。其中, 药效学研究通过建立能够模拟人类TBI病理特征的动物模型和体外模型, 用来预测候选药物的治疗潜力。理想的药效学模型应具备良好的重现性、可操作性和临床转化价值, 能够反映TBI后的行为障碍、组织损伤及分子机制变化。目前常用的动物模型包括液压冲击模型、控制性皮质撞击模型、重物坠落模型等, 它们分别适用于模拟不同类型的脑损伤机制。与此同时, 体外模型如机械拉伸、压缩损伤和化学损伤模型, 为在细胞水平研究TBI机制提供了重要补充。

尽管现有模型已在揭示TBI病理机制和药物筛选方面取得一系列进展, 但其预测临床疗效的能力仍存在明显局限。多数在临床前研究中显示有效的化合物在后续临床试验中未能展现出预期疗效, 反映出当前非临床评价体系在模型选择、指标选取及实验设计等方面尚存不足。因此, 构建标准化、系统化的非临床药效学评价体系, 整合多维度、多指标的评价方法, 对提升药物研发的成功率具有至关重要的意义。

本文旨在系统综述TBI的非临床药效学研究模型及其评价策略, 重点讨论常用动物模型与体外模型的建立方法、行为学与分子水平的关键评价指标, 并对当前研究中存在的问题与挑战进行分析。最后, 本文旨在推动TBI非临床药效学研究的规范化和标准化, 以期为该领域的深入研究与药物开发提供理论依据和方法学参考。

1　创伤性颅脑损伤动物模型

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建立科学可靠的TBI动物模型是开展相关研究的基础, 其可用于深入研究疾病的病理生理机制和评估潜在治疗策略。基于生物力学机制的不同, TBI动物模型主要可分为3类: 撞击性脑损伤模型(impact brain injury)、非撞击性加速脑损伤模型(non-impact acceleration brain injury)和爆炸冲击波诱导的脑损伤模型(blast wave-induced neurotrauma)^[5], 其分类如图1所示。建立并选择合理的动物模型, 有助于深入揭示TBI的发病机制、病理生理过程及验证潜在治疗策略, 为临床干预提供科学合理的试验依据。

1.1　撞击颅脑损伤模型

1.1.1　液压冲击损伤(fluid percussion injury, FPI)模型

FPI模型是研究TBI最具代表性且应用广泛的动物模型之一。该模型通过液压脉冲传递能量致伤, 可较好地模拟临床TBI中的弥漫性轴索损伤和颅内压变化。该模型最初应用于猫和兔, 后经改良已广泛用于大鼠和小鼠, 适用于研究神经元与胶质细胞的微观结构变化及相关行为学表现^{[6, 7]}。建模时, 通常在动物颅骨制备骨窗并植入液体接口, 通过击锤撞击液腔活塞产生可控的液压脉冲, 该脉冲经硬脑膜传导至颅内, 引发脑组织瞬间变形与位移。通过调节击锤高度可精确控制冲击压强, 从而控制损伤严重程度, 如中高强度冲击(约200 kPa)可引起局灶性及弥漫性损伤, 而轻度冲击(60~80 kPa)则以弥漫性损伤为主。该模型的优势在于: 在方法学上, 其损伤程度可控性强、重现性高且实验动物死亡率较低; 在生物学上, 它能较好地模拟人类TBI的关键病理特征。

当采用大鼠作为实验动物时, 根据骨窗位置不同, FPI模型可进一步分为中线FPI (位于矢状缝中心)^[8]、外侧FPI (位于中线旁3.5 mm处)^[9]及旁矢状FPI (位于中线旁小于3.5 mm处)^{[10, 11]}。不同位置的FPI可诱发不同类型的神经功能缺损, 如靠近中线的损伤更易导致认知功能障碍, 而外侧损伤则更多引起运动缺陷。值得注意的是, 该模型对手术操作及设备精度要求较高, 颅骨厚度和个体差异也可能影响实验结果的可重复性。

1.1.2　控制性皮层撞击(controlled cortical impact, CCI)模型

CCI模型通过气动或电磁驱动装置对暴露的脑组织进行精确深度和速度的撞击, 适用于模拟局灶性脑损伤, 如脑挫裂伤、硬膜下血肿等。该模型具有参数可控性强、重复性高、损伤梯度可调等特点, 被广泛用于研究TBI后的细胞凋亡、炎症反应及分子机制^{[12, 13]}。

CCI模型最初建立于雪貂, 现已成功应用于大鼠、小鼠、猪及非人灵长类动物^[14-16]。建模时通常需外科手术暴露硬脑膜, 撞击参数(如撞击速度、深度和停留时间)可通过计算机系统精确控制。如建立大鼠重度TBI模型常采用的撞击参数为: 撞击速度3.5 m·s^-1, 深度2~3 mm, 停留时间250 ms。该参数组合可导致明显的皮层挫伤、神经元变性及长期行为障碍^{[17, 18]}。近年来, 该模型广泛应用于轻度及重复性TBI研究, 显示出良好的适用性和灵活性^[19]。

1.1.3　高速投射物损伤模型

高速投射物损伤模型(high-velocity missile injury model)通过步枪、弩枪或弹道发射装置将小质量投射物高速射入动物颅腔, 从而模拟由撕裂效应、瞬时空腔形成和冲击波传递能量共同作用导致的脑损伤^[20]。为实现稳定且可重复的建模, 常通过调整投射物类型、速度及命中部位以控制损伤严重程度。有研究成功在颞骨区建立了从左至右的贯通性TBI模型, 其病理特征与临床枪伤高度相似^[21]。然而, 该模型的应用仍面临一定限制。首先, 常用实验动物(如犬、猪、猕猴等)的大脑结构与人类存在较大差异; 其次, 在技术层面, 高速投射物带来的高动能及由此产生的瞬时空腔效应导致动物存活率较低, 且常伴有显著的继发性病理改变, 损伤程度难以精确控制, 易导致不可逆损伤。

1.1.4　控制性脑震荡模型

控制性脑震荡模型(controlled cerebral concussion model)是研究闭合性脑损伤的常用方法, 与FPI不同, 该模型中动物的头部和身体不固定^[22]。通过落体撞击或气压/液压系统对头部给予瞬间冲击, 通过调控冲击参数(特定压力和时间)可建立不同严重程度的脑震荡模型。研究表明, 脑震荡所诱发的神经炎症、细胞死亡和神经可塑性改变是长期神经功能障碍的关键机制。一项研究显示, 多次脑震荡后可诱发神经炎症、提高脂质过氧化水平、破坏神经可塑性, 并损害记忆与认知功能, 具体表现为Morris水迷宫中逃避潜伏期的提高^[23]。组织病理学方面, 模型动物可出现神经元固缩、轴突丢失、髓鞘损伤及水肿等^[24]。目前, 该模型广泛应用于认知、情绪及运动行为研究, 许多临床前试验正基于该模型评估抗炎药、抗氧化剂及神经保护剂在促进神经功能恢复方面的潜力。

1.1.5　重物坠落模型(weight drop injury, WDI)

WDI模型是应用最广泛的TBI模型之一^[5]。该模型已在啮齿类动物中应用数十年, 促进了人们对TBI病理生理机制的深入理解。该模型通过垂直坠落的重物冲击引起局灶性和弥漫性脑损伤。在Feeney最早建立的WDI模型中, 需开颅并暴露硬脑膜, 随后释放重物以诱发皮质挫伤^{[25, 26]}、出血灶^[27]及血脑屏障破坏^{[28, 29]}等一系列病理改变。Shapira等^[30]在此基础上进行改良, 将动物头部固定于硬质平台以避免加速性弥漫损伤。磁共振成像(MRI)研究证实^[31], 小鼠WDI模型可导致神经行为异常、小胶质细胞和星形胶质细胞激活、神经变性和脑形态结构改变, 较好地模拟了临床闭合性脑损伤^[32]。本课题组采用的WDI建模标准如下: 大鼠经异氟烷麻醉后, 于右侧冠状缝后1.5 mm、中线旁2.5 mm处切开皮肤并钻取直径5 mm骨窗, 使用40 g重锤从25 cm高度自由坠落, 经撞杆以6 mm深度冲击脑组织, 术后缝合头皮^[33]。

目前采用改良Feeney法已评估多种天然产物及化合物对TBI的治疗作用。如姜黄素通过减轻TBI神经炎症和神经元凋亡^[34]、原花青素通过调节氧化应激^[35]、米诺环素(MINO)在超抗菌浓度能抑制TBI后的炎症和凋亡等^[36]。

1.2　非撞击性加速颅脑损伤模型

非撞击性加速脑损伤模型是一类通过快速的线性或旋转加速度, 在没有头部直接撞击的情况下诱发脑损伤的实验模型。其核心是模拟现实中高速度运动、跌落、摇晃、车辆加速/减速等导致的脑组织相对颅骨发生惯性位移, 从而触发的弥漫性损伤^[37]。临床TBI中, 超过70%的弥漫性轴突损伤(diffuse axonal injury, DAI)与非撞击性加速机制相关。由于造模过程不伴随直接撞击, 故可研究纯粹的加速度机械机制, 且其不需开颅, 损伤在轻度至中度之间, 具有良好的可控性。该模型选用成年雄性Sprague-Dawley大鼠(体重350~400 g), 于颅骨中线位置刚性固定一直径10 mm、厚2 mm的不锈钢盘, 动物俯卧于密度为14 kg·m^-3的泡沫垫上。通过重力驱动一圆柱形黄铜重物(通常450 g)沿有机玻璃管从设定高度(1~2 m)自由坠落, 撞击钢盘并引发头部加速-减速运动, 从而在无颅骨骨折的情况下诱导弥漫性轴索损伤、神经功能障碍及认知损害。损伤后需立即实施机械通气支持, 模型具备参数可调(重量、高度)及可叠加继发性损伤(如缺氧)的特点, 适用于脑损伤病理机制与干预策略的标准化研究。目前, 非撞击性加速模型是研究纯加速度诱导的DAI、神经炎症、突触功能缺损、神经退行性改变的关键工具^[38]。

1.3　爆炸冲击波致颅脑损伤模型

爆炸冲击波致脑损伤模型(blast wave-induced neurotrauma model)通过将动物固定后, 使用气动激波管或爆炸物驱动装置产生高压冲击波, 通过气压脉冲诱发脑损伤。该模型可再现爆炸相关TBI的多种病理特征, 包括弥漫性皮质充血、脑水肿和局灶性挫伤等, 目前已广泛应用于爆炸伤机制研究。爆炸波通过机械性破坏(如组织撕裂与空化效应)直接损伤神经血管单元, 导致急慢性脑血管功能障碍。动物模型显示, 这种损伤会引发慢性血管退行性过程, 并与年龄依赖性创伤后应激障碍(PTSD)样表型相关^[39]。此外, 爆炸波可能通过促进β淀粉样蛋白(Aβ)的积累, 增加阿尔茨海默病的发病风险^[40]。

1.4　颅脑损伤动物模型选择应注意的问题

表1对本文所述TBI动物模型进行总结。TBI动物模型的持续优化与发展, 不仅增进了对TBI病理机制的理解, 也加速了相关治疗策略的开发进程。然而, 鉴于临床TBI的病因多元、病理复杂, 单一模型难以全面模拟其复杂病理特征, 因此研究结果向临床转化存在一定局限性。此外, 动物模型在手术干预过程中引入的非生理性影响, 以及种属间解剖与生理差异, 也是在非临床研究中需慎重考虑的因素。

2　创伤性颅脑损伤体外模型

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体外模型在TBI研究中发挥着重要作用, 为在细胞和分子水平深入探索损伤机制、进行高通量药物筛选提供了高效、可控、标准化的平台。根据诱导损伤方式的不同, TBI体外模型可分为机械损伤法和化学损伤法两大类。机械损伤法旨在模拟外力导致的脑组织物理变形, 其核心在于再现原发性损伤机制; 而化学损伤法则通过施加神经毒性物质来模拟继发性损伤过程, 如兴奋性毒性、氧化应激和神经炎症。将两种模型结合使用, 可更为全面地再现TBI的复杂病理进程。

2.1　机械损伤法

2.1.1　拉伸损伤(stretch)

在TBI的体外机制研究中, 基于机械拉伸的损伤模型因其能够模拟创伤瞬间脑组织经历的快速形变(包括拉伸与剪切), 已成为应用最广泛的模型之一。该模型适用于研究TBI后的一系列核心病理过程, 如轴索损伤、血脑屏障功能障碍、神经炎症激活及线粒体损伤等。根据加载的应变模式, 体外拉伸模型主要分为单轴拉伸^[41]和等轴(多向均匀)拉伸^[42]。单轴拉伸模型多利用线性驱动器在数十毫秒内沿单一方向完成设定幅度(如5%~15%)的拉伸。该模型操作简便、通量高, 适用于药物初筛, 但其单向应变模式与体内真实多向力学环境存在差异。等轴拉伸模型通常基于气压脉冲装置, 通过向培养细胞的柔性硅胶膜施加可控压力与时长(如50 ms), 诱导膜发生球形对称鼓包, 从而实现多向均匀拉伸。该模型可产生分级可控的应变, 损伤程度与形变幅度及应变速率正相关, 能较好地模拟体内复杂的多向力学环境, 因此在揭示损伤的生物力学阈值及机制研究中应用广泛。目前, 商业化设备提升了模型的可重复性与通量, 但仍需注意应变分布不均可能掩盖局部细胞反应, 建议结合高内涵成像技术以提高检测敏感性。

2.1.2　压缩损伤(compression)

压缩损伤模型是TBI体外研究中一种重要的机械性损伤模型, 通过可控力学加载模拟脑组织受外力后的瞬时压迫、结构变形及细胞分子响应。模型通常对二维或三维培养的神经元、胶质细胞或类脑组织实施垂直压力, 以模拟撞击、挤压或颅内压骤升引起的组织损伤。根据加载机制, 模型主要分为位置控制驱动、弹簧辅助冲击和重力辅助冲击3类。其中, 位置控制驱动因可控性高、重复性好而被广泛应用。如线性音圈驱动器可精确设定压缩应变率(10 s^-1或75 s^-1)与应变幅度(如30%), 适用于水凝胶内神经元或三维类器官的加载^[43]。高自动化机械冲击仪则通过弹簧加速活塞撞击金属板, 经液体介质传递压力脉冲至胶质网络, 实现应变为330~905 μm·m^-1、应变率为3~15 s^-1的可重复冲击, 覆盖轻度TBI相关力学范围。细胞体系多采用原代混合胶质细胞(约80%星形胶质细胞、20%小胶质细胞)或三维胶原凝胶中的皮层神经元^[44]。结合高速显微镜与数字图像相关技术, 可实时量化基质或凝胶内的应变分布与动态响应。该模型能引起神经突萎缩、骨架重排、膜通透性增加、核浓缩等结构损伤, 并上调肿瘤坏死因子-α (TNF-α)、白细胞介素-1α (IL-1α)等炎症因子, 模拟TBI后神经炎症微环境。其具有良好的力学可控性、应变可量化及较高生理相关性, 适用于细胞机械响应机制研究、神经保护策略评估及药物筛选。

2.1.3　刮擦损伤(scratch)

刮擦损伤模型首先使用无菌移液枪头在细胞单层上制造机械性“划痕”, 模拟体内损伤微环境中细胞结构的破坏, 随后在一定时间内观察细胞向损伤区域的迁移情况。在TBI研究框架下, 划痕实验主要用于评估神经元、星形胶质细胞、少突胶质前体细胞及微血管内皮细胞在损伤后的迁移与修复能力。如星形胶质细胞的迁移与增殖是形成胶质瘢痕(glial scar)的核心过程, 而胶质瘢痕在不同阶段既可限制炎症扩散, 也可能阻碍轴突再生, 因此划痕实验常用于观察药物或信号通路调控对胶质细胞迁移的影响。

相比其他TBI细胞模型, 该方法操作简便、经济高效, 可用于初步评估药物干预效果^[45]。然而, 传统划痕方法可能因划痕宽度不一致、压力不稳定而导致实验重复性偏低, 且实验不能完全模拟体内三维环境、基质刚度变化及神经网络复杂结构。为此, 研究者开发了自动化细胞损伤装置(cell injury device, CID), 通过电动驱动与实时力反馈控制刮擦的力度、速度与路径, 将划痕面积的变异系数从手工操作的10.7%~19.6%显著降低至3.9%~8.4%, 并整合自动成像与分析功能, 支持在96孔板中进行高通量、标准化实验。该模型尤其适用于研究TBI后胶质瘢痕形成相关的细胞迁移行为, 以及筛选调节该过程的候选药物^[46]。

2.2　化学损伤法

化学损伤法通过外源性添加神经活性物质模拟TBI后的继发性损伤, 常见手段包括谷氨酸、活性氧(reactive oxygen species, ROS)和炎症因子刺激。

2.2.1　谷氨酸损伤

该模型通过给予谷氨酸过度激活N-甲基天冬氨酸(N-methyl aspartic acid, NMDA)受体, 引起钙内流、线粒体功能障碍和细胞死亡, 从而模拟TBI后的兴奋性毒性过程, 适用于研究TBI中的神经元过度激活现象^[47]。

2.2.2　氧化应激损伤

该模型常采用过氧化氢(H₂O₂)等试剂诱导ROS大量生成, 以模拟TBI后的氧化应激环境。为增强模型的病理相关性, 可将其与缺糖/缺氧(OGD)模型联用, 从而在体外更好地模拟TBI后脑局部的能量代谢障碍和再灌注损伤过程^[48]。

2.2.3　炎症因子损伤

通过在体外培养体系中添加重组TNF-α、白细胞介素-1β (IL-1β)或脂多糖(lipopolysaccharide, LPS)等促炎因子, 直接激活小胶质细胞并诱发炎症级联反应, 用于模拟TBI后的神经炎症过程。该模型是评价抗炎药物(如米诺环素、白藜芦醇)药效的有效工具, 常通过检测下游炎症介质如环氧合酶-2 (cyclooxygenase-2, COX-2)、诱导型一氧化氮合成酶(inducible nitric oxide synthase, iNOS)的表达和释放来评估干预效果^{[49, 50]}。化学损伤法相关总结见表2^[51-54]。

2.3　创伤性颅脑损伤体外模型细胞模型选择应注意的问题

化学损伤法虽不能完全模拟机械性原发损伤, 但可作为补充手段, 增强模型的病理复杂性和转化价值。

表3对本文所述TBI体外模型进行总结。作为TBI研究体系的重要组成部分, 体外模型与动物模型互为补充。机械损伤法能更好地模拟原发性损伤的生物力学特性, 而化学损伤法则在再现特定继发性损伤通路上具有独特优势。研究者可根据具体科学问题, 选择单一或联合模型策略。当前, 随着类器官^[55]、器官芯片(organ-on-a-chip)^[56]等新技术的发展, 更复杂、更接近人体生理条件的TBI体外模型正在不断涌现, 有望在未来基础机制研究和药物开发中发挥更大价值。

3　创伤性颅脑损伤药物的非临床药效学评价方法

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TBI可引发一系列复杂的病理生理学改变和多样化的临床症状。因此, 在药物非临床评价中, 需结合分子标志物(如炎症因子、凋亡相关蛋白)、影像学特征及行为学表型等多维度指标, 对TBI的病理进程与药物疗效进行系统、综合的评估。其中, 行为学实验主要针对TBI后的运动功能缺损、认知障碍、焦虑样行为等核心症状进行量化评估, 是评价药物整体疗效的关键环节。

3.1　运动功能与感觉运动整合评估

TBI常导致运动协调性、平衡能力及感觉运动整合功能受损^[57], 在术后1~7天时间内(急性期-亚急性期), 运动功能损害表现最为明显, 在该时间段内常采用以下行为学方法进行量化评估。

3.1.1　神经功能缺损评分(modified neurological severity score, mNSS)

神经功能缺损评分是一种广泛应用于啮齿类动物模型的评估感觉、运动、平衡及反射功能的综合量表。评分范围从0分(正常)至18分(严重神经功能缺损), 分数越高表明神经损伤损伤程度越重^[58]。该评分已广泛应用于脑卒中及TBI动物模型的神经功能评价。其优势在于能整合不同模型特有的行为缺陷, 实现全面且客观的损伤评估。如WD模型动物常表现为显著运动障碍, 在平衡木试验中得分较高; 而FPI模型动物则更多出现感觉与记忆障碍^{[59, 60]}。尽管有时总分相近, 其行为表现特征仍可区分, 体现出mNSS在辨别不同损伤类型与综合评估中的实用价值。

3.1.2　转棒实验(rotarod test)

转棒实验通过记录动物在加速旋转棒上的滞留时间(跌落潜伏期), 评价其运动协调与平衡能力。该方法无需施加外源刺激, 能客观反映TBI模型的运动功能缺损。该设备通常包括一个表面带有纹理的转动杆, 底部设有保护性软垫。为适应不同实验动物, 转动杆直径可调, 大鼠直径约3.5 cm, 小鼠约1.5 cm。正式实验前需进行3天适应性训练: 转速从5 r·min^-1起步, 180 s内匀速加速至25 r·min^-1 (大鼠)或40 r·min^-1 (小鼠), 每天训练3次, 每次间隔30 min。训练结束后筛选跌落潜伏期超过某一标准(如160 s)的动物用于正式实验。实验记录跌落时间(跌落潜伏期)若超过180 s则按180 s计^[61]。

3.1.3　黏合剂去除实验(adhesive removal test)

该实验通过记录动物发现并移除前爪粘贴胶带所需的时间, 评估其感觉运动功能及口腔灵敏度, 广泛应用于脑损伤模型的行为恢复评价^[62]。实验时将动物置于透明观察箱中, 双前爪对称粘贴相同尺寸的胶带, 分别记录两个关键时间指标: ①首次接触时间(time-to-contact), 即动物首次用口鼻部接触任一前爪胶带的时刻; ②完全移除时间(time-to-remove), 即动物成功移除双爪上所有胶带所需的总时长。为减少个体差异及焦虑干扰, 正式测试前需进行5~7天的适应性训练。

3.1.4　足误实验(foot fault test)

足误实验通过统计动物在行走中肢体从横梁滑落的次数, 以评估啮齿类动物的运动协调及肢体放置准确性。实验装置通常为一组平行的间隔横梁(小鼠间隔0.5~2.5 cm, 大鼠1~5 cm)。正式实验前, 动物需进行3天的适应性训练, 使其能够熟练地通过横梁。通过高速摄像记录动物行走过程, 根据爪滑情况评分: 完全踏空为0分, 严重滑落为1分, 轻微滑落为2分, 替换踏步为3分, 位置调整但未接触为4分, 部分正确放置为5分, 完全正确为6分。实验需在安静、光线稳定的环境中进行, 以避免外部干扰^{[63, 64]}。

3.2　认知功能评估

认知障碍是TBI后的一种常见后遗症^[65], 常表现为空间学习和记忆功能的明显缺陷, 考虑到其逐步发展的特点, 一般选择在术后10~21 d (亚急性期-慢性期)进行检测。最经典的评估方法之一是Morris水迷宫(Morris water maze, MWM)实验, 该方法在神经行为学研究领域被广泛应用, 常用于评估药物对啮齿类动物认知功能障碍的干预效果。

MWM是一种在神经行为学研究领域中广泛应用的经典实验方法, 常用于评估药物对啮齿类动物认知功能障碍的干预效果^[66]。该实验系统主要包括一个圆形水池、一个隐藏平台、图像采集装置及运动轨迹分析系统。水池的大小根据所用实验动物的体型进行调整, 内壁通常使用无反射涂层以排除干扰。池内区域一般被分为4个象限, 隐藏平台置于其中某一象限中心, 平台直径约为10~12 cm, 没于水面以下1~2 cm。为便于视频系统捕捉动物的运动, 水温应维持在21~23 ℃, 并通过添加奶粉或无毒染料使水体颜色与动物毛色形成对比。池外周围还设有多种视觉参考标志(如不同形状与颜色的图形), 以协助动物建立空间定位记忆。

该实验主要包括两个阶段: 定向导航训练和空间探测测试。定向导航阶段通过连续多日的训练使动物学会利用环境视觉线索记忆平台位置。每次训练开始时, 动物由池壁某一入水点释放, 实验人员记录其自入水至寻得平台所需时间。若动物在规定时间内成功登台, 则允许其在平台上停留片刻以巩固记忆; 若失败, 则由实验人员引导其登台并保持相同停留时间。该训练一般持续5~6天, 期间主要记录逃避潜伏期、游泳路径、速度、运动轨迹及搜索策略等指标。

空间探测实验通常在导航训练结束24 h后进行。此时撤除隐藏平台, 将动物自与训练阶段入水位置相对的象限释放, 记录其在原平台所在区域的穿越次数及停留时间等参数。部分实验方案会在空间探测后增设线索提示实验, 即将平台重新放置并标记醒目旗帜, 以检验动物是否能够依视觉提示直接定位平台。若动物在此阶段仍无法有效完成任务, 则需考虑其是否存在视觉、运动或动机等基础缺陷, 此类个体通常予以剔除, 以确保行为学差异的特异性。

3.3　焦虑样行为与情绪状态评估

TBI后常继发焦虑等情绪障碍^[67], 在动物模型中表现为焦虑样行为的增加, 其发生发展稍晚于认知功能损害, 常在术后14~28天(慢性期)进行检测, 以下为评价焦虑样行为和情绪状态的具体操作流程。

3.3.1　旷场实验(open field test, OFT)

旷场实验基于啮齿类动物在陌生开放环境中产生焦虑样行为的假设, 通过分析其在周边与中心区域的活动差异, 评估焦虑状态。高焦虑水平动物的活动轨迹明显集中于周边区域, 而低焦虑个体则更为频繁地探索中心区域。实验时, 将动物置于旷场装置中心或固定角落, 通过视频追踪或人工观察记录5~10 min内的总运动距离、速度、进入中心区域的次数与时间、站立理毛行为及排便数量等指标, 从而综合评价其焦虑水平^[68]。

3.3.2　高架十字迷宫实验(elevated plus maze, EPM)

高架十字迷宫利用啮齿类动物对开放与封闭空间的自然偏好来评估焦虑行为。焦虑水平高的动物更倾向于停留在封闭臂, 而较少探索开放臂。通过量化其在开放臂和封闭臂的停留时间及进入次数, 可评估其焦虑程度。实验开始时, 将动物置于中央平台并使其头部朝向开放臂, 记录5~10 min内进入各臂的次数与时间、中央平台停留时间及总活动量。开放臂进入比例(开放臂进入次数/总进入次数)越高, 表明焦虑水平越低。需注意的是, 若实验动物的总活动量显著减少, 应评估其是否存在运动功能障碍, 以排除该因素对结果的干扰^[69]。

3.4　脑水肿检测

TBI后继发的脑水肿是导致患者死亡和预后不良的主要因素之一^{[70, 71]}。根据发生机制的不同, TBI后脑水肿可分为4种类型: 细胞毒性水肿、离子性水肿、血管源性水肿和出血转化性水肿^{[72, 73]}。其中, 血管源性水肿最为常见, 其主要特征为血脑屏障破坏导致的液体外渗^[74]。作为TBI的重要病理表现, 脑水肿的进展可引起脑组织受压, 进而导致脑灌注及氧合水平下降, 加剧继发性损伤。因此, 脑组织含水量的变化已成为评估TBI严重程度与预后的关键指标之一。在临床前研究中, 干湿比重法作为检测动物脑组织含水量的经典技术被广泛应用^[75]。其标准操作流程如下: 断头处死动物、迅速取脑, 轻拭表面液体及血液后, 切取损伤侧脑组织并称录湿重。随后将样本置于100 ℃烘箱内干燥48 h, 再次称重获得干重, 最终按公式(湿重-干重) / 湿重× 100%计算脑组织含水量。

3.5　血脑屏障通透性检测

血脑屏障(blood-brain barrier, BBB)是由脑微血管内皮细胞、基底膜和神经胶质细胞终足共同构成的高度选择性屏障, 对维持中枢神经系统内环境稳态具有关键作用。它通过限制大分子及有害物质进入脑组织, 保护神经功能。在结构上, 脑微血管内皮细胞借助occludin、claudins和连接黏附分子(JAMs)等跨膜蛋白形成紧密连接, 并通过胞内闭锁小带蛋白-1 (ZO-1)与细胞骨架相连, 共同维持BBB的结构完整性与功能。ZO-1与occludin在调控屏障通透性中发挥核心作用。动物实验显示, TBI后可诱发血管源性水肿, 伴随occludin和claudin-5表达下调, 导致液体、蛋白及免疫细胞渗出, 并加剧神经炎症反应, 提示BBB结构和功能受损^[76]。这些发现也得到了临床影像学结果的支持^[77], 如MRI显示TBI患者在损伤灶及其周围区域出现明显的血管源性水肿特征, 包括FLAIR信号增高及白质弥散异常。动态对比增强MRI (DCE-MRI)和灌注成像进一步证实血脑屏障通透性升高, 表现为对比剂外渗, 以及容积传输常数K^trans值和渗透指数NPI的增加^[78]。此外, 有研究利用DCE-MRI并通过Patlak模型定量分析发现, 患有脑震荡后综合征的轻度创伤性脑损伤患者, 其血脑屏障存在广泛损伤。具体表现为双侧大脑皮层的通透性显著增加, 同时小脑白质和脑干的相对血容量显著降低^[79]。因此, 评估药物对TBI后BBB的保护作用具有重要意义。在非临床研究中, 常用伊文思蓝(Evans blue, EB)或胶体金微粒(colloidal gold, CG)进行BBB通透性检测。

3.5.1　EB检测法

EB是一种偶氮染料, 在体内可与血浆白蛋白结合, 形成的复合物因分子质量较大, 在正常情况下无法透过完整的BBB; 当BBB受损时, 该复合物则会发生外渗, 因此其脑内含量常用于评估BBB完整性。动物通过尾静脉注射4% EB溶液(3 mL·kg^-1), 之后麻醉动物并进行心脏灌流, 直至右心房流出液无色。取脑后称湿重, 置于甲酰胺中45 ℃孵育48 h以提取染料, 使用分光光度计在630 nm波长下测定提取液的吸光度。通过EB标准曲线计算脑组织中EB含量, 结果以单位脑湿重中的EB含量(μg·g^-1)表示^[33]。

3.5.2　胶体金微粒法

该方法基于以下原理: 氯金酸(HAuCl₄)在还原剂(如柠檬酸钠、抗坏血酸等)作用下形成带负电的胶体金纳米颗粒, 其可通过BBB受损部位进入脑组织。具体操作包括静脉注射不同粒径(如5、10、15 nm)的胶体金溶液, 后续检测可分为两种路径: 一是通过银增强显影反应, 通过光镜观察黑褐色颗粒, 从而对BBB通透性进行定性/半定量分析。因其操作简便、灵敏度高、结果直观, 该方法已被广泛应用于免疫组织化学、细胞生物学及病理学研究^[80]。二是经灌流取脑制备超薄切片, 通过透射电镜在纳米尺度下观察不同粒径金颗粒的分布, 以评估BBB超微结构变化^[81]。

3.6　药物作用机制的探索

非临床药效学不仅要阐明药物的有效性, 还要探索新药的作用机制。目前TBI发病机制的研究主要集中在继发性脑损伤所涉及的多种病理过程, 包括神经炎症、氧化应激和神经元损伤等, 因此对抗TBI药物作用机制的研究也集中在这几个方面。

3.6.1　神经炎症

神经炎症是TBI后的关键病理过程之一, 除血脑屏障破坏和脑水肿外, 其在疾病进展中扮演重要角色。炎症本是机体应对损伤的一种防御反应, 早期有助于清除有害物质并启动修复机制。然而, 过度或持续性的炎症反应可能导致继发性脑损伤, 进一步加重神经功能损害^[82]。动物实验表明, TBI后可迅速激活小胶质细胞, 释放TNF-α、IL-1β等炎症因子, 进而激活多条炎症信号通路, 触发级联放大反应。这一过程不仅破坏血脑屏障完整性、损伤线粒体功能, 还会引起兴奋性神经递质(如谷氨酸)的过量释放及中性粒细胞浸润, 最终加剧神经元损伤和神经功能障碍^[83]。因此, 神经炎症被公认为TBI继发性损伤的核心机制之一。在药物的非临床研究中, 常用的评价手段包括Western blot、ELISA检测炎症因子表达, 以及对TLR4-NF-κB、核因子E2相关因子2 (Nrf2)-血红素加氧酶-1 (HO-1)等信号通路中关键蛋白的表达分析, 以评估药物干预效果。

3.6.2　氧化应激

TBI触发的神经炎症可导致炎症因子的释放, 进而促进ROS大量积累, 打破细胞内氧化-抗氧化平衡, 引发氧化应激^[84]。大脑组织中的多不饱和脂肪酸含量较高, 易受自由基攻击及铁离子介导的脂质过氧化反应影响, 从而导致组织损伤并诱发铁死亡^[85]。近年研究表明, 抑制氧化应激和铁死亡已成为缓解TBI继发性损伤的重要策略^[86]。Nrf2是调控细胞抗氧化应答的关键转录因子。在正常状态或轻度应激下, Nrf2与其胞质抑制蛋白Keap1结合并通过泛素-蛋白酶体途径降解。在氧化或亲电子应激状态下, Nrf2-Keap1复合物构象发生改变, Nrf2入核与抗氧化反应元件(ARE)结合, 启动下游抗氧化基因如HO-1和谷胱甘肽过氧化物酶(GPx)的转录, 从而增强细胞抗氧化能力。在非临床研究中, 常通过检测脂质过氧化终产物丙二醛(MDA)水平、Nrf2及HO-1蛋白表达以及GPx酶活性等指标综合评价脑内氧化应激状态^[87]。

3.6.3　神经元损伤

神经元损伤是TBI后继发性损伤的另一关键机制, 其中弥漫性轴突损伤会导致Wallerian变性进而引起轴突连续性丧失和神经元功能障碍, 还会进一步导致淀粉样蛋白和tau蛋白过度磷酸化引起的毒性蛋白病^[88]。这些级联反应共同驱动了创伤后的神经退行性变进程, 最终导致神经元的不可逆死亡。随着神经炎症与氧化应激的加剧, TBI后可激活细胞凋亡信号通路, 包括外源性(死亡受体)和内源性(线粒体)途径, Caspase-3是这两条通路的关键效应分子。此外, 线粒体来源的凋亡诱导因子(AIF)也可介导非Caspase依赖性的神经元坏死。死亡神经元释放的ATP、核蛋白等物质进一步激活免疫反应, 加剧神经炎症和氧化应激, 形成恶性循环。因此, 在非临床研究中, 通常通过Western blot检测Caspase-3、Bcl-2及AIF等凋亡相关蛋白, 以评估TBI后的神经元损伤程度^[89]。

4　我国抗TBI药物的研究现状

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目前, 尽管多个研究团队针对不同作用靶点持续推进治疗TBI的药物研发, 但大多数在临床前模型中有效的候选化合物, 在Ⅲ期临床试验中未能展现出显著疗效。这一转化瓶颈仍是当前TBI药物开发面临的主要挑战。随着对TBI发病机制的不断深入研究, 潜在的治疗靶点逐渐被发掘, 为开发突破性治疗策略开辟了新途径。表4汇总了目前处于Ⅱ期和Ⅲ期临床试验阶段的抗TBI候选药物。

5　小结

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创伤性颅脑损伤是神经外科中常见且病理机制复杂的疾病, 目前尚缺乏高效的特异性治疗手段。其治疗药物的研发一直是神经科学领域的重点研究方向。尽管众多候选药物在临床前动物实验中显示出显著疗效, 但其临床转化成功率仍然较低, 反映出当前非临床研究阶段所采用的动物模型与评价体系存在一定局限性。这些问题可能源于以下几方面因素: 实验动物与人类在生理结构及病理应答机制方面存在固有差异, 部分非临床研究在实验设计严谨性不足, 以及疗效评判标准缺乏统一性或临床相关性。因此, 在药物研发早期建立科学、规范且标准化的药效评价体系, 对于提高转化成功率和优化资源配置具有重要意义。同时, 进一步深入探讨TBI的发病机制, 明确疾病进程中的关键分子通路与细胞事件, 也将为靶点发现与创新药物设计提供坚实依据。单一动物模型往往难以全面模拟人体中复杂的病理反应, 其适用性常受造模方式、损伤部位及疾病分期等多因素影响, 但鉴于TBI病理机制尚未完全阐明, 行为学测试及相关神经功能评分量表目前仍是药效评价的重要组成部分。研究者应基于不同模型的特点, 合理化建模策略或联合多种模型体系, 以实现对药物疗效的多维度综合评价。随着动物模型的持续优化与创新治疗策略的不断涌现, TBI的药物治疗有望取来重要突破, 为改善患者神经功能预后开辟新的途径。

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2026年第61卷第6期

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doi: 10.16438/j.0513-4870.2025-1492

接收时间：2025-12-11
首发时间：2026-09-21
出版时间：2026-06-12

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收稿日期：2025-12-11
修回日期：2026-01-15

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¹中国医学科学院北京协和医学院, 药物研究所, 北京 100050

²国家药品监督管理局药品审评中心, 北京 100076

³中国食品药品检定研究院, 北京 102629

通讯作者:

唐婧姝，E-mail: tangjingshu@imm.ac.cn

王庆利，E-mail: wangql@nifdc.org.cn

彭英，E-mail: ypeng@imm.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Model name	Injury mechanism	Simulated clinical type	Key pathological feature	Advantage	Disadvantage
Fluid percussion injury (FPI)	Fluid pulse pressure transmission	Diffuse injury, elevated intracranial pressure (ICP), post-traumatic epilepsy	Diffuse axonal injury, cerebral edema, neuroinflammation	High reproducibility, controllable severity, suitable for diffuse injury	Requires craniotomy, non-physiological mechanism, surgical variability
Controlled cortical impact (CCI)	Mechanical impactor	Focal contusion/laceration	Cortical necrosis, hematoma, blood-brain barrier (BBB) disruption	Precise parameter control, excellent reproducibility, ideal for focal studies	Requires craniotomy, injury is localized, high surgical skill required
High-velocity projectile	High-speed penetration	Penetrating injury, simulates gunshot wounds	Diffuse injury, BBB disruption, edema	Often uses large animals, clinically relevant for ballistic trauma	High equipment cost, low survival rate
Controlled concussion	Head inertial motion	Mild TBI/concussion, chronic traumatic encephalopathy (CTE)	Microstructural damage, transient functional impairment	Reproducible mild injury	Injury is mild/transient, difficult to detect
Weight-drop injury (WDI)	Gravitational impact	Severe TBI/concussion, simulates open-head injury from falling objects	Diffuse injury, BBB disruption, edema	Simple equipment	Poor parameter control, low reproducibility, risk of cervical spine injury
Blast overpressure	High-pressure shockwave	Blast-related TBI	Diffuse vascular injury, BBB disruption	Simulates unique biomechanics, high military relevance	Complex equipment, high cost, complex mechanisms difficult to parse
Non-impact acceleration	Acceleration-deceleration forces	Diffuse axonal injury (DAI)	Axonal breakage/swelling, APP positive, pronounced injury in white matter tracts (corpus callosum, brainstem, internal capsule)	Studies pure inertial/acceleration mechanics, high controllability	Rarely causes severe contusion or major hemorrhage, not suitable for studying mixed TBI

Model name

Injury mechanism

Simulated clinical type

Key pathological feature

Advantage

Disadvantage

Fluid percussion injury (FPI)

Fluid pulse pressure transmission

Diffuse injury, elevated intracranial pressure (ICP), post-traumatic epilepsy

Diffuse axonal injury, cerebral edema, neuroinflammation

High reproducibility, controllable severity, suitable for diffuse injury

Requires craniotomy, non-physiological mechanism, surgical variability

Controlled cortical impact (CCI)

Mechanical impactor

Focal contusion/laceration

Cortical necrosis, hematoma, blood-brain barrier (BBB) disruption

Precise parameter control, excellent reproducibility, ideal for focal studies

Requires craniotomy, injury is localized, high surgical skill required

High-velocity projectile

High-speed penetration

Penetrating injury, simulates gunshot wounds

Diffuse injury, BBB disruption, edema

Often uses large animals, clinically relevant for ballistic trauma

High equipment cost, low survival rate

Controlled concussion

Head inertial motion

Mild TBI/concussion, chronic traumatic encephalopathy (CTE)

Microstructural damage, transient functional impairment

Reproducible mild injury

Injury is mild/transient, difficult to detect

Weight-drop injury (WDI)

Gravitational impact

Severe TBI/concussion, simulates open-head injury from falling objects

Diffuse injury, BBB disruption, edema

Simple equipment

Poor parameter control, low reproducibility, risk of cervical spine injury

Blast overpressure

High-pressure shockwave

Blast-related TBI

Diffuse vascular injury, BBB disruption

Simulates unique biomechanics, high military relevance

Complex equipment, high cost, complex mechanisms difficult to parse

Non-impact acceleration

Acceleration-deceleration forces

Diffuse axonal injury (DAI)

Axonal breakage/swelling, APP positive, pronounced injury in white matter tracts (corpus callosum, brainstem, internal capsule)

Studies pure inertial/acceleration mechanics, high controllability

Rarely causes severe contusion or major hemorrhage, not suitable for studying mixed TBI

Modeling agent	Typical concentration	Exposure duration	Primary target cell	Simulated TBI pathology phase/mechanism
Hydrogen peroxide (H₂O₂)^[51]	100-300 µmol·L^-1	2-6 h	Neurons, astrocytes, oligodendrocytes	Early secondary injury: Oxidative stress, ROS burst, mitochondrial dysfunction, lipid peroxidation, DNA damage
Glutamate^[52]	10-100 µmol·L^-1 (chronic); 100 µmol·L^-1-1 mmol·L^-1 (acute)	30 min-6 h	Neurons (especially cortical and hippocampal neurons)	Early secondary injury: Excitotoxicity, NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor overactivation, Ca²⁺ influx, mitochondrial damage
NMDA^[52]	10-50 µmol·L^-1	30 min-4 h	Neurons	Specific simulation of NMDA receptor-mediated excitotoxicity
TNF-α^[53]	10-20 μg·L^-1	6–48 h	Neurons, glial cells	Mid secondary injury: Inflammation amplification, apoptotic signaling tumor necrosis factor receptor 1 (TNFR1)-Caspase pathway
IL-1β^[53]	5-10 ng·mL^-1	6-48 h	Neurons, astrocytes	Mid secondary injury: Neuroinflammation, glial activation, synaptic dysfunction
LPS^[54]	10-1 000 μg·L^-1 (commonly 100 μg·L^-1)	12-72 h	Microglia (BV2 cell line, primary microglia)	Immune-inflammatory phase: TLR4-NF-κB activation, simulating the post-TBI inflammatory cascade
Cytokine cocktail (TNF-α + IL-1β + IFN-γ) ^[53]	5-20 μg·L^-1 each	12-48 h	Neuron-glia co-culture	Closer to in vivo secondary injury environment: Synergistic inflammation, apoptosis, and synaptic damage

Modeling agent

Typical concentration

Exposure duration

Primary target cell

Simulated TBI pathology phase/mechanism

Hydrogen peroxide (H₂O₂)^[51]

100-300 µmol·L^-1

2-6 h

Neurons, astrocytes, oligodendrocytes

Early secondary injury: Oxidative stress, ROS burst, mitochondrial dysfunction, lipid peroxidation, DNA damage

Glutamate^[52]

10-100 µmol·L^-1 (chronic); 100 µmol·L^-1-1 mmol·L^-1 (acute)

30 min-6 h

Neurons (especially cortical and hippocampal neurons)

Early secondary injury: Excitotoxicity, NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor overactivation, Ca²⁺ influx, mitochondrial damage

NMDA^[52]

10-50 µmol·L^-1

30 min-4 h

Neurons

Specific simulation of NMDA receptor-mediated excitotoxicity

TNF-α^[53]

10-20 μg·L^-1

6–48 h

Neurons, glial cells

Mid secondary injury: Inflammation amplification, apoptotic signaling tumor necrosis factor receptor 1 (TNFR1)-Caspase pathway

IL-1β^[53]

5-10 ng·mL^-1

6-48 h

Neurons, astrocytes

Mid secondary injury: Neuroinflammation, glial activation, synaptic dysfunction

LPS^[54]

10-1 000 μg·L^-1 (commonly 100 μg·L^-1)

12-72 h

Microglia (BV2 cell line, primary microglia)

Immune-inflammatory phase: TLR4-NF-κB activation, simulating the post-TBI inflammatory cascade

Cytokine cocktail (TNF-α + IL-1β + IFN-γ) ^[53]

5-20 μg·L^-1 each

12-48 h

Neuron-glia co-culture

Closer to in vivo secondary injury environment: Synergistic inflammation, apoptosis, and synaptic damage

Model name	Method	Simulated pathological process	Advantage	Disadvantage
Mechanical Injury	Mechanical stretch	Application of strain force to cells	Primary mechanical injury, diffuse axonal injury	High-throughput, good controllability, commercial instruments available	Difficult to simulate complex in vivo mechanical environment
	Compression Injury	Compression of cell layers/3D cultures	Focal contusion	Applicable to 3D culture, relatively simple setup	Strain rate control is imprecise
	Scratch injury	Creating a "scratch" on a cell monolayer	Wound healing, partially simulates secondary inflammation	Simple, economical, well-established, suitable for cell migration studies	Injury level cannot be precisely controlled
Chemical injury	Application of excitotoxins/ROS/inflammatory factors	Excitotoxicity, oxidative stress, neuroinflammation	Simple operation, high-throughput, clear mechanisms	Lacks primary mechanical injury, concentration difficult to control

Model name

Method

Simulated pathological process

Advantage

Disadvantage

Mechanical Injury

Mechanical stretch

Application of strain force to cells

Primary mechanical injury, diffuse axonal injury

High-throughput, good controllability, commercial instruments available

Difficult to simulate complex in vivo mechanical environment

Compression Injury

Compression of cell layers/3D cultures

Focal contusion

Applicable to 3D culture, relatively simple setup

Strain rate control is imprecise

Scratch injury

Creating a "scratch" on a cell monolayer

Wound healing, partially simulates secondary inflammation

Simple, economical, well-established, suitable for cell migration studies

Injury level cannot be precisely controlled

Chemical injury

Application of excitotoxins/ROS/inflammatory factors

Excitotoxicity, oxidative stress, neuroinflammation

Simple operation, high-throughput, clear mechanisms

Lacks primary mechanical injury, concentration difficult to control

Compound	Mechanism	Research stage	Company	Clinical trial information
Ronopterin	Nitric oxide synthase inhibitors	STAGE-3	veriNOS operations GmbH	NCT02794168
Biperiden hydrochloride	Muscarinic acetylcholine receptor antagonists	STAGE-3	Instituto Doutor José Frota	NCT04945213
Bezisterim	Extracellular-signal-regulated kinase 1 inhibitors	STAGE-3	Biovie	NCT05970575
Izaflortaucipir F-18	Positron-emission tomography enhancers	STAGE-3	Ac Immune	NCT05337774
Angiontensin (1-7)	Angiotensin Ⅱ receptor agonists	STAGE-3	Constant Therapeutics LLC	NCT06282965
Adezunap	Cannabinoid CB1 receptor agonists	STAGE-3	CannaXan GmbH	NCT06071949
Buntanetap	Microtubule-associated protein tau inhibitors	STAGE-3	Annovis Bio Inc.	NCT07284784
PRV-002	Neuroprotectants	STAGE-2	Avance Clinical Pty Ltd.	NCT06870240
Amantadine hydrochloride	Glutamate [NMDA] receptor antagonists	STAGE-2	SHINKEI Therapeutics Inc.	NCT02566720
AST-004	Adenosine A1 receptor agonists	STAGE-2	Astrocyte Pharmaceuticals Inc.	Astrocyte Pharmaceuticals Presents Update on AST-004 Clinical Program at 2022 Military Health System Research Symposium
ABX-101	Calmodulin inhibitors	STAGE-2	Abalonex LLC	NCT06096415
Synthetic ghrelin	Ghrelin/growth hormone secretagogue receptor agonists	STAGE-2	Oxeia Biopharmaceuticals Inc.	NCT04558346
Ibogaine	Mu opioid receptor agonists	STAGE-2	University of Sao Paulo	NCT05029401
Mebufotenin	Serotonin 1a (5-HT1a) receptor agonists	STAGE-2	GH Research Ireland Ltd.	NCT05800860
Pittsburgh compound B	Beta amyloid A4 protein inhibitors	STAGE-2	University of Pittsburgh	NCT01723553

Compound

Mechanism

Research stage

Company

Clinical trial information

Ronopterin

Nitric oxide synthase inhibitors

STAGE-3

veriNOS operations GmbH

NCT02794168

Biperiden hydrochloride

Muscarinic acetylcholine receptor antagonists

STAGE-3

Instituto Doutor José Frota

NCT04945213

Bezisterim

Extracellular-signal-regulated kinase 1 inhibitors

STAGE-3

Biovie

NCT05970575

Izaflortaucipir F-18

Positron-emission tomography enhancers

STAGE-3

Ac Immune

NCT05337774

Angiontensin (1-7)

Angiotensin Ⅱ receptor agonists

STAGE-3

Constant Therapeutics LLC

NCT06282965

Adezunap

Cannabinoid CB1 receptor agonists

STAGE-3

CannaXan GmbH

NCT06071949

Buntanetap

Microtubule-associated protein tau inhibitors

STAGE-3

Annovis Bio Inc.

NCT07284784

PRV-002

Neuroprotectants

STAGE-2

Avance Clinical Pty Ltd.

NCT06870240

Amantadine hydrochloride

Glutamate [NMDA] receptor antagonists

STAGE-2

SHINKEI Therapeutics Inc.

NCT02566720

AST-004

Adenosine A1 receptor agonists

STAGE-2

Astrocyte Pharmaceuticals Inc.

Astrocyte Pharmaceuticals Presents Update on AST-004 Clinical Program at 2022 Military Health System Research Symposium

ABX-101

Calmodulin inhibitors

STAGE-2

Abalonex LLC

NCT06096415

Synthetic ghrelin

Ghrelin/growth hormone secretagogue receptor agonists

STAGE-2

Oxeia Biopharmaceuticals Inc.

NCT04558346

Ibogaine

Mu opioid receptor agonists

STAGE-2

University of Sao Paulo

NCT05029401

Mebufotenin

Serotonin 1a (5-HT1a) receptor agonists

STAGE-2

GH Research Ireland Ltd.

NCT05800860

Pittsburgh compound B

Beta amyloid A4 protein inhibitors

STAGE-2

University of Pittsburgh

NCT01723553