药学学报

Index	pMCAO	tMCAO	dMCAO
Blood flow recovery	No reperfusion (permanent occlusion)	With reperfusion (filament removal or ligation release after occlusion)	Usually no reperfusion (partial models allow reperfusion)
Infarct range	Largest (involves ischemic core and surrounding areas) (Figure 2 left)	Large (core infarct region, ischemic penumbra) (Figure 2 middle)	Small (confined to the perfusion area of distal MCA branches, e.g., cortex) (Figure 2 right)
Surgical method	Electrocoagulation or ligation of proximal MCA, filament method	Filament method: insert filament to the origin of MCA, withdraw after a certain time	Transcranial electrocoagulation or filament occlusion of distal MCA branches
Pathological mechanism	Persistent ischemia leading to energy failure and cell necrosis	Ischemia-reperfusion injury (free radical burst, inflammatory response)	Focal ischemia causing selective neuronal injury
Research focus	Irreversible injury mechanism, long-term neural repair	Reperfusion injury, efficacy of neuroprotective agents	Cortical function recovery, small molecule drug screening
Mortality rate	High (massive infarction prone to cerebral edema)	Moderate (risk of cerebral edema after reperfusion)	Low (small infarct range, high survival rate)
Advantages & disadvantages	Advantages: simulates patients not receiving reperfusion therapy; disadvantages: severe surgical trauma, high mortality	Advantages: simulates pathological process after thrombolysis; disadvantages: precise control of reperfusion time window required	Advantages: localized injury, suitable for long-term behavioral evaluation; disadvantages: high surgical technique requirements, large infarct heterogeneity

Index	pMCAO	tMCAO	dMCAO
Blood flow recovery	No reperfusion (permanent occlusion)	With reperfusion (filament removal or ligation release after occlusion)	Usually no reperfusion (partial models allow reperfusion)
Infarct range	Largest (involves ischemic core and surrounding areas) (Figure 2 left)	Large (core infarct region, ischemic penumbra) (Figure 2 middle)	Small (confined to the perfusion area of distal MCA branches, e.g., cortex) (Figure 2 right)
Surgical method	Electrocoagulation or ligation of proximal MCA, filament method	Filament method: insert filament to the origin of MCA, withdraw after a certain time	Transcranial electrocoagulation or filament occlusion of distal MCA branches
Pathological mechanism	Persistent ischemia leading to energy failure and cell necrosis	Ischemia-reperfusion injury (free radical burst, inflammatory response)	Focal ischemia causing selective neuronal injury
Research focus	Irreversible injury mechanism, long-term neural repair	Reperfusion injury, efficacy of neuroprotective agents	Cortical function recovery, small molecule drug screening
Mortality rate	High (massive infarction prone to cerebral edema)	Moderate (risk of cerebral edema after reperfusion)	Low (small infarct range, high survival rate)
Advantages & disadvantages	Advantages: simulates patients not receiving reperfusion therapy; disadvantages: severe surgical trauma, high mortality	Advantages: simulates pathological process after thrombolysis; disadvantages: precise control of reperfusion time window required	Advantages: localized injury, suitable for long-term behavioral evaluation; disadvantages: high surgical technique requirements, large infarct heterogeneity

Evaluation index	Experiment name	Specific detection indicator	Training required	Evaluation time
Neurological deficit	Bederson score	The Bederson score assesses neurological dysfunction, with higher scores indicating more severe impairment 0: When suspended by the tail, the animal extends both forelimbs naturally, with no flexion or movement asymmetry 1: The forelimb contralateral to the injury shows flexion (e.g., wrist or elbow bending), without additional abnormalities such as circling or reduced resistance to lateral push 2: In addition to forelimb flexion, the animal exhibits significantly reduced resistance to lateral push toward the impaired side 3: The animal displays spontaneous circling behavior toward the injured side, in addition to the above signs 4: The animal shows longitudinal rotation or is unable to move independently	No	Acute phase (0-72 h)
Longa score	This scale evaluates motor behavior and neurological deficits, ranging from 0 (no deficit) to 4 (severe deficit or death). It includes assessments of somatosensory function, placement response, grip strength, reflexes, balance, and coordination	No	Acute phase (0-72 h)
mNSS	1. Suspended tail test: elevate the tail above the ground and observe any deviation or flexion of the head and limbs 2. Normal gait, inability to walk in a straight line, circling towards the affected side, and leaning towards the affected side are scored 0-3 points respectively 3. Sensory testing: visual testing; tactile testing; proprioceptive testing 4. Balance beam test 5. Reflex testing: auricular reflex; corneal reflex; startle reflex; epileptic seizures, muscle spasms, abnormal muscle tone mNSS ranges from 0 to 18 points, with higher scores indicating more severe neurological impairment	No	Acute phase (0-72 h), subacute phase (3-14 d), recovery phase (> 14 d)
Garcia neurobehavioral scale	1. General activity in the test cage 2. Symmetry of forelimb movement 3. Ability to extend forepaws 4. Climbing ability on a wire 5. Tactile response to body stimulation (cotton swab) 6. Response to vibrissae touch Each test is scored from 0 to 3, with a maximum total of 18 points. Higher scores indicate milder neurological injury	No	Acute phase (0-72 h), subacute phase (3-14 d)
Motor coordination & balance	Rotarod test	A rotating cylinder (diameter: 3.0-3.2 cm) with multiple compartments allows simultaneous testing of several mice. The latency to fall from the rod is recorded as a measure of motor coordination and balance	Yes (2-3 d, 2-3 rounds)	Acute phase (0-72 h), subacute phase (3-14 d)
Wire hanging test	The animal grasps a horizontal wire with its forepaws and hangs until fatigue. The latency to fall is recorded to assess motor function and neuromuscular strength	Yes (2-3 times)	Acute phase (0-72 h), subacute phase (3-14 d)
Pole test	Motor recovery is evaluated by observing the animal's coordination, speed, and success in climbing down a pole. The time taken to descend from top to bottom serves as an indicator of motor speed and control	Yes (2-3 times)	Subacute phase (3-14 d)
Grid walk test	An elevated metal grid (41 cm × 41 cm, grid size: 3.5 cm² × 3.5 cm², height: 41 cm) is placed in a dim, soundproof room. After each trial, the grid is cleaned with 70% ethanol. A camera records the movement of mouse, and data analysis includes total forepaw steps and average step distance	No	Subacute phase (3-14 d), recovery phase (> 14 d)
Limb use symmetry & exploratory behavior	Cylinder test	A glass cylinder (diameter: 9-10 cm, height: 15 cm) is used to assess vertical exploration. The number of forelimb contacts with the cylinder wall is recorded. A mirror or camera ensures both forelimbs are visible during scoring	No	Recovery phase (> 14 d)
Forelimb placing test	This test evaluates motor function and recovery through seven specific tasks: 1. Forelimb extension when the mouse is held above a table 2. Retrieval and placement of forelimbs after gentle pulling 3. Same as task 2, but with the head elevated at 45° 4. Lateral placement of forelimbs along the table edge 5. Lateral placement of hindlimbs along the table edge 6. Hindlimb grip strength when pulled from the edge 7. Grip ability when gently pushed from behind toward the table edge Each item is scored 0-2, with a total maximum of 14 points. Lower scores indicate more severe dysfunction	No	Acute phase (0-72 h), subacute phase (3-14 d)
Corner turn test	Two cardboard walls form a 30° angle with a small opening at the corner to attract the animal. The animal is placed facing the corner, and vibrissae stimulation prompts a turning response. The direction of the turn is recorded to assess sensory and motor asymmetry	No	Subacute phase (3-14 d), recovery phase (> 14 d)
Adhesive removal test	Small adhesive patches (rectangular: 0.3 cm × 0.4 cm; or circular: diameter 0.3 cm) are placed on each forepaw. The mouse is placed in a transparent box and allowed to acclimate. The time to contact and remove each patch is recorded, reflecting sensorimotor integration and limb functionality	No	Subacute phase (3-14 d), recovery phase (> 14 d)

Evaluation index	Experiment name	Specific detection indicator	Training required	Evaluation time
Neurological deficit	Bederson score	The Bederson score assesses neurological dysfunction, with higher scores indicating more severe impairment 0: When suspended by the tail, the animal extends both forelimbs naturally, with no flexion or movement asymmetry 1: The forelimb contralateral to the injury shows flexion (e.g., wrist or elbow bending), without additional abnormalities such as circling or reduced resistance to lateral push 2: In addition to forelimb flexion, the animal exhibits significantly reduced resistance to lateral push toward the impaired side 3: The animal displays spontaneous circling behavior toward the injured side, in addition to the above signs 4: The animal shows longitudinal rotation or is unable to move independently	No	Acute phase (0-72 h)
Longa score	This scale evaluates motor behavior and neurological deficits, ranging from 0 (no deficit) to 4 (severe deficit or death). It includes assessments of somatosensory function, placement response, grip strength, reflexes, balance, and coordination	No	Acute phase (0-72 h)
mNSS	1. Suspended tail test: elevate the tail above the ground and observe any deviation or flexion of the head and limbs 2. Normal gait, inability to walk in a straight line, circling towards the affected side, and leaning towards the affected side are scored 0-3 points respectively 3. Sensory testing: visual testing; tactile testing; proprioceptive testing 4. Balance beam test 5. Reflex testing: auricular reflex; corneal reflex; startle reflex; epileptic seizures, muscle spasms, abnormal muscle tone mNSS ranges from 0 to 18 points, with higher scores indicating more severe neurological impairment	No	Acute phase (0-72 h), subacute phase (3-14 d), recovery phase (> 14 d)
Garcia neurobehavioral scale	1. General activity in the test cage 2. Symmetry of forelimb movement 3. Ability to extend forepaws 4. Climbing ability on a wire 5. Tactile response to body stimulation (cotton swab) 6. Response to vibrissae touch Each test is scored from 0 to 3, with a maximum total of 18 points. Higher scores indicate milder neurological injury	No	Acute phase (0-72 h), subacute phase (3-14 d)
Motor coordination & balance	Rotarod test	A rotating cylinder (diameter: 3.0-3.2 cm) with multiple compartments allows simultaneous testing of several mice. The latency to fall from the rod is recorded as a measure of motor coordination and balance	Yes (2-3 d, 2-3 rounds)	Acute phase (0-72 h), subacute phase (3-14 d)
Wire hanging test	The animal grasps a horizontal wire with its forepaws and hangs until fatigue. The latency to fall is recorded to assess motor function and neuromuscular strength	Yes (2-3 times)	Acute phase (0-72 h), subacute phase (3-14 d)
Pole test	Motor recovery is evaluated by observing the animal's coordination, speed, and success in climbing down a pole. The time taken to descend from top to bottom serves as an indicator of motor speed and control	Yes (2-3 times)	Subacute phase (3-14 d)
Grid walk test	An elevated metal grid (41 cm × 41 cm, grid size: 3.5 cm² × 3.5 cm², height: 41 cm) is placed in a dim, soundproof room. After each trial, the grid is cleaned with 70% ethanol. A camera records the movement of mouse, and data analysis includes total forepaw steps and average step distance	No	Subacute phase (3-14 d), recovery phase (> 14 d)
Limb use symmetry & exploratory behavior	Cylinder test	A glass cylinder (diameter: 9-10 cm, height: 15 cm) is used to assess vertical exploration. The number of forelimb contacts with the cylinder wall is recorded. A mirror or camera ensures both forelimbs are visible during scoring	No	Recovery phase (> 14 d)
Forelimb placing test	This test evaluates motor function and recovery through seven specific tasks: 1. Forelimb extension when the mouse is held above a table 2. Retrieval and placement of forelimbs after gentle pulling 3. Same as task 2, but with the head elevated at 45° 4. Lateral placement of forelimbs along the table edge 5. Lateral placement of hindlimbs along the table edge 6. Hindlimb grip strength when pulled from the edge 7. Grip ability when gently pushed from behind toward the table edge Each item is scored 0-2, with a total maximum of 14 points. Lower scores indicate more severe dysfunction	No	Acute phase (0-72 h), subacute phase (3-14 d)
Corner turn test	Two cardboard walls form a 30° angle with a small opening at the corner to attract the animal. The animal is placed facing the corner, and vibrissae stimulation prompts a turning response. The direction of the turn is recorded to assess sensory and motor asymmetry	No	Subacute phase (3-14 d), recovery phase (> 14 d)
Adhesive removal test	Small adhesive patches (rectangular: 0.3 cm × 0.4 cm; or circular: diameter 0.3 cm) are placed on each forepaw. The mouse is placed in a transparent box and allowed to acclimate. The time to contact and remove each patch is recorded, reflecting sensorimotor integration and limb functionality	No	Subacute phase (3-14 d), recovery phase (> 14 d)

Drug name	Target/mechanism	Development phase	Preclinical animal experimental model
Y-3 for injection	Targets PSD95-nNOS coupling—a key pathological mechanism in stroke and mood disorders—and modulates GABA_A receptors, enabling synergistic treatment of ischemic stroke and prevention of post-stroke depression	Completed phase Ⅲ clinical trial	MCAO^[60]
Nitronezine (TBN) for injection	Crosses the blood-brain barrier efficiently, inhibits calcium overload, scavenges free radicals, and suppresses neuroinflammation. Enhances mitochondrial function and boosts ATP production via activation of the AMPK/PGC-1α/Nrf2/HO-1 signaling axis; promotes autophagic clearance of toxic proteins through mTOR pathway inhibition	Phase Ⅲ clinical trial	MCAO^[61]
GD-11 for injection	GLUT-1	Phase Ⅲ clinical trial	Not disclosed
JX10 for injection	Anti-inflammatory thrombolytic agent	Phase Ⅱ/Ⅲ clinical trial	Not disclosed
Recombinant staphylokinase (r-Sak) for injection	Forms a complex with plasminogen to activate the fibrinolytic system, promoting the conversion of plasminogen to plasmin, selectively degrading fibrin, and dissolving thrombi	Phase Ⅱ clinical trial	Thromboembolic model^[62]
LT3001 for injection	Synthetic polypeptide, promoting vascular recanalization and mitigating reperfusion injury	Phase Ⅱ clinical trial	MCAO^[63]
ZHB206 injection	Reduces HMGB1-mediated apoptosis and inflammation after ischemic stroke, enhancing cell survival and glial migration, thereby attenuating ischemic brain damage	Phase Ⅱ clinical trial	Not disclosed
HRS8179	Inhibits the SUR1/TRPM4 ion channel to reduce sodium influx, alleviating cytotoxic edema and lowering mortality	Phase Ⅱ clinical trial	Not disclosed
BAY 3018250	Anti α2AP protein therapy. Significantly reduces microvascular thrombosis, ischemic injury, brain swelling, hemorrhagic transformation, and mortality following thromboembolic stroke	Phase Ⅱ clinical trial	Not disclosed
Y-6 sublingual tablets	Novel sublingual compound tablet. Combines two marketed drugs targeting cerebral vasculature and neural tissue, respectively. Inhibits vascular smooth muscle proliferation, protects endothelial integrity, reduces bleeding risk, preserves blood-brain barrier function, suppresses inflammatory cytokine release, and mitigates glutamate excitotoxicity. Improves neurological outcomes and cerebral metabolism, enhances cognitive function in cerebrovascular disease, and reduces white matter hyperintensity volume. The components exhibit synergistic efficacy with a clear dose-response relationship. Suitable for acute and recovery phases of stroke, as well as long-term management of cerebral small vessel disease	Phase Ⅱ clinical trial	MCAO^[64]
AD108 injection	Exogenous KLK-1 supplementation improves neurological deficits, reduces disability, and enhances daily functioning in acute ischemic stroke. AD108 injection enables sustained release via subcutaneous administration, maintaining stable plasma levels and therapeutic effect while avoiding hypotension risks associated with intravenous delivery	Phase Ⅰ clinical trial	Not disclosed
NR-20201 injection	Promote vascular regeneration and functional repair of ischemic brain tissue via homing mechanisms and synergistic interaction with endothelial cells	Phase Ⅰ clinical trial	Not disclosed
Xing lou cheng qi granules	Clears heat from the fu-organs, activates blood circulation, and removes meridian obstruction	Phase Ⅰ clinical trial	Not disclosed
PMS-001 for injection	AMPA receptor modulator (ionotropic glutamate receptor AMPA modulator)	Phase Ⅰ clinical trial	Not disclosed
ZHB103 injection	Mechanism not disclosed	Phase Ⅰ clinical trial	Not disclosed
Asarone emulsion injection	Elevates cortical electrical stimulation threshold, inhibits synaptic transmission of epileptiform activity, and prevents seizure propagation	Phase Ⅰ clinical trial	Not disclosed

Drug name	Target/mechanism	Development phase	Preclinical animal experimental model
Y-3 for injection	Targets PSD95-nNOS coupling—a key pathological mechanism in stroke and mood disorders—and modulates GABA_A receptors, enabling synergistic treatment of ischemic stroke and prevention of post-stroke depression	Completed phase Ⅲ clinical trial	MCAO^[60]
Nitronezine (TBN) for injection	Crosses the blood-brain barrier efficiently, inhibits calcium overload, scavenges free radicals, and suppresses neuroinflammation. Enhances mitochondrial function and boosts ATP production via activation of the AMPK/PGC-1α/Nrf2/HO-1 signaling axis; promotes autophagic clearance of toxic proteins through mTOR pathway inhibition	Phase Ⅲ clinical trial	MCAO^[61]
GD-11 for injection	GLUT-1	Phase Ⅲ clinical trial	Not disclosed
JX10 for injection	Anti-inflammatory thrombolytic agent	Phase Ⅱ/Ⅲ clinical trial	Not disclosed
Recombinant staphylokinase (r-Sak) for injection	Forms a complex with plasminogen to activate the fibrinolytic system, promoting the conversion of plasminogen to plasmin, selectively degrading fibrin, and dissolving thrombi	Phase Ⅱ clinical trial	Thromboembolic model^[62]
LT3001 for injection	Synthetic polypeptide, promoting vascular recanalization and mitigating reperfusion injury	Phase Ⅱ clinical trial	MCAO^[63]
ZHB206 injection	Reduces HMGB1-mediated apoptosis and inflammation after ischemic stroke, enhancing cell survival and glial migration, thereby attenuating ischemic brain damage	Phase Ⅱ clinical trial	Not disclosed
HRS8179	Inhibits the SUR1/TRPM4 ion channel to reduce sodium influx, alleviating cytotoxic edema and lowering mortality	Phase Ⅱ clinical trial	Not disclosed
BAY 3018250	Anti α2AP protein therapy. Significantly reduces microvascular thrombosis, ischemic injury, brain swelling, hemorrhagic transformation, and mortality following thromboembolic stroke	Phase Ⅱ clinical trial	Not disclosed
Y-6 sublingual tablets	Novel sublingual compound tablet. Combines two marketed drugs targeting cerebral vasculature and neural tissue, respectively. Inhibits vascular smooth muscle proliferation, protects endothelial integrity, reduces bleeding risk, preserves blood-brain barrier function, suppresses inflammatory cytokine release, and mitigates glutamate excitotoxicity. Improves neurological outcomes and cerebral metabolism, enhances cognitive function in cerebrovascular disease, and reduces white matter hyperintensity volume. The components exhibit synergistic efficacy with a clear dose-response relationship. Suitable for acute and recovery phases of stroke, as well as long-term management of cerebral small vessel disease	Phase Ⅱ clinical trial	MCAO^[64]
AD108 injection	Exogenous KLK-1 supplementation improves neurological deficits, reduces disability, and enhances daily functioning in acute ischemic stroke. AD108 injection enables sustained release via subcutaneous administration, maintaining stable plasma levels and therapeutic effect while avoiding hypotension risks associated with intravenous delivery	Phase Ⅰ clinical trial	Not disclosed
NR-20201 injection	Promote vascular regeneration and functional repair of ischemic brain tissue via homing mechanisms and synergistic interaction with endothelial cells	Phase Ⅰ clinical trial	Not disclosed
Xing lou cheng qi granules	Clears heat from the fu-organs, activates blood circulation, and removes meridian obstruction	Phase Ⅰ clinical trial	Not disclosed
PMS-001 for injection	AMPA receptor modulator (ionotropic glutamate receptor AMPA modulator)	Phase Ⅰ clinical trial	Not disclosed
ZHB103 injection	Mechanism not disclosed	Phase Ⅰ clinical trial	Not disclosed
Asarone emulsion injection	Elevates cortical electrical stimulation threshold, inhibits synaptic transmission of epileptiform activity, and prevents seizure propagation	Phase Ⅰ clinical trial	Not disclosed

抗缺血性脑卒中药物非临床药效学评价体系探索

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杨艳 ¹ , 赵春阳 ² , 渠凯 ¹ , 吴镭 ¹ , 王庆利 ³^,^* , 彭英 ¹^,^*

药学学报 | 综述 2026,61(2): 332-348

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药学学报 |综述 2026 , 61 (2) : 332 -348

抗缺血性脑卒中药物非临床药效学评价体系探索

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杨艳¹, 赵春阳², 渠凯¹, 吴镭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:

王庆利, E-mail: wangql@cde.org.cn

彭英, E-mail: ypeng@imm.ac.cn

作者简介:

共同第一作者.

作者贡献:

杨艳和赵春阳负责相关文献的收集、文章作图及综述的撰写; 吴镭和渠凯对综述的构思提出建议并进行修改和检查; 彭英和王庆利是综述框架的构思者及负责人, 指导综述写作并对综述进行修改和检查。全体作者都阅读并同意最终的文本。

Exploration of non-clinical pharmacodynamics evaluation system of ischemic stroke

Yan YANG¹, Chun-yang ZHAO², Kai QU¹, Lei WU¹, 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 Institute for Food and Drug Control, Beijing 102629, China

出版时间: 2026-02-12 doi: 10.16438/j.0513-4870.2025-0869

文章导航

摘要

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缺血性脑卒中(ischemic stroke, IS) 是导致全球成年人致死致残的主要病因之一, 其核心病理机制为脑血流中断引发的神经元缺血缺氧性损伤。目前, 临床治疗以静脉溶栓和血管内取栓为主, 但这些手段受限于严格的时间窗(通常≤ 4.5 h) 及部分患者面临潜在出血风险, 且对血管再通后神经损伤的修复作用有限。尽管近年来神经保护剂研发活跃, 但在临床转化中因疗效不足或安全性问题相继失败, 凸显了疾病机制的复杂性与临床前评价体系的局限性。因此, 针对IS的病理级联反应, 开发脑细胞保护药物, 成为突破当前治疗瓶颈的关键方向。本文结合近年来国内外治疗IS药物的研发进展, 主要从模型构建、药效评价指标及相关分子机制方面对治疗IS药物非临床药效学评价体系进行归纳总结, 为IS非临床药效学评价体系的构建与优化提供有益探索, 有助于推动IS治疗药物的研发进程, 为临床治疗提供更多有效的治疗策略。

关键词

缺血性脑卒中 / 非临床药效学评价 / 动物模型 / 药物研发 / 分子机制

Abstract

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Ischemic stroke (IS) remains a leading cause of mortality and disability among adults worldwide, with its core pathological mechanism rooted in ischemic-hypoxic neuronal injury triggered by cerebral blood flow interruption. Current clinical treatments primarily rely on intravenous thrombolysis and endovascular thrombectomy. However, these approaches are constrained by strict therapeutic time windows (typically ≤ 4.5 h), bleeding risks in certain patients, and limited neurorestorative effects following reperfusion. Despite active development of neuroprotective agents and anti-inflammatory drugs in recent years, most have failed in clinical translation due to insufficient efficacy or safety concerns, highlighting the complexity of disease mechanisms and limitations of preclinical evaluation systems. Consequently, developing therapeutics that simultaneously address reperfusion protection and neural repair by targeting the pathological cascade of IS has emerged as a critical direction to overcome current therapeutic bottlenecks. This review synthesizes recent advances in domestic and international IS drug development in recent years, focusing on model establishment, efficacy evaluation metrics, and relevant molecular mechanisms to systematically summarize the preclinical pharmacodynamic evaluation framework for IS therapeutics. The findings provide valuable insights for optimizing preclinical evaluation systems, accelerating drug development, and expanding effective clinical treatment strategies for IS.

Key words

ischemic stroke / non-clinical pharmacodynamic evaluation / animal model / drug discovery / molecular mechanism

引用本文

杨艳, 赵春阳, 渠凯, 吴镭, 王庆利, 彭英. 抗缺血性脑卒中药物非临床药效学评价体系探索. 药学学报, 2026 , 61 (2) : 332 -348 . DOI: 10.16438/j.0513-4870.2025-0869

Yan YANG, Chun-yang ZHAO, Kai QU, Lei WU, Qing-li WANG, Ying PENG. Exploration of non-clinical pharmacodynamics evaluation system of ischemic stroke[J]. Acta Pharmaceutica Sinica, 2026 , 61 (2) : 332 -348 . DOI: 10.16438/j.0513-4870.2025-0869

正文

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缺血性脑卒中(ischemic stroke, IS) 是由于脑局部血液循环障碍所导致的神经功能缺损综合征, 症状持续时间至少24 h或存在经影像学证实的新发脑梗死灶。据《中国脑卒中防治报告(2023)》及全球研究数据, 中国IS发病人数为200~240例/10万人, 是我国居民死亡的首要原因。近年来, 发病率呈逐年上升趋势, 年均增长率约1.5%~3%, 发病人群呈现逐渐年轻化趋势^{[1, 2]}。IS发病机制复杂且多因素参与, 核心在于脑血管阻塞导致脑血流中断, 进而引发脑组织缺血缺氧性损伤。在临床治疗实践中, 对其病因和梗死部位进行准确分类, 制定针对性的治疗策略, 可降低患者的死亡率和致残率。

急性IS治疗的早期目标是恢复缺血区域的血流灌注, 即血管再通治疗, 主要措施是静脉溶栓和血管内介入治疗, 静脉溶栓药物主要包括阿替普酶、替奈普酶、瑞替普酶及尿激酶。其中, 阿替普酶是一种重组组织型纤溶酶原激活剂(recombinant tissue plasminogen activator, rt-PA)。根据美国心脏协会指南, 发病4.5 h内接受rt-PA溶栓可显著改善预后, 每延迟1 h溶栓, 患者获得良好功能结局的可能性降低20%^[3]。然而, 中国仅5%的患者能在4.5 h内接受溶栓, 远低于发达国家。同时, 溶栓和血管内介入治疗的出血风险需严格筛选患者。多项大型临床试验研究表明, 尽管现有的溶栓取栓治疗在急性IS患者中显示出一定的疗效, 但治疗窗口狭窄, 受益患者比例有限^[4], 且存在脑水肿或出血性转化。

随缺血持续时间延长, 患者神经功能受损逐渐加重。神经保护是IS治疗中极具价值但尚未突破的领域, 过去几十年间, 尽管逾千种神经保护剂在非临床研究中通过不同机制证实了明确的神经保护作用, 其向临床试验的转化却持续遇阻, 至今未突破有效性验证瓶颈。本文通过总结整合国内外的相关文献报道, 期望构建系统完善的IS非临床药效学评价体系(图1), 深入探究IS的病理机制, 更准确地评估IS创新药物的治疗潜力, 为临床前阶段的药物筛选和优化提供强有力的科学支持, 改善IS患者的临床治疗结局。

1　IS的动物模型

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目前, 动物模型是研究IS病理生理机制和新药筛选的首选工具, 根据缺血类型、损伤机制及研究终点的差异, 当前应用的IS经典动物模型可系统分为以下几类。

1.1　大脑中动脉闭塞模型

大脑中动脉闭塞(middle cerebral artery occlusion, MCAO) 模型通过短暂性或永久性血管阻断, 高度模拟人类IS的病理过程。该模型技术实施门槛低, 操作流程易于标准化, 其结果兼具可靠性和可重复性。目前是常使用的IS动物模型之一。Longa等^[5]最早在1989年建立了此模型。该模型需要暴露颈部的颈动脉血管并临时阻塞颈总动脉及颈外动脉、枕动脉和翼腭动脉, 以便将线栓推进至阻塞大脑中动脉。插入线栓后, 可将其永久放置, 或在一定时间段后取出, 以模拟大脑中动脉的再灌注。MCAO模型通常使用大鼠和小鼠等啮齿类动物进行造模, 以下是建议的标准化实验步骤^[6]。

1.1.1　大鼠MCAO模型

使用3%异氟烷麻醉大鼠。将大鼠仰卧位置于恒温板上, 保持体温37 ℃。将大鼠置右侧卧位, 颈部正中切口约25 mm, 在右侧的肩胛舌骨和胸锁乳突肌形成的三角处暴露颈总动脉及其分支颈外动脉和颈内动脉。结扎颈外动脉的分支枕动脉、甲状腺上动脉及颈外动脉终末支, 分离颈内动脉的颅外分支翼颚动脉。将颈外动脉结扎并剪断, 把颈外动脉远心端拉直与颈内动脉成直线, 并在分叉处轻轻结扎一根4-0丝线。用动脉夹夹闭颈总动脉和颈内动脉, 在颈外动脉上剪一个小切口。将一根长4.0~4.5 cm, 直径0.24~0.28 mm的圆头非硅胶栓线由颈外动脉插入颈内动脉, 扎紧分叉处的丝线, 移去颈内动脉上动脉夹, 缓慢地将尼龙线插入颈内动脉约18 mm, 进颅内至大脑中动脉起始处。移去颈总动脉上的动脉夹。使用激光多普勒血流监测仪监测脑血流, 脑血流迅速下降至先前基线值的42% ± 16%^[7]。实验过程中, 持续监测心率和血压。插线时, 动作要轻柔, 避免穿破血管。缝合颈部的手术切口。大鼠苏醒后, 观察其行为学变化, 以判断大脑中动脉是否阻断成功。

1.1.2　小鼠MCAO模型

其操作过程与大鼠模型基本一致, 只是手术难度略有增加。需要特别指出的是所用栓线直径与小鼠体重要相互匹配。小鼠体重一般在20~30 g。栓线头端经钝化处理并包被硅化树脂后, 直径约在0.18~0.22 mm。栓线插入颈内动脉深度约为9~10 mm。该模型个体间差异较大, 特别在使用转基因小鼠时, 对照组应尽量匹配同品系小鼠以控制遗传背景变量。

MCAO模型通过血管内介入实现脑缺血诱导, 可避免开颅手术对颅骨结构的破坏, 显著降低操作创伤性, 同时诱导梗死的成功率达88%~100%^[8]。MCAO模型诱导的梗死范围包括纹状体和覆盖在其上的额顶叶和颞叶皮质及部分枕叶皮质, 深部梗死灶可累及丘脑和下丘脑^[9]。MCAO模型可分为永久性大脑中动脉闭塞(permanent MCAO, pMCAO)、短暂性大脑中动脉闭塞(transient MCAO, tMCAO) 和远端大脑中动脉闭塞(distal MCAO, dMCAO)。pMCAO模型通过持续缺血模拟不可逆性脑梗死, 形成稳定的核心梗死区, 进而导致能量衰竭、钙超载、细胞坏死等, 该模型尤其适用于抗血栓药物评价及长期神经功能恢复研究。tMCAO模型通过按预设时间窗移除线栓实现再灌注, 急性期研究通常阻断1~2 h, 观察长期行为学变化研究的阻断时间可根据实际情况调整为30、45 min或1 h, 以控制损伤程度。tMCAO模型可特征性地形成核心梗死区和可逆性半暗带, 通常适用于研究溶栓治疗时间窗、神经保护剂和再灌注损伤机制等。dMCAO模型通过选择性阻断大脑中动脉远端分支, 诱导局灶性皮层梗死, 其损伤范围较小, 死亡率明显低于近端MCAO模型。dMCAO模型的梗死区域局限于皮层, 较少累及基底节, 主要适用于皮层功能重塑研究、小分子药物筛选及长期神经行为学评估。三者详细的区别、应用及优缺点见表1, 同时使用2, 3, 5-三苯基氯化四唑(2, 3, 5-triphenyltetrazolium chloride, TTC) 染色的大脑切片见图2。

MCAO模型中, 纹状体由于豆纹动脉(终末动脉) 缺乏侧支循环而首先缺血, 覆盖其上的皮质区则因软脑膜侧支代偿, 梗死进展相对延迟。再灌注后120 min内, 皮质脑血流量(cerebral blood flow, CBF) 可恢复至基线, 而纹状体CBF仍持续低灌注。在小鼠中, 梗死涉及大脑半球的大部分(大部分皮质、纹状体、丘脑、海马和室下区), 与大鼠进行MCAO后的梗死相似。小鼠对于缺血性阻塞持续时间非常敏感, 有文献^[10]报道称, 15和30 min MCAO之间的差异导致梗死体积增加5倍(9 ± 2 mm³ vs 56 ± 6 mm³)。MCAO模型的优势在于高度模拟人类卒中临床特征, 且缺血时间及范围可控, 适用于药效评价。然而, 其局限性亦不容忽视, 手术难度高, 尤其是小鼠模型易因血管损伤导致蛛网膜下腔出血; 啮齿类动物与人类脑侧支循环存在差异, 人类大脑前动脉近端汇合成单一正中动脉。而啮齿类动物翼腭动脉可提供颅外侧支连接, 且其脑侧支循环更丰富, 使缺血模型缺血程度较轻, 低估神经保护剂疗效。

1.2　光化学法诱导脑血栓模型

光化学法诱导脑血栓模型是一种广泛应用于啮齿类动物的局灶性脑缺血模型, 通过光敏剂介导的血管内皮损伤实现精准血栓诱导。

首先, 将光敏染料通过静脉注射到动物体内。然后, 使用特定波长的光照射大脑, 光照激活染料, 产生单线态氧和超氧化物, 这些自由基会损伤血管内皮细胞, 导致血小板活化、聚集, 最终形成血栓。血栓的形成导致照射区域(通常是大脑皮层) 的血管阻塞, 从而引起缺血性细胞死亡, 最终形成脑梗死^[11]。动物实验前禁食6~8 h (仅提供水)。实验操作过程中, 一般采用戊巴比妥钠(50~60 mg·kg^-1) 腹腔注射或异氟烷吸入麻醉动物, 确保手术过程中动物无痛感及生理指标稳定。光敏剂常用孟加拉玫瑰红, 其激发波长范围为530~560 nm。静脉注射需按物种调整剂量: 大鼠推荐20~40 mg·kg^-1 (溶解于生理盐水, 浓度1%~2%), 小鼠为15~20 mg·kg^-1, 均经尾静脉给药。通过颅骨开窗暴露大脑中动脉, 采用YAG激光(355 nm波长) 或氙灯(540 nm波长) 照射, 光斑直径约2 mm, 持续10~20 min, 注射后需避光处理, 防止非靶区光激活^[12]。为防止灯光热度, 在非照射的其他部位采取冷却措施, 如用白色反光物品遮盖住非照射部位。其栓塞程度(大小或位置) 取决于照射光的波长、强度及照射时间。C57BL/6小鼠对光化学损伤耐受性较高, 而SD大鼠模型成功率可达90%。该模型可控性强, 可通过选择特定的大脑区域进行照射, 从而精确控制梗死灶的位置和大小; 操作简单, 梗死灶的大小和位置具有高度可重复性。相比其他脑缺血模型, 该模型的死亡率较低, 但其生理病理特征和梗死灶的分布与人类脑卒中存在差异: 该模型血栓形成集中于照射核心区, 周围区域缺乏典型半暗带结构; 缺血区域血脑屏障的破坏速度超过人类, 导致细胞毒性水肿和血管源性水肿几乎同时发生; 梗死灶局限于皮层, 而人类常见梗死部位累及大脑中动脉供血区域。

1.3　血栓栓塞模型

血栓栓塞模型是常用的造模方法之一, 其通过模拟临床血栓形成或栓塞过程, 诱导局部脑组织缺血损伤。通过向脑动脉(通常经颈内动脉或大脑中动脉) 注射微栓子悬液、凝血酶, 诱导血栓形成, 从而诱发脑血管阻塞。实验方法首先是暴露颈总动脉, 结扎远端, 近端插入导管至颈内动脉分叉处, 用于注射栓子或凝血酶, 常用明胶海绵颗粒(直径50~200 μm)、聚苯乙烯微球或自体血凝块。将明胶海绵剪碎后过筛, 悬浮于生理盐水或含造影剂的溶液中(浓度约每毫升1×10⁵颗粒)。或通过颈动脉注射凝血酶(100 U·kg^-1) 联合氯化铁局部涂抹, 激活内源性凝血途径形成血栓。注射方法分为单次注射及分次注射, 单次注射是缓慢推注微栓子悬液(大鼠0.2~0.3 mL), 避免过快导致栓子反流至颈外动脉; 分次注射适用于模拟多发性栓塞, 间隔10~15 min分次注入, 实时监测CBF变化^[13]。此外, 还可使用自体血栓, 取动物静脉血注入旋转的圆环装置, 模拟血流剪切力。通过旋转(17.5 r·min^-1) 促进血小板聚集和纤维蛋白形成, 生成体外血栓。经颈外动脉插管将血栓注入颈内动脉, 最终阻塞大脑中动脉形成栓塞模型。此模型能更接近地模拟人类脑卒中^[14]。其中, 兔子栓塞卒中模型造模需选16~20周龄、3.0~3.7 kg雄性新西兰白兔, 术前2天取耳缘静脉血, 与凝血酶混合注入导管形成4 mm × 1.4 mm自体血栓。术时先镇静麻醉, 监测生理参数, 于右侧股动脉切口插导管至颈内动脉远端, 注入血栓阻塞大脑中动脉, 造影确认阻塞。48 h通过磁共振成像(magnetic resonance imaging, MRI) 验证梗死与血脑屏障情况, 7天TTC染色量化梗死^[15]。该模型的特点是不需要实施开颅手术, 手术时间短, 仅需一次麻醉。其核心优势在于诱导的梗死灶具有高度可重复性, 且死亡率显著降低^[16]。

1.4　自发性卒中模型

自发性卒中模型是指动物在无外界直接干预(手术或药物诱导) 的情况下, 因遗传缺陷、慢性病理状态或衰老等因素自然发生脑血管事件的模型。这类模型在模拟人类卒中自然病理过程、解析遗传易感性及慢性血管病变机制方面具有独特优势。高血压是人类脑卒中的首要可干预风险因素, 利用高血压动物模型开展卒中研究可更准确模拟临床病理生理过程, 增强研究结果的临床转化价值。最常用的高血压动物模型是易卒中自发性高血压大鼠(spontaneous hypertensive rat-stroke prone strain, SHR-SP), 在6周大时喂食高盐饮食的情况下就会发展成高血压, 并在12周时迅速恶化成恶性高血压(> 240 mmHg), 伴随脑血管内皮功能障碍、血管壁增厚及纤维素样坏死, 最终导致自发性脑出血或脑梗死。到大约20周时, 在动物前额叶、顶叶和枕叶区域发生皮质卒中和大出血^[17]。典型病变包括基底节区出血、皮质微梗死及白质损伤, 与人类高血压性脑卒中的病理表现高度相似^[18]。该模型常用于研究慢性高血压对脑血管的影响、血脑屏障破坏机制及抗高血压药物的疗效评估。此外, 还有其他自发卒中模型, 如Ⅳ型胶原蛋白α1链(collagen type Ⅳ alpha 1 chain, Col4a1) 基因突变小鼠模型。Col4a1基因突变可导致脑血管基底膜结构异常, 引发自发性脑出血或微血管病变, 突变小鼠表现为血管壁变薄、微动脉瘤形成及自发性脑出血, 尤其在应激(高血压) 下更易发生。该转基因小鼠模型适用于研究遗传性脑血管病及小血管病相关的卒中机制^[19]。

1.5　其他模型

除前述模型外, 缺血性卒中动物模型还包括: ①内皮素-1化学闭塞模型, 内皮素-1是一种具有强效和持久血管收缩作用的肽类物质。通过立体定位向动物目标脑区微量注射内皮素-1, 引起血管收缩, 导致血流减少, 进而引起下游缺血, 诱导可控性局灶缺血^[20]; ②微线圈法, 是经血管介入将微小的线圈放置在大脑中动脉周围, 造成物理性血管阻塞; ③全脑缺血模型, 涉及动物全脑血液供应的阻断, 包括双血管闭塞/低压模型和四血管闭塞模型, 四血管闭塞模型是同时阻塞大鼠的两侧颈总动脉和椎动脉, 双血管闭塞/低压模型是通过结扎双侧颈总动脉, 同时降低平均动脉压至50 mmHg。该类模型主要用于研究全脑缺血的病理生理机制。上述IS动物模型多采用啮齿类动物造模, 因其在脑血管解剖、缺血级联反应及神经血管单元病理机制方面与人类有相似之处, 同时成本较低, 易于操作^[7]。此外, 多种非啮齿类动物可用于缺血性卒中药物的药效评价。犬的大脑血管解剖结构与人相似, 可进行脑血管造影、血栓切除术等研究。但犬的颅内血管迂曲, 增加了手术难度和并发症风险。猪的大脑血管结构也与人类相似, 可构建各种卒中模型, 包括血栓栓塞、血管内导管闭塞等。但猪存在网状丛结构, 限制了血管内导管闭塞的应用。羊的大脑血管结构简单, 可进行各种卒中模型的构建, 包括血栓栓塞、血管内导管闭塞等。但羊的大脑相对较小, 限制了某些研究的应用。非人灵长类(non-human primates, NHPs) 如狨猴、恒河猴的脑血管解剖和功能与人类高度相似, 是研究复杂脑卒中机制的理想模型^[21]。老年狨猴是一种自发性卒中模型, 随年龄增长出现脑淀粉样血管病和微出血, 可模拟人类老年性卒中^[22]; 或可通过开颅手术或血管内介入法阻断大脑中动脉血流, 或注入自体血栓或微球(直径300~500 μm) 以阻塞大脑中动脉, 建立狨猴缺血性卒中模型。NHP模型虽在神经功能长期评估中具有不可替代性, 但其高昂的成本、严苛的饲养条件及复杂的伦理审查流程, 限制了大规模应用^[23]。选择非啮齿动物缺血性卒中模型需要综合考虑研究目的、实验技术、病理解剖相似性和资源可行性等因素^[24], 如研究血管再通技术可选犬或NHP模型; 研究缺血性卒中的病理生理机制可选猪或羊模型^[25]; 操作层面血管内介入可选犬或NHP (血管迂曲度低) 模型, 开颅手术则可选猪或羊(颅骨厚度可调) 模型。

2　IS的细胞模型

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体外缺氧/复氧(hypoxia/reoxygenation, H/R) 模型是模拟机体组织器官在缺血脑卒中的经典实验模型, 主要包括物理缺氧法或化学缺氧法, 物理缺氧法最常用的是氧糖剥夺, 将正常培养基更换为低糖/无糖培养基, 将纯N₂或者氩气等惰性气体与CO₂混合后充入密闭装置中形成一个密闭的缺氧环境, 以模拟脑缺血时神经细胞所在的物理微环境, 更能接近实际IS状态。化学缺氧法可用氯化钴, Co²⁺进入细胞并置换催化酶上的Fe²⁺导致酶失效, 使细胞内活性氧(reactive oxygen species, ROS) 增多、缺氧诱导因子-1α激活、核转录因子及信号蛋白改变, 导致细胞周期停滞和细胞凋亡。还可用速效神经毒剂氰化钠和氰化钾, 抑制细胞色素C氧化酶对电子传递链的作用, 阻断线粒体呼吸导致线粒体ROS产生和细胞毒性增加, 最终引发细胞功能障碍。此外, 使用过氧化氢和谷氨酸也可模拟缺血后的损伤环境, 过氧化氢易于穿过细胞膜, 短期内引发细胞产生更多ROS。ROS在细胞内积聚后迅速引起细胞内物质过氧化, 导致膜通透性增加、细胞功能紊乱甚至死亡。细胞外谷氨酸浓度过高会导致神经细胞持续去极化, 钙离子大量涌入细胞, ROS增多, 最终引起神经细胞死亡。缺氧结束后可更换为正常培养基进行复氧以模拟IS后的再通微环境。

在IS的传统动物模型中, 因物种差异难以准确模拟人脑病理变化, 而二维细胞模型则无法再现三维微环境的结构功能特征。近年来, 脑类器官三维模型技术迅速发展, 脑类器官是由人类多能干细胞(胚胎干细胞或诱导多能干细胞) 通过定向分化发育而成的三维组织结构, 其遗传背景、细胞类型及药物反应均能更真实地反映人脑特性。脑类器官自发形成类似于人脑发育早期的分层结构, 包含神经元、星形胶质细胞、少突胶质细胞等多种细胞类型, 并建立功能性神经网络, 这种复杂三维结构能更真实地模拟缺血缺氧条件下细胞间相互作用及微环境变化。将源自人多能干细胞的人类脑类器官移植到卒中小鼠的梗死核心和梗死周围区域的交界处, 几个月后发现移植的类器官在梗死核心存活良好, 分化为靶神经元, 修复梗死组织, 消除了卒中小鼠的感觉运动缺陷行为^[26]。有研究者从成体真皮成纤维细胞来源的神经干细胞生成自组织的3D人类神经类器官。这些人类神经类器官中的放射状神经胶质细胞表现出人类大脑皮层趋势的特征, 包括内层(心室区) 和外层(早期和晚期皮质区)。同时, 研究者对类器官进行缺氧损伤以研究神经元损伤和再生, 发现复氧恢复了神经元细胞增殖^[27]。目前, 主要是基于人多能干细胞构建类器官, 包括胚胎干细胞或诱导多能干细胞, 将人多能干细胞解聚为单细胞, 在低吸附孔板中离心聚集(约9 000~11 000个细胞/孔), 加入Rho相关卷曲螺旋蛋白激酶抑制剂(Y-27632) 抑制凋亡, 24 h内形成胚状体。通过阻断骨形态发生蛋白/转化生长因子-β信号联合Wnt激活剂, 诱导神经外胚层分化形成神经玫瑰花结, 模拟神经管发育。再使用3D基质包埋, 胚状体转移至Matrigel液滴中, 提供细胞外基质支持, 促进神经上皮极化和腔室形成^[28]。

3　IS动物模型药效学评价指标

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IS动物模型的药效学评价需覆盖全病程阶段, 涵盖急性期(0~72 h)、亚急性期(3~14 d) 和恢复期(> 14 d) 等多个阶段。在急性期, 可采用Bederson评分和改良神经功能缺损评分(modified neurological severity score, mNSS) 快速判断神经功能损伤等级, 结合TTC染色或MRI测定梗死体积, 同时通过干湿重法检测脑组织含水量评估脑水肿程度, 可全面反映药物对急性缺血损伤的抑制作用。亚急性期: 通过mNSS评分评估综合神经功能恢复趋势, 借助转棒测试、爬杆测试评价运动协调能力改善情况, 明确药物对神经功能恢复的作用。恢复期: 采用圆筒测试、黏合剂去除测试评估肢体使用对称性及感觉运动整合能力, 揭示药物对神经重塑的促进机制。以下将详细综述IS动物模型药效学评价指标(表2) 及示意图(图3), 这些指标对药效学整体评价至关重要。

3.1　神经功能缺损评分

Bederson评分通过4项标准化行为测试评估大鼠急性缺血性卒中中的神经功能缺损: 提尾实验、身体对称性、前肢屈曲和旋转行为。评分采用0~4分区间量表(0 =无缺损, 4 =严重障碍), 分数越高, 表示神经功能障碍越严重。

Zea-Longa评分包括对动物的行为和运动能力的观察, 如提尾实验、前肢屈曲、身体侧偏和自主运动。通常采用5级量表(0~4分), 从0 (无缺陷) 到4 (严重缺陷或死亡), 可用于评估大鼠急性缺血性运动功能障碍。

mNSS是一种综合性的神经功能评估方法, 广泛应用于大鼠脑损伤模型。包括运动/感觉/反射/平衡四大维度11项测试, 如本体感觉、放置反应、抓握力、反射、平衡和协调能力。mNSS的总分为0~18分, 分数越高, 表示神经功能损伤越严重。上述神经功能评分系统是IS动物模型的评价核心, 通过对神经功能缺损的定量化, 为药物的非临床药效学评价提供金标准。每种评分系统都有其特定的应用模型和应用场景, 因此在选择时应考虑实验的具体需求和目标。

Garcia神经行为学量表是一种标准化的啮齿类动物神经功能评估工具, 尤其适用于小鼠IS模型的急性期神经功能损伤的评价。Garcia评分包含6项行为学测试, 用于定量分析运动、感觉和反射缺陷, 每项评分范围0~3分(3分表示功能正常), 总分最高18分表征神经功能完整。评分与神经功能缺损程度呈显著负相关, 评分越高说明神经功能损伤越轻, 详细评价指标见表2。Garcia评分不需要对动物进行预训练, 但需要进行环境适应: 实验前3天将小鼠置于测试间, 每日30 min熟悉实验者气味及操作台。正式测试在隔音避光条件下, 使用标准化开放场装置(30 cm × 30 cm × 35 cm), 确保实验期间仅单一操作者在场以排除干扰。

3.2　运动协调与平衡评价指标

3.2.1　转棒测试

转棒测试(rotarod test) 是一种常用于评估啮齿类动物运动功能和共济失调的测试方法, 通过测量动物在旋转杆上的潜伏时间(从放置到跌落) 来评估其运动能力。对于小鼠, 转棒通常由一个直径为3.0~3.2 cm的旋转圆柱和一个带有多个隔间的附件组成, 隔间之间由薄壁隔开, 可同时测试多只小鼠。在进行正式测试前, 需对小鼠进行训练, 以使其适应旋转的杆。训练通常需要2~3天时间, 每天进行2~3轮测试, 训练目的是让小鼠学会在旋转的杆上保持平衡, 并减少其跌落次数。将小鼠放置在旋转的杆上, 并记录其跌落潜伏期。跌落潜伏期越长, 说明小鼠的运动功能和共济失调程度越好。转棒测试的优势在于操作标准化程度高, 易于实施; 可重复性好, 结果可靠; 对多种卒中模型敏感。但缺点是预训练需要时间, 小鼠的学习能力会影响测试结果, 无法区分不同类型神经损伤导致的运动功能障碍, 且对单纯感觉缺陷不敏感。

3.2.2　悬线测试

悬线测试(wire hanging test) 是一种简单且有效的行为学测试方法, 常用于评估动物在IS后皮质脊髓束介导的神经肌肉功能异常。该方法通过检测动物在金属丝上悬挂的时间来评估其握力、平衡性和耐力。悬线测试要求动物用前爪抓住一根水平金属丝, 保持身体悬挂在空中, 直到无法继续支撑体重为止。悬线测试通过记录动物掉落前的潜伏期, 评估其运动功能和神经肌肉功能。在IS研究中, 脑卒中小鼠在悬线测试中的潜伏期显著缩短, 表明运动皮层-皮质脊髓束通路介导的抓握力与肌耐力受到了影响^[29]。该测试被广泛用于评估动物在脑卒中后的运动功能恢复和肌力改善情况, 以及评估不同治疗药物在脑卒中小鼠模型中的神经保护作用。

3.2.3　爬杆测试

爬杆测试(pole test) 是一种常用的动物行为学测试方法, 通过量化动物头向下转身落地的潜伏期, 来评估IS后基底节-丘脑-皮层通路介导的运动协调性。该方法一般选择直径1~3 cm、高度50~100 cm的木杆或金属杆, 通过观察动物在爬杆过程中的协调性、速度和完成度, 来评估其运动功能的恢复情况。对于结果判定, 在IS早期, 实验动物在爬杆时往往表现出动作不协调, 转身困难, 甚至跌落, 反映了脑部损伤导致的运动功能障碍。随着康复时间的增加和治疗的介入, 动物的动作逐渐变得协调, 能顺利转身并完成爬杆任务, 表明其运动功能逐渐恢复。通过记录动物从杆顶完全转身至四爪触底的潜伏期, 定量评估其运动速度。对同一动物在不同时间点的爬杆速度进行对比, 速度的提升通常表明其上肢和下肢的力量及运动控制能力有所增强。有研究表明, 爬杆测试通常在IS亚急性期能够明显反映运动功能^[30]。

3.2.4　网格行走测试

网格行走测试(grid walk test) 通过记录啮齿类动物跨越网格时的足部失误情况, 能敏感检测IS等神经系统疾病导致的感觉运动协调障碍。在实验中, 通常使用高架金属方形网格(41 cm × 41 cm, 每个网格单元3.5 cm × 3.5 cm, 高度40 cm)。网格装置位于光线昏暗的隔音室中, 每次实验后用70%乙醇清洗装置。实验过程中, 通过摄像机记录小鼠在网格上的行为, 随后采用盲法让其他实验者进行分析。数据分析主要包括前爪步总数、平均前爪步距等指标^[31]。

3.3　肢体使用对称性和探索行为评价指标

3.3.1　圆筒测试

圆筒测试(cylinder test) 也称为自发前肢使用不对称测试, 通过观察在受限圆筒里的自发探索行为, 评估小鼠IS后的前肢使用不对称性。此方法操作简便, 但观测期较长, 可类比于某些卒中患者可能出现的肢体失用症。当大脑的特定区域(感觉运动皮层、皮质脊髓束和纹状体) 受损时, 可能会导致慢性前肢使用不对称性。圆筒测试通过观察小鼠在垂直探索圆柱时前肢的使用情况来评估这种不对称性^[32]。实验使用一个直径为9~10 cm、高度为15 cm的玻璃圆柱, 将小鼠放入圆柱中, 记录小鼠在垂直探索圆柱时前肢的接触次数, 可使用镜子或摄像头来确保观察者或相机能够始终看到两只前肢。该测试的不对称评分计算公式最初由Schallert等^[32]基于大鼠实验建立, 具体为(非受损前肢接触次数-受损前肢接触次数) / (非受损前肢接触次数+受损前肢接触次数+双侧接触次数)。

3.3.2　前肢放置测试

前肢放置测试(forelimb placing test) 通过观察动物在特定任务中的行为表现, 评估其运动功能的损伤程度和恢复情况。该测试主要用于评估大脑皮层和皮层下结构损伤导致的运动功能障碍。在IS模型中, 前肢放置测试可有效评估运动功能的损伤程度。在大鼠MCAO模型中, 研究人员发现脑卒中后大鼠的前肢放置能力明显下降。通过对康复治疗前后动物的前肢放置行为进行比较, 评估康复治疗的效果。还可通过分析前肢放置行为的变化, 探讨IS后运动功能恢复的神经生物学机制。

3.3.3　转角测试

转角测试(corner turn test) 是一种用于评估脑卒中后小鼠感觉运动功能恢复情况的行为学测试^[33]。该实验通过观察小鼠在特定环境中转身的行为, 来评估其运动、感觉和姿势功能是否存在不对称性。实验装置由两个相连的硬纸板墙组成, 形成一个30度的角, 在墙的连接处留有一个小开口, 以吸引小鼠进入角落。将小鼠放置在装置的中间, 面向角落, 通常会进入角落, 此时其触须受到刺激, 小鼠会抬头并转向一侧(左侧或右侧), 记录小鼠的转向行为。在假手术组中没有观察到侧向偏好, 但脑卒中模型小鼠则更倾向于转向非损伤侧(同侧)。在皮质损伤的小鼠中, 小鼠则更倾向于转向损伤侧(对侧)。该测试可用于检测亚急性期和恢复期的运动功能情况, 且对于损伤严重的模型, 该实验更为敏感^[34]。

3.3.4　黏合剂去除测试

黏合剂去除测试(the adhesive removal test) 是一种用于评估脑卒中后小鼠感觉运动功能的行为学测试。该实验通过观察小鼠接触和移除黏合剂的时间, 来评估其前肢运动和协调功能是否存在缺陷, 通常用于评估单侧感觉运动皮层、皮质脊髓束和纹状体损伤后的感觉运动损伤^[35]。黏合剂贴片的大小和形状可以是矩形(0.3 cm × 0.4 cm) 或圆形(直径0.3 cm)。将小鼠放入透明塑料盒中, 让其自由探索并适应环境。记录小鼠接触和移除每只前爪上黏合剂贴片的时间。通常情况下, 小鼠会先接触并尝试移除非损伤侧(同侧) 前爪上的黏合剂贴片, 然后才会接触并移除损伤侧(对侧) 前爪上的黏合剂贴片。脑卒中后, 小鼠由于前肢运动和协调功能受损, 移除损伤侧前爪上黏合剂贴片的时间会更长。

3.4　脑梗死体积评价

在IS动物模型中, 脑梗死体积的评价方法包括传统的染色技术(TTC和甲苯胺蓝染色) 和现代成像技术(磁共振成像)。

TTC染色是一种评估脑梗死体积的常用染色方法。该方法利用活细胞线粒体脱氢酶将TTC还原为红色甲臜, 使存活组织呈红色, 而酶活性缺失的梗死组织不着色, 从而区分梗死区与存活组织, 并可量化脑梗死体积。将脑组织切片置于1%~2% TTC溶液中, 37 ℃避光孵育30 min, 每侧染15 min, 按照顺序摆片, 拍照留存。与TTC染色类似^[36], 甲苯胺蓝染色是另一种用于脑梗死体积的传统方法。这种方法通过特异性染色神经元胞质内的尼氏体, 使正常神经元和胶质细胞显色, 而梗死区神经元因结构崩解不着色, 从而量化梗死区域。

MRI是一种非侵入性成像技术, 可精确评估IS后的脑梗死体积。其高分辨率图像能提供关于神经元损伤的详细时空信息, 对理解病情和指导研究/治疗策略至关重要。CT扫描是评估急性脑卒中的首选影像学方法, 尤其能有效识别出血性转化^[37]。CT扫描可显示脑梗死区域的水肿和低密度改变, 对早期缺血性损伤的敏感性较低, 但能有效识别出血性转化^[36]。MRI在检测脑梗死早期阶段的敏感性优于CT。其多模态成像能力可提供包括T1加权、T2加权和弥散加权成像(diffusion weighted imaging, DWI) 在内的详细脑组织结构信息, 其中DWI对早期缺血性损伤具有高度特异性。DWI能够检测到缺血后细胞毒性水肿引起的水分子扩散受限(表现为表观扩散系数降低), 是诊断早期脑梗死的关键标准^[38]。灌注加权成像(perfusion weighted imaging, PWI) 可定量评估脑组织的血流动力学状态, 通过检测脑血流量和脑血容量等参数变化, 准确识别缺血半暗带, 即虽然灌注不足但尚未发生不可逆损伤的脑组织区域。这一区域是临床治疗干预的关键目标。功能性磁共振成像可精准评估脑卒中对大脑功能的影响。通过解析任务态下脑区激活模式与认知行为表现的关联性, 能帮助研究脑卒中后认知功能障碍的神经机制。磁共振光谱成像能提供脑组织代谢的详细信息, 通过检测N-乙酰天门冬氨酸(神经元完整性标志物)、胆碱(细胞膜代谢标志物) 和乳酸(无氧代谢产物) 等特定代谢物的谱峰变化, 精确评估神经元损伤和脑组织代谢状态。正电子发射断层扫描是一种分子影像学技术, 通过检测不同的放射性示踪剂在脑内的分布和代谢动力学, 可定量评估脑部代谢状态和神经元活动。正电子发射断层扫描可提供关于脑部葡萄糖代谢、氧消耗和血流量等信息^[39]。

3.5　脑水肿评价指标

在脑卒中模型中, 脑水肿指标对于理解疾病进展、评估治疗效果及预后具有重要意义。CT是临床评估脑水肿的常规影像学手段。通过测量脑室系统压缩程度、脑实质密度变化等参数, 评估脑水肿的严重程度。MRI在评估脑水肿方面具有较高的敏感性和特异性。常用的序列包括T1WI、T2WI、FLAIR和DWI, 通过综合分析不同序列的特征性表现, 计算病变区域的体积和信号强度, 评估脑水肿的程度和病理类型。此外, 还可通过测定脑组织含水量评价脑水肿的程度, 常用的方法有干湿重法、冷冻干燥法等。在脑水肿的病理进程中, 肿瘤坏死因子-α、白细胞介素-1β等促炎因子通过诱导神经炎症反应破坏血脑屏障, 水通道蛋白4通过调控细胞内外水分子转运, 共同促进脑水肿的发生发展。定量测定这些细胞因子的水平有助于评价脑水肿的程度, 为相关分子机制研究提供依据。

3.6　血脑屏障损伤评价指标

在动物模型研究中, 血脑屏障(blood-brain barrier, BBB) 功能的评价依赖于多种互补性指标, 这些指标共同揭示了BBB在生理和病理条件下的结构完整性和功能状态。屏障通透性是评价BBB完整性的最直接指标, 反映了物质从血液循环进入脑实质的能力。在动物模型中, 主要通过外源性示踪剂的脑内渗透量和清除率来量化评估。也可通过伊文思蓝示踪技术评估血脑屏障的通透性, 该染料静脉注射后与血浆白蛋白结合, 在血脑屏障开放区域外渗至脑实质, 通过分光光度法测定脑组织中的染料含量, 可反映血脑屏障的受损程度。除伊文思蓝外, 不同分子量(NaF、^99mTc-DTPA、¹⁴C-蔗糖) 的标记物被用于评估不同孔径的BBB开放程度, 通过测定脑-血浆比率或脑组织放射活性, 可计算渗透系数或脑摄取指数。此外, 紧密连接蛋白、基质金属蛋白酶、转运蛋白等在脑组织中的表达量和亚细胞定位是构成BBB物理屏障的核心分子组件。

4　动物模型选择及药效学评价原则

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4.1　动物模型选择的考虑因素

虽然IS动物模型在研究脑缺血性卒中的病理生理机制和药物研发方面具有重要价值, 但现有模型仍存在显著的种属差异和病理生理学局限性, 制约了其临床转化潜力及对人类IS病理过程的完全模拟。小鼠和人类在许多基因的表达谱存在差异, 这种转录组层面的种属差异直接导致二者炎症刺激的反应也不同^[40]。此外, 啮齿类动物的脑白质含量仅为10%^[41], 而人类大脑白质比例高达50%^[42], 而脑白质损伤在人类脑缺血性卒中预后中起着重要作用^[43], 这种结构性差异导致啮齿类动物模型难以完全模拟人类缺血性卒中后白质束损伤对预后的关键影响。啮齿类动物的大脑侧支循环也较差, 而人类大脑具有丰富的侧支循环, 可部分代偿缺血区域的血流, 从而影响梗死灶的大小和预后。在免疫系统方面, 小鼠和人类白细胞的比例不同, 且白细胞在脑缺血性卒中的作用也存在差异^[44]。最重要的一点是传统模型(线栓法MCAO模型) 多基于年轻健康动物, 难以模拟临床患者常伴发的代谢紊乱(糖尿病、高血压)。老龄(> 18月龄) SD大鼠联合高脂饮食诱导的胰岛素抵抗模型, 可更真实反映卒中后血脑屏障渗漏加剧、神经炎症持续等病理特征。在卒中临床亚型对应动物模型选择方面, 大动脉粥样硬化型IS临床以大脑中动脉、颈内动脉等大动脉粥样硬化狭窄或闭塞为主要特征, 可选用大鼠/小鼠pMCAO模型, 模拟大动脉持续闭塞导致的永久性缺血, 其梗死范围累及纹状体及皮质, 与临床大动脉粥样硬化型IS的梗死部位及病理特征高度契合。心源性栓塞型IS临床多由心房颤动等心源性疾病导致血栓脱落栓塞脑血管引起, 首选大鼠/小鼠血栓栓塞模型(注射自体血凝块或微栓子), 该模型通过向颈内动脉注射自体血栓, 模拟血栓随血流栓塞大脑中动脉的过程, 梗死灶具有随机性且易出现多发性梗死, 与心源性栓塞型IS的临床特点一致, 可用于评价溶栓药物及抗栓药物的疗效。小动脉闭塞型(腔隙性) IS主要因脑内小动脉(豆纹动脉) 闭塞导致, 推荐采用小鼠dMCAO模型或SHR-SP大鼠自发性小梗死模型。该模型通过阻断大脑中动脉远端分支, 诱导局限于皮质的小梗死灶, 类似临床腔隙性梗死的病灶特点, SHR-SP大鼠在高血压基础上易出现脑内小动脉纤维素样坏死, 自发形成皮质微梗死及白质损伤, 可用于研究小动脉闭塞型IS的病理机制及抗高血压联合脑保护药物的评价。此外, 人类卒中流行病学研究显示, 绝经后女性因雌激素水平下降导致卒中风险较同龄男性升高15%~20%, 且对治疗反应存在性别差异(女性对rt-PA溶栓的出血转化率更高)。因此, 动物实验设计需综合考虑, 如使用性成熟雌、雄双性别动物, 明确雌性动物动情周期阶段(因其影响血脑屏障通透性) 等。忽略性别因素可能导致神经保护剂的疗效评估偏差, 美国国立卫生研究院自2016年起要求资助的临床前研究需纳入雌雄两性动物(除非有明确理由仅用单一性别), 以避免性别偏见并提高研究准确性。总之, IS模型的共同问题在于其与人类脑缺血性卒中的相似性有限, 这可能会影响实验结果的可靠性和可重复性。因此, 在选择IS动物模型时需要谨慎考虑这些共同问题。

4.2　模型动物纳入/排除标准

在IS动物模型研究中, 啮齿类动物常用品系包括SD大鼠、C57BL/6小鼠等, 需明确动物品系来源及遗传背景。动物体重范围需严格控制, 如线栓法, 应根据动物体重来选择合适的线栓规格。由于实验动物存在显著的个体差异性, 即使采用标准化的造模方法, 不同个体在术后仍可能表现出脑缺血程度、梗死体积及功能损伤的明显差异。这可能导致实验组内数据离散度增大, 进而影响候选化合物药效评价的准确性。因此, 在模型制备完成后、实验分组前, 需客观地对动物进行严格筛选, 确保纳入样本的病理特征与实验目标高度匹配, 从而提升研究结果的可靠性。首先, 通过激光散斑血流成像或激光多普勒血流仪, 连续记录大脑中动脉供血区的脑血流量, 仅选择血流灌注量下降至基线水平一定值的个体, 通常为50%~70%, 具体阈值根据模型类型调整^[5]。其次, 在术后24~48 h, 通过TTC染色或MRI T2加权成像计算梗死体积, 设定体积阈值(啮齿类动物梗死体积需占同侧半球体积的20%~50%), 可剔除体积过小(< 10%) 的样本。在未经脑血流验证的动物模型中, 约30%个体实际缺血程度不符合实验标准要求。此外, 标准化筛选还能减少实验所需动物数量, 符合动物伦理的“3R原则”(减少、优化、替代)。

4.3　随机化及盲法

在IS动物模型中, 随机化是遵循随机原则分组, 避免人为或系统性误差。以上提到的行为学评价(Zea-Longa评分、转棒测试) 常依赖观察者主观判断。盲法可避免评估者对分组信息的潜在影响, 手术及给药人员不知动物分组, 避免操作差异(线栓插入深度、给药剂量偏差), 行为学评分者不知动物所属组别, 防止对“治疗组”动物评分更宽松或严格。统计人员不知分组信息, 避免选择性报告结果。盲法可显著减少假阳性/假阴性风险, 提高数据可信度^[45]。此外, 在脑卒中临床前研究向临床转化的过程中, 研究失败的原因涉及多维度、多层次的复杂因素, 主要包括以下几方面关键问题: 试验设计; 样本量设置; 终点指标的选择; 试验数据的统计及建立非临床研究的事先注册与过程监督机制; 开展多中心的随机对照非临床试验等方面可参考本实验室前期发表的文章^[46]。

4.4　识别缺血半暗带

在IS的病理机制研究中, 缺血半暗带作为核心概念具有关键研究价值。缺血半暗带是指脑组织血流减少但仍能维持能量代谢的区域, 其中的细胞尚未发生不可逆损伤, 这种介于正常灌注区与核心梗死区之间的动态过渡带, 其存在时间窗为临床干预提供了关键治疗靶点^[47]。Astrup等^[48]基于CBF的双阈值理论定义了缺血半暗带。他们在麻醉狒狒中确定了两个关键阈值: 缺血上限阈值是20 mL·100 g^-1·min^-1, 此时细胞出现电衰竭, 但能量代谢仍然持续, 细胞外钾离子浓度降低; 缺血下限阈值6 mL·100 g^-1·min^-1, 细胞外钾离子浓度升高, 同时出现电衰竭和能量衰竭。在这两个阈值之间的区域被称为“半暗带”, 恢复血流到功能阈值以上可逆转功能障碍而不会造成永久性损害。

在开展药物非临床药效学评价时, 给药后对缺血半暗带的影响需结合影像学、分子生物学、代谢和功能评估等多维度指标, 以动态监测药物干预对半暗带的保护、修复或潜在损伤作用。如PWI和DWI是两种常用的影像学技术。PWI可显示脑组织的灌注状态, 而DWI则检测水分子在组织中的布朗运动, 从而识别出梗死区域。基于DWI (显示梗死核心) 与PWI (显示低灌注区域) 的不匹配区, 如比较PWI和DWI, 可识别出DWI上没有明显梗死信号, 但PWI显示灌注不足的区域, 这通常被认为是缺血半暗带区域^[49]。此外, 生物标志物也是评价缺血半暗带的重要手段, 这些生物标志物可反映脑组织损伤的程度和修复潜力, 包括炎症相关蛋白、氧化应激标志物和细胞凋亡相关蛋白等。

因此, 缺血半暗带的发现对于IS的治疗策略具有重要意义。缺血半暗带的识别为急性IS的治疗提供了新的靶点^[50]。这一概念的提出推动了两大治疗方向的发展: 通过血管再通恢复血流(溶栓治疗) 和通过细胞保护维持神经元存活(神经保护剂)^[51]。多模态影像技术(DWI-PWI不匹配) 的应用, 使临床医生能精准识别可挽救的脑组织, 为个体化治疗决策提供科学依据。该理论至今仍是卒中治疗时间窗决策的基础依据。

5　IS相关分子机制及检测指标

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5.1　兴奋性毒性

兴奋性毒性是指过量兴奋性氨基酸(谷氨酸) 引起的神经元损伤。在缺血条件下, 能量代谢受阻, 导致谷氨酸无法正常回收, 进而过度激活谷氨酸受体, 引起钙离子大量内流, 导致细胞内钙超载, 最终引发神经元死亡^[52]。兴奋性毒性的核心机制是突触间隙谷氨酸过度释放, 导致神经元过度兴奋。通过微透析技术或脑脊液检测谷氨酸水平可评估其毒性作用。N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid, NMDA) 受体是谷氨酸的主要作用靶点, 其亚基的表达上调或磷酸化状态变化可反映受体过度激活。此外, α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体介导快速突触传递, 其亚基的异常表达与兴奋性毒性相关。还可通过荧光探针检测神经元内钙离子水平, 钙超载是神经元凋亡的关键触发因素。钙调蛋白依赖性激酶的活性增强可反映钙信号通路的异常激活, 与突触可塑性损伤相关。兴奋性毒性的检测需采用多学科交叉的研究策略: 在基础研究层面, 可通过高效液相色谱定量分析突触间隙谷氨酸浓度动态变化, 结合荧光探针(Fluo-4) 实时监测细胞内钙离子水平; 在转化医学层面, 可运用磁共振波谱无创检测脑内谷氨酸含量, 并通过扩散张量成像评估由此导致的神经纤维束损伤。这种多层次检测体系能完整揭示兴奋性毒性级联反应。

5.2　氧化应激

氧化应激是缺血再灌注损伤的重要机制之一。在缺血再灌注后, 线粒体电子传递链解耦联, 产生大量ROS, 引发脂质过氧化、DNA损伤和蛋白质失活^[53]。对于氧化应激的检测指标需涵盖氧化损伤产物和抗氧化系统的动态变化。丙二醛(malondialdehyde, MDA) 和8-异前列腺素F2α (8-isoprostaglandin F2α, 8-iso-PGF2α) 作为脂质过氧化的特异性标志物, 在IS患者血浆中显著升高, 并与神经功能缺损程度呈正相关^[54]。8-羟基脱氧鸟苷酸(8-hydroxy-2'-deoxy-guanosine, 8-OHdG) 是DNA氧化损伤的标志物, 可作为氧化应激严重程度的灵敏指标, 尤其在进展性脑梗死患者中, 其水平在急性期持续升高^[55]。谷胱甘肽过氧化物酶(glutathione peroxidase, GPx) 是清除过氧化氢的关键酶, 其活性在卒中患者中显著降低, 与氧化应激程度呈负相关。研究显示, 缺血性卒中患者的GPx水平高于出血性卒中, 可能反映不同病理机制下的抗氧化能力差异。超氧化物歧化酶(superoxide dismutase, SOD) 是清除超氧自由基的主要酶类, 卒中后脑组织中SOD活性降低。总之, IS氧化应激的检测需综合脂质过氧化、DNA损伤、抗氧化酶活性等指标^[56]。

5.3　神经炎症和免疫

神经炎症是中枢神经系统对损伤或疾病的免疫反应, 其机制涉及多种细胞类型和分子途径。小胶质细胞是中枢神经系统中的主要免疫细胞, 在神经炎症中起关键作用。神经炎症的相关检测指标需涵盖炎症细胞活化、促炎/抗炎因子释放、氧化应激及信号通路激活等多个层面。肿瘤坏死因子α (tumor necrosis factor α, TNF-α) 是中枢神经系统中促炎反应的核心促炎因子, 缺血后由小胶质细胞和星形胶质细胞释放, 促进血脑屏障破坏和神经元凋亡。老年IS患者血清TNF-α水平显著升高, 并与神经功能缺损评分正相关。白细胞介素1β (interleukin-1β, IL-1β) 通过激活核因子κB (nuclear factor kappa-B, NF-κB) 通路加剧炎症反应, 而白细胞介素6 (interleukin-6, IL-6) 参与急性期炎症调控。两者在IS患者血清和脑脊液中均显著升高, 且与梗死体积相关^[57]。缺血后小胶质细胞向促炎表型(M1) 或抗炎表型(M2) 极化, M1型标志物和M2型标志物的比值可反映炎症平衡状态^[58]。中性粒细胞通过释放中性粒细胞胞外诱捕网促进血栓形成和炎症扩散, 其DNA水平在IS患者外周血中升高, 与梗死面积和死亡率相关。调节性T细胞(regulatory T cells, Tregs) 通过分泌白细胞介素10 (interleukin-10, IL-10) 抑制炎症, 促进神经修复。Treg细胞数量减少与IS预后不良相关^[59]。

5.4　细胞凋亡

细胞凋亡即程序性细胞死亡, 是细胞在特定信号诱导下发生的一种有序的死亡过程, 对维持组织稳态和清除受损细胞至关重要。细胞凋亡的主要机制包括线粒体途径和内质网应激途径。线粒体途径是线粒体膜电位的丧失会导致细胞色素C的释放。细胞色素C进一步激活下游的含半胱氨酸的天冬氨酸蛋白水解酶3 (cysteinyl aspartate specific proteinase 3, Caspase-3), 引发细胞凋亡。Caspase家族是执行凋亡的关键酶类, 其中Caspase-3的激活是凋亡过程中的关键步骤。对于细胞凋亡的相关指标检测, 首先是形态学指标, 可通过光学显微镜或电子显微镜观察: 核固缩(染色质致密化)、核碎裂(染色质颗粒状分解) 及凋亡小体形成(膜包被囊泡), 以及进一步的核孔解聚、线粒体空泡化等超微结构改变。使用Annexin-V结合磷脂酰丝氨酸的荧光探针(Annexin-V-FITC) 可检测早期凋亡细胞, 同时联合核酸荧光染料碘化丙啶区分晚期凋亡或坏死细胞。其次是线粒体功能相关指标, 可通过荧光探针精准检测, 其中膜电位的下降可反映线粒体功能损伤。通过Western blot或免疫荧光检测胞质中细胞色素C水平升高, 提示线粒体膜通透性改变。此外, Caspase-3/7活性检测(荧光底物法) 是凋亡执行阶段的关键指标。TUNEL染色可以标记DNA断裂的3'-OH末端, 特异性识别凋亡细胞。彗星实验常用于检测DNA片段化程度。

5.5　血小板聚集

在IS机制研究中评价血小板聚集机制需结合实验室检测指标与影像学评估。可通过光学比浊法或阻抗法测定血小板对诱导剂(花生四烯酸、ADP和胶原) 的反应, 评估血小板聚集能力。通过检测凝血反应时间、最大振幅等, 综合评估血小板功能及纤维蛋白形成动态。流式细胞术检测血小板表面标志物。P-选择素升高反映血小板活化状态, 血小板糖蛋白是血小板聚集的最终通路, 可作为药物靶点评估指标。免疫比浊法或ELISA法检测D-二聚体, 它是纤维蛋白降解产物, 其水平升高提示血栓形成或溶解过程。纤维蛋白原水平 > 3.5 g·L^-1与血栓形成风险增加相关, 是评估高凝状态的重要指标。检测凝血酶原时间(prothrombin-time, PT) 与活化部分凝血活酶时间(activated partial thromboplastin time, APTT), PT延长提示外源性凝血途径异常, APTT延长提示内源性凝血途径异常, 联合分析可指导抗凝治疗。

6　我国IS药物研究现状

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近年来, 我国在抗血小板治疗、溶栓药物研发、脑保护剂创新及中医药现代化等领域取得显著突破, 形成了具有中国特色的卒中防治体系。

目前, 我国针对急性IS已上市的自主创新溶栓药物有注射用替奈普酶(tenecteplase, TNK), 这是一种通过基因工程技术改良的重组人组织型纤溶酶原激活剂改构体。其作用机制是通过激活纤溶酶原转化为纤溶酶, 溶解血栓。相比传统阿替普酶, 其半衰期延长可至20 min, 溶栓时间窗可扩展至24 h, 出血风险更低。尿激酶原直接激活纤溶酶原, 溶解纤维蛋白血栓。此外还有瑞替普酶, 是新一代溶栓药物, 在2025年国际卒中大会的网状Meta分析中, 显示其对美国国立卫生研究院卒中量表评分 < 15分患者的疗效优于其他溶栓药物, 但需警惕高评分患者的出血风险。脑细胞保护剂包括依达拉奉右莰醇(先必新), 清除自由基, 抑制脂质过氧化和神经炎症, 保护血脑屏障。丁苯酞(恩必普) 能通过促进脑微循环改善线粒体能量代谢等多靶点作用。此外, 还有许多处于临床试验的针对IS的药物(表3^[60-64])。

7　小结

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IS作为全球第二大死亡原因及致残性疾病, 其治疗药物的研发面临非临床药效学评价体系的局限性: 动物模型与人类病理生理机制的差异导致部分临床前有效药物转化失败、神经保护剂的多靶点协同机制尚未完全解析、传统功能评价指标对长期功能恢复的敏感性不足、中医药现代化需结合分子药理学证据提升国际认可度等方面存在不足等, 因此亟需构建涵盖更全面、精准的非临床药效学评价体系。本文系统综述了IS非临床药效学评价体系的构建与优化, 重点围绕动物模型、药效学评价指标、分子机制及我国药物研发现状展开分析。未来需结合神经血管单元保护理念, 优化模型筛选标准(血流阈值、梗死体积筛选), 按照系统化、标准化、多维度的原则, 并探索天然药物的分子机制研究, 以提升IS治疗药物的研发效率与临床转化成功率。

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2026年第61卷第2期

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doi: 10.16438/j.0513-4870.2025-0869

接收时间：2025-07-23
首发时间：2026-09-21
出版时间：2026-02-12

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收稿日期：2025-07-23
修回日期：2025-10-10

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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:

王庆利, E-mail: wangql@cde.org.cn

彭英, E-mail: ypeng@imm.ac.cn

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https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2025-0869

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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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Index	pMCAO	tMCAO	dMCAO
Blood flow recovery	No reperfusion (permanent occlusion)	With reperfusion (filament removal or ligation release after occlusion)	Usually no reperfusion (partial models allow reperfusion)
Infarct range	Largest (involves ischemic core and surrounding areas) (Figure 2 left)	Large (core infarct region, ischemic penumbra) (Figure 2 middle)	Small (confined to the perfusion area of distal MCA branches, e.g., cortex) (Figure 2 right)
Surgical method	Electrocoagulation or ligation of proximal MCA, filament method	Filament method: insert filament to the origin of MCA, withdraw after a certain time	Transcranial electrocoagulation or filament occlusion of distal MCA branches
Pathological mechanism	Persistent ischemia leading to energy failure and cell necrosis	Ischemia-reperfusion injury (free radical burst, inflammatory response)	Focal ischemia causing selective neuronal injury
Research focus	Irreversible injury mechanism, long-term neural repair	Reperfusion injury, efficacy of neuroprotective agents	Cortical function recovery, small molecule drug screening
Mortality rate	High (massive infarction prone to cerebral edema)	Moderate (risk of cerebral edema after reperfusion)	Low (small infarct range, high survival rate)
Advantages & disadvantages	Advantages: simulates patients not receiving reperfusion therapy; disadvantages: severe surgical trauma, high mortality	Advantages: simulates pathological process after thrombolysis; disadvantages: precise control of reperfusion time window required	Advantages: localized injury, suitable for long-term behavioral evaluation; disadvantages: high surgical technique requirements, large infarct heterogeneity

Index

pMCAO

tMCAO

dMCAO

Blood flow recovery

No reperfusion (permanent occlusion)

With reperfusion (filament removal or ligation release after occlusion)

Usually no reperfusion (partial models allow reperfusion)

Infarct range

Largest (involves ischemic core and surrounding areas) (Figure 2 left)

Large (core infarct region, ischemic penumbra) (Figure 2 middle)

Small (confined to the perfusion area of distal MCA branches, e.g., cortex) (Figure 2 right)

Surgical method

Electrocoagulation or ligation of proximal MCA, filament method

Filament method: insert filament to the origin of MCA, withdraw after a certain time

Transcranial electrocoagulation or filament occlusion of distal MCA branches

Pathological mechanism

Persistent ischemia leading to energy failure and cell necrosis

Ischemia-reperfusion injury (free radical burst, inflammatory response)

Focal ischemia causing selective neuronal injury

Research focus

Irreversible injury mechanism, long-term neural repair

Reperfusion injury, efficacy of neuroprotective agents

Cortical function recovery, small molecule drug screening

Mortality rate

High (massive infarction prone to cerebral edema)

Moderate (risk of cerebral edema after reperfusion)

Low (small infarct range, high survival rate)

Advantages & disadvantages

Advantages: simulates patients not receiving reperfusion therapy; disadvantages: severe surgical trauma, high mortality

Advantages: simulates pathological process after thrombolysis; disadvantages: precise control of reperfusion time window required

Advantages: localized injury, suitable for long-term behavioral evaluation; disadvantages: high surgical technique requirements, large infarct heterogeneity

Evaluation index	Experiment name	Specific detection indicator	Training required	Evaluation time
Neurological deficit	Bederson score	The Bederson score assesses neurological dysfunction, with higher scores indicating more severe impairment 0: When suspended by the tail, the animal extends both forelimbs naturally, with no flexion or movement asymmetry 1: The forelimb contralateral to the injury shows flexion (e.g., wrist or elbow bending), without additional abnormalities such as circling or reduced resistance to lateral push 2: In addition to forelimb flexion, the animal exhibits significantly reduced resistance to lateral push toward the impaired side 3: The animal displays spontaneous circling behavior toward the injured side, in addition to the above signs 4: The animal shows longitudinal rotation or is unable to move independently	No	Acute phase (0-72 h)
Longa score	This scale evaluates motor behavior and neurological deficits, ranging from 0 (no deficit) to 4 (severe deficit or death). It includes assessments of somatosensory function, placement response, grip strength, reflexes, balance, and coordination	No	Acute phase (0-72 h)
mNSS	1. Suspended tail test: elevate the tail above the ground and observe any deviation or flexion of the head and limbs 2. Normal gait, inability to walk in a straight line, circling towards the affected side, and leaning towards the affected side are scored 0-3 points respectively 3. Sensory testing: visual testing; tactile testing; proprioceptive testing 4. Balance beam test 5. Reflex testing: auricular reflex; corneal reflex; startle reflex; epileptic seizures, muscle spasms, abnormal muscle tone mNSS ranges from 0 to 18 points, with higher scores indicating more severe neurological impairment	No	Acute phase (0-72 h), subacute phase (3-14 d), recovery phase (> 14 d)
Garcia neurobehavioral scale	1. General activity in the test cage 2. Symmetry of forelimb movement 3. Ability to extend forepaws 4. Climbing ability on a wire 5. Tactile response to body stimulation (cotton swab) 6. Response to vibrissae touch Each test is scored from 0 to 3, with a maximum total of 18 points. Higher scores indicate milder neurological injury	No	Acute phase (0-72 h), subacute phase (3-14 d)
Motor coordination & balance	Rotarod test	A rotating cylinder (diameter: 3.0-3.2 cm) with multiple compartments allows simultaneous testing of several mice. The latency to fall from the rod is recorded as a measure of motor coordination and balance	Yes (2-3 d, 2-3 rounds)	Acute phase (0-72 h), subacute phase (3-14 d)
Wire hanging test	The animal grasps a horizontal wire with its forepaws and hangs until fatigue. The latency to fall is recorded to assess motor function and neuromuscular strength	Yes (2-3 times)	Acute phase (0-72 h), subacute phase (3-14 d)
Pole test	Motor recovery is evaluated by observing the animal's coordination, speed, and success in climbing down a pole. The time taken to descend from top to bottom serves as an indicator of motor speed and control	Yes (2-3 times)	Subacute phase (3-14 d)
Grid walk test	An elevated metal grid (41 cm × 41 cm, grid size: 3.5 cm² × 3.5 cm², height: 41 cm) is placed in a dim, soundproof room. After each trial, the grid is cleaned with 70% ethanol. A camera records the movement of mouse, and data analysis includes total forepaw steps and average step distance	No	Subacute phase (3-14 d), recovery phase (> 14 d)
Limb use symmetry & exploratory behavior	Cylinder test	A glass cylinder (diameter: 9-10 cm, height: 15 cm) is used to assess vertical exploration. The number of forelimb contacts with the cylinder wall is recorded. A mirror or camera ensures both forelimbs are visible during scoring	No	Recovery phase (> 14 d)
Forelimb placing test	This test evaluates motor function and recovery through seven specific tasks: 1. Forelimb extension when the mouse is held above a table 2. Retrieval and placement of forelimbs after gentle pulling 3. Same as task 2, but with the head elevated at 45° 4. Lateral placement of forelimbs along the table edge 5. Lateral placement of hindlimbs along the table edge 6. Hindlimb grip strength when pulled from the edge 7. Grip ability when gently pushed from behind toward the table edge Each item is scored 0-2, with a total maximum of 14 points. Lower scores indicate more severe dysfunction	No	Acute phase (0-72 h), subacute phase (3-14 d)
Corner turn test	Two cardboard walls form a 30° angle with a small opening at the corner to attract the animal. The animal is placed facing the corner, and vibrissae stimulation prompts a turning response. The direction of the turn is recorded to assess sensory and motor asymmetry	No	Subacute phase (3-14 d), recovery phase (> 14 d)
Adhesive removal test	Small adhesive patches (rectangular: 0.3 cm × 0.4 cm; or circular: diameter 0.3 cm) are placed on each forepaw. The mouse is placed in a transparent box and allowed to acclimate. The time to contact and remove each patch is recorded, reflecting sensorimotor integration and limb functionality	No	Subacute phase (3-14 d), recovery phase (> 14 d)

Evaluation index

Experiment name

Specific detection indicator

Training required

Evaluation time

Neurological deficit

Bederson score

The Bederson score assesses neurological dysfunction, with higher scores indicating more severe impairment
0: When suspended by the tail, the animal extends both forelimbs naturally, with no flexion or movement asymmetry
1: The forelimb contralateral to the injury shows flexion (e.g., wrist or elbow bending), without additional abnormalities such as circling or reduced resistance to lateral push
2: In addition to forelimb flexion, the animal exhibits significantly reduced resistance to lateral push toward the impaired side
3: The animal displays spontaneous circling behavior toward the injured side, in addition to the above signs
4: The animal shows longitudinal rotation or is unable to move independently

Acute phase (0-72 h)

Longa score

This scale evaluates motor behavior and neurological deficits, ranging from 0 (no deficit) to 4 (severe deficit or death). It includes assessments of somatosensory function, placement response, grip strength, reflexes, balance, and coordination

Acute phase (0-72 h)

mNSS

1. Suspended tail test: elevate the tail above the ground and observe any deviation or flexion of the head and limbs
2. Normal gait, inability to walk in a straight line, circling towards the affected side, and leaning towards the affected side are scored 0-3 points respectively
3. Sensory testing: visual testing; tactile testing; proprioceptive testing
4. Balance beam test
5. Reflex testing: auricular reflex; corneal reflex; startle reflex; epileptic seizures, muscle spasms, abnormal muscle tone
mNSS ranges from 0 to 18 points, with higher scores indicating more severe neurological impairment

Acute phase (0-72 h), subacute phase (3-14 d), recovery phase (> 14 d)

Garcia neurobehavioral scale

1. General activity in the test cage
2. Symmetry of forelimb movement
3. Ability to extend forepaws
4. Climbing ability on a wire
5. Tactile response to body stimulation (cotton swab)
6. Response to vibrissae touch
Each test is scored from 0 to 3, with a maximum total of 18 points. Higher scores indicate milder neurological injury

Acute phase (0-72 h), subacute phase (3-14 d)

Motor coordination & balance

Rotarod test

A rotating cylinder (diameter: 3.0-3.2 cm) with multiple compartments allows simultaneous testing of several mice. The latency to fall from the rod is recorded as a measure of motor coordination and balance

Yes (2-3 d, 2-3 rounds)

Acute phase (0-72 h), subacute phase (3-14 d)

Wire hanging test

The animal grasps a horizontal wire with its forepaws and hangs until fatigue. The latency to fall is recorded to assess motor function and neuromuscular strength

Yes (2-3 times)

Acute phase (0-72 h), subacute phase (3-14 d)

Pole test

Motor recovery is evaluated by observing the animal's coordination, speed, and success in climbing down a pole. The time taken to descend from top to bottom serves as an indicator of motor speed and control

Yes (2-3 times)

Subacute phase (3-14 d)

Grid walk test

An elevated metal grid (41 cm × 41 cm, grid size: 3.5 cm² × 3.5 cm², height: 41 cm) is placed in a dim, soundproof room. After each trial, the grid is cleaned with 70% ethanol. A camera records the movement of mouse, and data analysis includes total forepaw steps and average step distance

Subacute phase (3-14 d), recovery phase (> 14 d)

Limb use symmetry & exploratory behavior

Cylinder test

A glass cylinder (diameter: 9-10 cm, height: 15 cm) is used to assess vertical exploration. The number of forelimb contacts with the cylinder wall is recorded. A mirror or camera ensures both forelimbs are visible during scoring

Recovery phase (> 14 d)

Forelimb placing test

This test evaluates motor function and recovery through seven specific tasks:
1. Forelimb extension when the mouse is held above a table
2. Retrieval and placement of forelimbs after gentle pulling
3. Same as task 2, but with the head elevated at 45°
4. Lateral placement of forelimbs along the table edge
5. Lateral placement of hindlimbs along the table edge
6. Hindlimb grip strength when pulled from the edge
7. Grip ability when gently pushed from behind toward the table edge
Each item is scored 0-2, with a total maximum of 14 points. Lower scores indicate more severe dysfunction

Acute phase (0-72 h), subacute phase (3-14 d)

Corner turn test

Two cardboard walls form a 30° angle with a small opening at the corner to attract the animal. The animal is placed facing the corner, and vibrissae stimulation prompts a turning response. The direction of the turn is recorded to assess sensory and motor asymmetry

Subacute phase (3-14 d), recovery phase (> 14 d)

Adhesive removal test

Small adhesive patches (rectangular: 0.3 cm × 0.4 cm; or circular: diameter 0.3 cm) are placed on each forepaw. The mouse is placed in a transparent box and allowed to acclimate. The time to contact and remove each patch is recorded, reflecting sensorimotor integration and limb functionality

Subacute phase (3-14 d), recovery phase (> 14 d)

Drug name	Target/mechanism	Development phase	Preclinical animal experimental model
Y-3 for injection	Targets PSD95-nNOS coupling—a key pathological mechanism in stroke and mood disorders—and modulates GABA_A receptors, enabling synergistic treatment of ischemic stroke and prevention of post-stroke depression	Completed phase Ⅲ clinical trial	MCAO^[60]
Nitronezine (TBN) for injection	Crosses the blood-brain barrier efficiently, inhibits calcium overload, scavenges free radicals, and suppresses neuroinflammation. Enhances mitochondrial function and boosts ATP production via activation of the AMPK/PGC-1α/Nrf2/HO-1 signaling axis; promotes autophagic clearance of toxic proteins through mTOR pathway inhibition	Phase Ⅲ clinical trial	MCAO^[61]
GD-11 for injection	GLUT-1	Phase Ⅲ clinical trial	Not disclosed
JX10 for injection	Anti-inflammatory thrombolytic agent	Phase Ⅱ/Ⅲ clinical trial	Not disclosed
Recombinant staphylokinase (r-Sak) for injection	Forms a complex with plasminogen to activate the fibrinolytic system, promoting the conversion of plasminogen to plasmin, selectively degrading fibrin, and dissolving thrombi	Phase Ⅱ clinical trial	Thromboembolic model^[62]
LT3001 for injection	Synthetic polypeptide, promoting vascular recanalization and mitigating reperfusion injury	Phase Ⅱ clinical trial	MCAO^[63]
ZHB206 injection	Reduces HMGB1-mediated apoptosis and inflammation after ischemic stroke, enhancing cell survival and glial migration, thereby attenuating ischemic brain damage	Phase Ⅱ clinical trial	Not disclosed
HRS8179	Inhibits the SUR1/TRPM4 ion channel to reduce sodium influx, alleviating cytotoxic edema and lowering mortality	Phase Ⅱ clinical trial	Not disclosed
BAY 3018250	Anti α2AP protein therapy. Significantly reduces microvascular thrombosis, ischemic injury, brain swelling, hemorrhagic transformation, and mortality following thromboembolic stroke	Phase Ⅱ clinical trial	Not disclosed
Y-6 sublingual tablets	Novel sublingual compound tablet. Combines two marketed drugs targeting cerebral vasculature and neural tissue, respectively. Inhibits vascular smooth muscle proliferation, protects endothelial integrity, reduces bleeding risk, preserves blood-brain barrier function, suppresses inflammatory cytokine release, and mitigates glutamate excitotoxicity. Improves neurological outcomes and cerebral metabolism, enhances cognitive function in cerebrovascular disease, and reduces white matter hyperintensity volume. The components exhibit synergistic efficacy with a clear dose-response relationship. Suitable for acute and recovery phases of stroke, as well as long-term management of cerebral small vessel disease	Phase Ⅱ clinical trial	MCAO^[64]
AD108 injection	Exogenous KLK-1 supplementation improves neurological deficits, reduces disability, and enhances daily functioning in acute ischemic stroke. AD108 injection enables sustained release via subcutaneous administration, maintaining stable plasma levels and therapeutic effect while avoiding hypotension risks associated with intravenous delivery	Phase Ⅰ clinical trial	Not disclosed
NR-20201 injection	Promote vascular regeneration and functional repair of ischemic brain tissue via homing mechanisms and synergistic interaction with endothelial cells	Phase Ⅰ clinical trial	Not disclosed
Xing lou cheng qi granules	Clears heat from the fu-organs, activates blood circulation, and removes meridian obstruction	Phase Ⅰ clinical trial	Not disclosed
PMS-001 for injection	AMPA receptor modulator (ionotropic glutamate receptor AMPA modulator)	Phase Ⅰ clinical trial	Not disclosed
ZHB103 injection	Mechanism not disclosed	Phase Ⅰ clinical trial	Not disclosed
Asarone emulsion injection	Elevates cortical electrical stimulation threshold, inhibits synaptic transmission of epileptiform activity, and prevents seizure propagation	Phase Ⅰ clinical trial	Not disclosed

Drug name

Target/mechanism

Development phase

Preclinical animal experimental model

Y-3 for injection

Targets PSD95-nNOS coupling—a key pathological mechanism in stroke and mood disorders—and modulates GABA_A receptors, enabling synergistic treatment of ischemic stroke and prevention of post-stroke depression

Completed phase Ⅲ clinical trial

MCAO^[60]

Nitronezine (TBN) for injection

Crosses the blood-brain barrier efficiently, inhibits calcium overload, scavenges free radicals, and suppresses neuroinflammation. Enhances mitochondrial function and boosts ATP production via activation of the AMPK/PGC-1α/Nrf2/HO-1 signaling axis; promotes autophagic clearance of toxic proteins through mTOR pathway inhibition

Phase Ⅲ clinical trial

MCAO^[61]

GD-11 for injection

GLUT-1

Phase Ⅲ clinical trial

Not disclosed

JX10 for injection

Anti-inflammatory thrombolytic agent

Phase Ⅱ/Ⅲ clinical trial

Not disclosed

Recombinant staphylokinase (r-Sak) for injection

Forms a complex with plasminogen to activate the fibrinolytic system, promoting the conversion of plasminogen to plasmin, selectively degrading fibrin, and dissolving thrombi

Phase Ⅱ clinical trial

Thromboembolic model^[62]

LT3001 for injection

Synthetic polypeptide, promoting vascular recanalization and mitigating reperfusion injury

Phase Ⅱ clinical trial

MCAO^[63]

ZHB206 injection

Reduces HMGB1-mediated apoptosis and inflammation after ischemic stroke, enhancing cell survival and glial migration, thereby attenuating ischemic brain damage

Phase Ⅱ clinical trial

Not disclosed

HRS8179

Inhibits the SUR1/TRPM4 ion channel to reduce sodium influx, alleviating cytotoxic edema and lowering mortality

Phase Ⅱ clinical trial

Not disclosed

BAY 3018250

Anti α2AP protein therapy. Significantly reduces microvascular thrombosis, ischemic injury, brain swelling, hemorrhagic transformation, and mortality following thromboembolic stroke

Phase Ⅱ clinical trial

Not disclosed

Y-6 sublingual tablets

Novel sublingual compound tablet. Combines two marketed drugs targeting cerebral vasculature and neural tissue, respectively. Inhibits vascular smooth muscle proliferation, protects endothelial integrity, reduces bleeding risk, preserves blood-brain barrier function, suppresses inflammatory cytokine release, and mitigates glutamate excitotoxicity. Improves neurological outcomes and cerebral metabolism, enhances cognitive function in cerebrovascular disease, and reduces white matter hyperintensity volume. The components exhibit synergistic efficacy with a clear dose-response relationship. Suitable for acute and recovery phases of stroke, as well as long-term management of cerebral small vessel disease

Phase Ⅱ clinical trial

MCAO^[64]

AD108 injection

Exogenous KLK-1 supplementation improves neurological deficits, reduces disability, and enhances daily functioning in acute ischemic stroke. AD108 injection enables sustained release via subcutaneous administration, maintaining stable plasma levels and therapeutic effect while avoiding hypotension risks associated with intravenous delivery

Phase Ⅰ clinical trial

Not disclosed

NR-20201 injection

Promote vascular regeneration and functional repair of ischemic brain tissue via homing mechanisms and synergistic interaction with endothelial cells

Phase Ⅰ clinical trial

Not disclosed

Xing lou cheng qi granules

Clears heat from the fu-organs, activates blood circulation, and removes meridian obstruction

Phase Ⅰ clinical trial

Not disclosed

PMS-001 for injection

AMPA receptor modulator (ionotropic glutamate receptor AMPA modulator)

Phase Ⅰ clinical trial

Not disclosed

ZHB103 injection

Mechanism not disclosed

Phase Ⅰ clinical trial

Not disclosed

Asarone emulsion injection

Elevates cortical electrical stimulation threshold, inhibits synaptic transmission of epileptiform activity, and prevents seizure propagation

Phase Ⅰ clinical trial

Not disclosed