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海马定位注射单次长时间应激小鼠脑脊液可诱发创伤后应激障碍样行为

丁可可¹, 陈道康¹, 杨绍杰², 朱国旗^1,*

¹安徽中医药大学分子生物学(脑病)重点实验室，合肥 230012；²安徽中医药大学第二附属医院，合肥230061

摘要

本文旨在探明海马定位注射单次长时间应激(single-prolonged stress, SPS)模型小鼠脑脊液(cerebrospinal fluid, CSF)对正常小鼠神经行为的影响及可能的机制。提取SPS小鼠CSF (SPS-CSF)，高效液相色谱-电化学法(HPLC-ECD)检测其去甲肾上腺素(norepinephrine, NE)水平；通过套管直接注射SPS-CSF到海马脑区，高架十字迷宫、恐惧记忆消退等行为学实验评价小鼠焦虑及恐惧记忆变化；苏木精和伊红染色评价海马神经元病理变化；Western blot 和免疫荧光检测星形胶质细胞及突触相关蛋白的表达变化；透射电镜评估突触结构的变化；评价腹腔注射氯胺酮(ketamine, KET)对SPS-CSF诱导的小鼠行为变化的影响。结果显示，SPS小鼠表现出焦虑样行为和恐惧记忆消退异常，且SPS小鼠CSF中NE水平较正常动物显著升高；连续注射6 次SPS-CSF造成正常小鼠焦虑样行为，并降低恐惧记忆消退；小鼠接受SPS-CSF后，海马出现神经元损伤、胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)表达减弱以及突触蛋白表达减少；有趣的是，SPS-CSF造成小鼠行为功能的变化能持续数周，同时SPS-CSF 造成小鼠行为的改变能被KET所阻断。上述结果表明海马区连续注射SPS-CSF 可诱发正常小鼠产生焦虑样行为和恐惧记忆异常，其机制与调控星形胶质细胞及突触相关，提示SPS-CSF具有“传染性”，为未来创伤后应激障碍研究提供了新的着力点。

关键词： 创伤后应激障碍; 脑脊液; 单次长时间应激; 突触; 星形胶质细胞

Hippocampal stereotactic injection of cerebrospinal fluid from single-prolonged stress mice induces PTSD-like behavior

DING Ke-Ke¹, CHEN Dao-Kang¹, YANG Shao-Jie², ZHU Guo-Qi^1,*

¹Key Laboratory of Molecular Biology (Brain Diseases), Anhui University of Chinese Medicine, Hefei 230012, China；²The Second Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230061, China

Abstract

This study aimed to investigate the impact of cerebrospinal fluid (CSF) from single-prolonged stress (SPS) model mice on the neurobehavioral outcomes in normal mice, and to explore the underlying mechanisms. CSF was extracted from SPS mice (SPS-CSF), and norepinephrine (NE) levels were measured using high-performance liquid chromatography-electrochemical detection (HPLC-ECD). A cannula was used for direct hippocampal injection of SPS-CSF, followed by behavioral experiments such as the elevated plus maze and fear extinction tests to assess changes in anxiety and fear memory in SPS-CSF injected mice. Hematoxylin and eosin (HE) staining was employed to evaluate neuronal pathology in the hippocampus. Western blotting and immunofluorescence were used to detect changes in the expression of astrocytic and synaptic proteins. Transmission electron microscopy (TEM) was adopted for synaptic ultrastructural analysis. Additionally, the effect of intraperitoneal ketamine (KET) injection on the behavioral changes induced by SPS-CSF was assessed. The results showed that SPS mice exhibited typical anxiety-like behavior, and NE levels in the CSF of SPS mice were significantly higher than those in control animals. Repeated injections of SPS-CSF into normal mice led to anxiety-like behavior and impaired fear memory extinction. After SPS-CSF administration, neuronal damage was observed in the hippocampus, along with decreased glial fibrillary acidic protein (GFAP) expression and reduced synaptic protein levels. Interestingly, the behavioral changes induced by SPS-CSF persisted for several weeks, and these changes could be blocked by KET. These findings suggest that stereotactic injection of SPS-CSF into the hippocampus can induce anxiety-like behavior and abnormal fear memory in normal mice, with mechanisms involving the regulation of astrocytes and synapses. This model indicates a potential "infectious" nature of SPS-CSF and provides a novel approach for future research on post-traumatic stress disorder (PTSD).

Key words: post-traumatic stress disorder; cerebrospinal fluid; single-prolonged stress; synapses; astrocytes

收稿日期：　　录用日期：

通讯作者：朱国旗　　E-mail:

DOI: 10.13294/j.aps.2025.0068

引用本文：

丁可可, 陈道康, 杨绍杰, 朱国旗. 海马定位注射单次长时间应激小鼠脑脊液可诱发创伤后应激障碍样行为[J]. 生理学报 2025; 77 (6): 1157-1170.

DING Ke-Ke, CHEN Dao-Kang, YANG Shao-Jie, ZHU Guo-Qi. Hippocampal stereotactic injection of cerebrospinal fluid from single-prolonged stress mice induces PTSD-like behavior. Acta Physiol Sin 2025; 77 (6): 1157-1170 (in Chinese with English abstract).