目的:探讨小鼠青春期香烟暴露对前额叶小胶质细胞介导的炎症反应及其对成年后学习记忆功能的影响。方法:健康2周龄雄性昆明小鼠72只，每只体重(11。0±1。5)g。随机分为对照组与香烟暴露组，每组各36只。香烟暴露组每日被动吸烟6支，持续10周。分别在对照组与香烟暴露组小鼠4周龄(少年期)、8周龄(青年期)、12周龄(成年期)三个时点，每组各取6只检测其神经行为学与病理学变化。具体如下:通过跳台测试、三箱交互实验、新奇事物识别实验检测小鼠学习记忆能力及认知行为学功能变化;免疫荧光实验检测各组小鼠前额叶皮质小胶质细胞的形态、突触数量以及小胶质细胞周围炎性因子凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD，ASC)的表达;Western blot实验检测各组小鼠大脑皮质突触小泡蛋白(synaptophysin，SYP)和突触后致密蛋白95(postsynaptic density protein-95，PSD-95)的表达水平;酶联免疫吸附测定(enzyme-linked immunosorbent assay，ELISA)实验检测前额叶皮质中肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)、白细胞介素1β(interleukin-1β，IL-1β)、IL-6的蛋白表达。结果:(1)与同龄对照组相比，8周、12周龄香烟暴露小鼠跳台潜伏期显著缩短、跳台错误次数显著增多(P＜0。01)，社交识别时间以及对新事物探索时间延长(P＜0。05)。(2)免疫荧光实验表明，8周龄、12周龄香烟暴露组小鼠前额叶皮质SYP阳性斑点减少，Iba1阳性小胶质细胞数量增多，呈现激活状态;小胶质细胞周围ASC阳性斑点增多。Western blot实验进一步证明8周龄、12周龄香烟暴露组小鼠大脑皮质突触素蛋白SYP、PSD-95表达降低(P＜0。05)。(3)ELISA实验表明8周龄、12周龄香烟暴露组小鼠前额叶皮质炎性因子TNF-α、IL-1β和IL-6含量增高(P＜0。05)。结论:小鼠青春期香烟暴露可能会通过激活前额叶皮质小胶质细胞，导致炎症因子分泌增多，影响小胶质细胞功能并造成突触损伤，进而影响小鼠成年后的学习记忆功能及认知能力。
Smoke exposure during adolescence affects learning and memory abili-ties of mice in adulthood via prefrontal microglia-mediated inflammation
AIM:To investigate the effects of cigarette smoke exposure during adolescence on the inflammato-ry response mediated by microglia in the prefrontal cortex of mice,and its impact on learning and memory functions in adulthood.METHODS:72 two-week-old healthy male Kunming mice,with each weighing(11.0±1.5)g,were random-ly divided into control and cigarette exposure groups(n=36 per group).The mice in the cigarette exposure group were pas-sively exposed to 6 cigarettes daily for 10 weeks.At three time points of 4-week-old(infancy),8-week-old(adoles-cence),and 12-week-old(adulthood),six mice were selected from each group to have their neurobehavioral and patholog-ical changes examined.In particular,the step-down test,three-chamber social interaction test,and novel object recogni-tion test were used to detect changes in learning and memory abilities and cognitive behavior.Immunofluorescence testing was performed to detect the morphology,number of synapses,and expression of inflammatory factor apoptosis-associated speck-like protein containing a CARD(ASC)around the microglial cells in the prefrontal cortex of mice in each group.Western blot was performed to assess the expression levels of synaptophysin(SYP)and postsynaptic density protein-95(PSD-95)in the cerebral cortex of mice in each group.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the protein expression of tumor necrosis factor-α(TNF-α),interleukin 1β(IL-1β),and IL-6 in the prefrontal cor-tex.RESULTS:(1)In the step-down test,the latency of mice at 8 and 12 weeks of age was significantly shortened,and the number of errors was significantly increased in the cigarette exposure group compared with the age-matched control group(P＜0.01).In addition,the social recognition time and exploration time for novel objects were prolonged(P＜0.05).(2)Immunofluorescence assays revealed that exposure to cigarette smoke in mice,at both 8 and 12 weeks of age,resulted in a reduction of SYP-positive puncta within the prefrontal cortex.Concurrently,there was an observed increase in the number of Iba1-positive microglia,which exhibited an activated phenotype,as well as an elevation in ASC-positive puncta in proximity to the microglia.Western blot further revealed reduced expression of synaptophysin protein SYP and PSD-95 in the cerebral cortex of the mice at 8 and 12 weeks of age in the cigarette exposure group(P＜0.05).(3)ELISA showed increased levels of inflammatory factors TNF-α,IL-1β,and IL-6 in the prefrontal cortex of the mice at 8 and 12 weeks of age in the cigarette exposure group(P＜0.05).CONCLUSION:Exposure to cigarette smoke during adoles-cence in mice may result in the enhanced secretion of inflammatory factors through the activation of microglia in the pre-frontal cortex.This activation can alter microglial function and induce synaptic damage,consequently impairing learning,memory,and cognitive abilities in adulthood.
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