小鼠脑出血后不同时间点小胶质细胞极化状态的实验研究

doi:10.3969/j.issn.1673-5765.2020.10.012

摘要/Abstract

摘要：

目的观察小鼠脑出血后不同时间点小胶质细胞M1及M2型的转化，为促炎型M1型小胶质细胞向抗炎修复型M2型小胶质细胞的转化，减轻脑出血后神经功能损伤提供理论依据。方法选取健康雄性ICR小鼠48只，随机分为假手术组、脑出血组，每组按术后时间点不同随机分为1 d、3 d、7 d三个时间点，每个时间点8只。通过立体定位仪用微量注射器向尾状核注射Ⅳ型胶原酶0.5 U制备脑出血模型，假手术组注射等量生理盐水。各组于术后对应时间点参照改良Garcia 评分量表进行神经功能缺损评分后灌注取脑，采用蛋白免疫印迹检测M1型小胶质细胞标志物肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素6（interleukin-6，IL-6），M2型小胶质细胞标志物脑源性神经营养因子（brai n-derived neurotrophic factor，BDNF）、胰岛素样生长因子1（insulinlike growth factor 1，IGF-1）的含量；采用免疫荧光染色标记小胶质细胞M1型（Iba1+CD80）、M2型（Iba1+CD206），评价出血后血肿周围组织小胶质细胞活化状态。结果脑出血组1 d、3 d、7 d各时间点Garci a评分均较假手术组低，TNF-α、IL-6、BDNF及I GF-1的蛋白表达量均较假手术组增多（均P＜0.01）。脑出血后1 d时M1型高于M2型小胶质细胞数量（38.33±1.53 vs 23.00±3.00，P =0.01）；3 d时M1型同样高于M2型（66.33±3.06 vs 57.33±2.52，P =0.02）；7 d时M1 型低于M2型（33.67±1.15 vs 52.33±0.58，P＜0.01）。结论脑出血急性期（1～3 d）以M1型小胶质细胞为主，脑出血亚急性期（7 d）以M2型小胶质细胞为主。

文章导读： 本研究显示脑出血后小胶质细胞活化，且不同活化类型的小胶质细胞呈动态演变，M1型小胶质细胞以脑出血后早期为主，M2型以出血后恢复期为主。

关键词: 脑出血; 小胶质细胞极化; 小胶质细胞Ⅰ型; 小胶质细胞Ⅱ型

Abstract:

Objective To investigate the transformation of microglia type Ⅰ and type Ⅱ at different time points after intracerebral hemorrhage (ICH) in mice, and to provide a theoretical basis for reducing the neurological damage after ICH by transforming pro-inflammatory microglia type Ⅰ (M1) into anti-inflammatory repair microglia type Ⅱ (M2). Methods Forty-eight healthy male ICR mice were randomly divided into sham operation group and ICH group. Each group was randomly divided into three subgroups according to the different postoperation time points of day 1, day 3, and day 7, with 8 mice in each subgroup. The ICH model was prepared by injecting Ⅳ collagenase 0.5 U into caudate nucleus through a stereotactic locator, while the mice in sham group were injected with the same amount of normal saline. The neurological deficit of the mice of two groups were assessed using the modified Garcia score at each time point, then the brain was perfused and brain tissue was taken. Western blot was used to detect the contents of microglia M1 markers tumor necrosis factor alpha (TNF-α), interleukin-6 (IL- 6), and M2 markers brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1);microglia M1 (Iba1+CD80) and M2 (Iba1+CD206) were labeled by immunofluorescence staining to evaluate the activation state of microglia around the hematoma after hemorrhage. Results At day 1, day 3 and day 7, the Garcia score of the ICH group was lower than that of the sham operation group, and the protein expression of TNF-α, IL-6, BDNF and IGF-1 were higher than that of the sham operation group (all P <0.01). The amount of microglia M1 was higher than M2 at day 1 (38.33±1.53 vs 23.00±3.00, P =0.01) and day 3 (66.33±3.06 vs 57.33±2.52, P =0.02), while lower than M2 at day 7 (33.67±1.15 vs 52.33±0.58, P <0.01). Conclusions Microglia was mainly type Ⅰ in acute phase of ICH (1-3 day after ICH), and mainly type Ⅱ in subacute phase (7 day after ICH).

Key words: Intracerebral hemorrhage; Microglial polarization; Microglia type Ⅰ; Microglia type Ⅱ

武翠梅，王改青，要振佳. 小鼠脑出血后不同时间点小胶质细胞极化状态的实验研究[J]. 中国卒中杂志, 2020, 15(10): 1094-1100.

WU Cui-Mei, WANG Gai-Qing, YAO Zhen-Jia. Experimental Study on the Polarization State of Microglia at Different Time Points after Intracerebral Hemorrhage in Mice[J]. Chinese Journal of Stroke, 2020, 15(10): 1094-1100.

参考文献

[1] ZHU H M，WANG Z Q，YU J X，et al. Role and

mechanisms of cytokines in the secondary brain

injury after intracerebral hemorrhage[J/OL]. Prog

Neurobiol，2019，178：101610[2020-02-01].

https：//doi.org/10.1016/j.pneurobio.2019.03.003.

[2] VERGARA D，NIGRO A，ROMANO A，et al.

Distinct protein expression networks are activated in

microglia cells after stimulation with IFN-γ and IL-

4[J]. Cells，2019，8（6）：580.

[3] WANG J，XING H Y，WAN L，et al. Treatment

targets for M2 microglia polarization in ischemic

stroke[J/OL]. Biomed Pharmacother，2018，105：

518-525[2020-02-01]. https：//doi.org/10.1016/

j.biopha.2018.05.143.

[4] WOLF S A，BODDEKE H W，KETTENMANN H.

Microglia in physiology and disease[J/OL]. Annu

Rev Physiol，2017，79：619-643[2020-02-01].

https：//doi.org/10.1146/annurev-physiol-022516-

034406.

[5] ZHANG Z，ZHANG Z，LU H，et al. Microglial

polarization and inflammatory mediators after

intracerebral hemorrhage[J]. Mol Neurobiol，2017，

54（3）：1874-1886.

[6] YAO K，ZU H B. Microglial polarization：novel

therapeutic mechanism against Alzheimer's

disease[J]. Inflammopharmacology，2020，28（1）：

95-110.

[7] KIM J Y，BAE H J. Spontaneous intracerebral

hemorrhage：management[J]. J Stroke，2017，19（1）：

28-39.

[8] XIONG X Y，LIU L，YANG Q W. Functions

and mechanisms of microglia/macrophages in

neuroinflammation and neurogenesis after stroke[J/

OL]. Prog Neurobiol，2016，142：23-44[2020-02-01].

https：//doi.org/10.1016/j.pneurobio.2016.05.001.

[9] MIAO H S，LI R M，HAN C，et al. Minocycline

promotes posthemorrhagic neurogenesis via M2 microglia polarization via upregulation of the TrkB/

BDNF pathway in rats[J]. J Neurophysiol，2018，120

（3）：1307-1317.

[10] CHEN Z Q，YU H，LI H Y，et al. Negative

regulation of glial Tim-3 inhibits the secretion of

inflammatory factors and modulates microglia to

antiinflammatory phenotype after experimental

intracerebral hemorrhage in rats[J]. CNS Neurosci

Ther，2019，25（6）：674-684.

[11] ROSENBERG G A，MUN-BRYCE S，WESLEY M，

et al. Collagenase-induced intracerebral hemorrhage

in rats[J]. Stroke，1990，21（5）：801-807.

[12] WANG G Q，MANAENKO A，SHAO A W，et al.

Low-density lipoprotein receptor-related protein-1

facilitates heme scavenging after intracerebral

hemorrhage in mice[J]. J Cereb Blood Flow Metab，

2017，37（4）：1299-1310.

[13] GUZMAN-MARTINEZ L，MACCIONI R B，

ANDRADE V，et al. Neuroinflammation as a

common feature of neurodegenerative disorders[J/

OL]. Front Pharmacol，2019，10：1008[2020-02-01].

https：//doi.org/10.3389/fphar.2019.01008.

[14] CHEN S，DONG Z P，CHENG M，et al.

Homocysteine exaggerates microglia activation and

neuroinflammation through microglia localized

STAT3 overactivation following ischemic stroke[J]. J

Neuroinflammation，2017，14（1）：187.

[15] LAN X，HAN X N，LI Q，et al. Pinocembrin

protects hemorrhagic brain primarily by inhibiting

toll-like receptor 4 and reducing M1 phenotype

microglia[J/OL]. Brain Behav Immun，2017，61：

326-339[2020-02-01]. https：//doi.org/10.1016/

j.bbi.2016.12.012.

[16] WAN S，CHENG Y Y，JIN H，et al. Microglia

activation and polarization after intracerebral

hemorrhage in mice：the role of protease-activated

receptor-1[J]. Transl Stroke Res，2016，7（6）：478-

487.