热射病小鼠脑组织小胶质细胞活化的变化

Changes of microglial activation after heat stroke in mice brain

目的研究热射病小鼠脑组织中小胶质细胞活化的变化情况,初步探讨小胶质细胞在热射病脑损伤中的作用。方法采用小动物环境模拟舱建立温度(41.2±0.5)℃,相对湿度(60±2)%的热暴露损伤模型,将57只BALB/c雄性小鼠分为正常组和热射病1、6、24 h组。Real-time PCR检测大脑皮层M1型极化标志物TNF-α、CD45和CD11b的表达,以及M2型极化标志物Arg1、FIZZ和CD206的表达。Western blot检测各组大脑皮层CD45、CD11b、FIZZ和CD206的表达。免疫组织荧光技术检测CD45和CD206的分布和共定位表达。结果 Real-time PCR检测结果显示M1型极化标志物TNF-α、CD45和CD11b在热损伤后1 h达高峰,而M2型极化标志物Arg1、FIZZ和CD206在热损伤后24 h达高峰(P<0.01)。Western blot检测M1型极化标志物CD45及CD11b的蛋白表达量,在1 h后显著升高,随后逐渐下降,24 h后最低;M2型极化标志物FIZZ与CD206的表达趋势并不完全一致,但二者均在24 h后显著升高并达峰值(P<0.01)。免疫组织荧光染色结果显示CD45和CD206共定位表达于热射病小鼠大脑皮层,且分别在热损伤后1 h和24 h荧光光密度增强。结论热射病后小鼠脑组织小胶质细胞出现明显活化,其活化的规律表现为热射病后1 h主要为M1型极化,而在热射病后24 h则主要表现为M2型极化,这种变化形式可能与热射病后出现的中枢神经损伤有关。

Objective To explore the role of microglia played in mice brain after heat stroke by observing the effect of acute injury-related heat exposure on microglial activation. Methods Fifty-seven adult male BALB / c mice were randomly divided into normal control group and 3 heat stroke groups after heat exposure of different time periods（ 1,6,and 24 h）. The heat stroke groups were established by placing the mice in small animal environmental stimulation chambers with air temperature of 41. 2 ± 0. 5 ℃ and relative humidity of（ 60 ± 2） %. M1 polarization markers including TNF-α,CD45,and CD11 b as well as M2 polarization markers including Arg1,FIZZ,and CD206 were measured using real-time reverse transcriptascpolymerase chain reaction（ RT-PCR）. Protein expression of CD45,CD11 b,FIZZ,and CD206 was detected by Western blotting. Distribution and co-localization of CD45 and CD206 were assessed by immunofluorescence staining. Results RT-PCR analysis showed that the m RNA levels of M1 polarization markers（ TNF-α,CD45,and CD11b） and M2 polarization markers（ Arg1,FIZZ,and CD206） reached the peak at 1 and 24 h after heat exposure（ P〈0. 01）,respectively. Western blot results revealed that the expression of M1 polarization markers CD45 and CD11 b was significantly increased after 1 h followed by gradually declining to the base level 24 h after heat exposure. M2 polarization markers FIZZ and CD206showed different expression trends,but both of their expression were significantly increased and reached the maximum at 24 h（ P〈0. 01）. Immunofluorescence staining detected the co-localization of CD45 and CD206,exhibiting strong fluorescence signals at 1 and 24 h,respectively,after heat exposure in the cerebral cortex of mice. Conclusion Microglia are significantly activated in response to brain injury after heat exposure,which is characterized by prominent polarization of M1 at 1 h and M2 at 24 h after heat stroke. This polarized profile of microglia may be associated with central nervous system injury after heat stroke.