中度烧伤小鼠大脑免疫细胞的浸润及神经细胞凋亡

Infiltration of immune cells and neuron apoptosis in brain after moderate burn injury： experiment with mice

目的 观察中度烧伤小鼠大脑免疫细胞的浸润情况以及神经细胞凋亡.方法 BALB/C雄性小鼠随机分为烫伤组（n=15）和假伤组（n=15）,其中烫伤组按照15%总体表面积（total body surface area,TBSA）浸于94℃热水中8 s造成深Ⅱ度烫伤,假伤组动物浸于37℃水中.烫伤后24 h经心脏对各组中12只小鼠大脑进行生理盐水灌注,冲去脑血管内血液,Percoll梯度离心法分离脑内单个核细胞,以CD3＋CD45 high标记T细胞,CD11b＋CD45 high标记单核细胞,CD11b＋CD45int为激活的小胶质细胞,进行流式细胞染色和分析.每组剩余3只小鼠,大脑经4%多聚甲醛固定和蔗糖脱水后进行冰冻切片,免疫荧光法标记脑内免疫细胞,TUNEL法检测细胞凋亡.用含DAPI的抗荧光淬灭剂封片,共聚焦显微镜拍照.结果 烧伤组小鼠脑组织中CD45＋CD3＋T细胞所占比例较假伤组明显增高[（1.103 ±0.674）% vs.（0.385 ± 0.109）%,P〈0.05].烧伤组CD45int CD11b＋较假伤组比例增高[（1.017±0.35）% vs.（0.502±0.358）%,P〈0.05],提示烧伤组大脑中小胶质细胞激活程度较假伤组增高.免疫荧光染色可见烫伤组血管周围及脉络丛处有免疫反应阳性的T细胞、CD45high细胞,假伤组未见阳性.此外,烧伤小鼠与假伤组相比,在大脑皮层和脑室周围的脑实质中可见到明显增多的凋亡细胞.结论 中度烧伤后24 h大脑即可出现免疫细胞的浸润及神经细胞凋亡,可能和此后出现的神经精神障碍有关.

Objective To observe the infiltration of immune cells into the brain and the apoptosis of brain neurons after moderate burn injury. Method Thirty BALB/C male mice were randomly divided into two equal groups： burn group with their backs immersed in water at 94°C for 8s so as to cause second-degree burn in 15% of the total surface area （TBSA）,and sham burn group with their backs immersed in water at 37℃..12 brains of mice from each group were removed after intracardiac normal saline perfusion 24 hours after scald.Mononuclear cells from the brain were isolated by discontinuous Percoll gradient centrifugation. For flow cytometry analysis, T cells were labeled with CD3＋CD45high,monocytes with CD11b＋CD45 high and activated microglia with CD11b＋CD45int.The remaining 3 brains of each group were fixed and dehydrated.Then the frozen sections of brain tissue were stained by immunofluorescence for the immune cells,and with TUNEL method for the apoptotic cells.The sections were sealed with mounting medium for fluorescence with DAPI and photographed under confocal microscopy. Results The proportion of T cells in the brain tissue of burn group was[（1.103 ±0.674）%,significantly higher than that of the sham injury group[（0.385 ± 0.109）%,P〈0.05]. The proportion of microglia（CD45intCD11b＋）of the burn group was（1.017±0.35）%,significantly higher than that of the sham injury group [（0.502 ± 0.358）%,P〈0.05],suggesting a higher degree of activation of microglia. Immunofluorescence staining showed an increased distribution of T cells and CD45high cells in the perivascular and choroid plexus of the burn group, compared with the sham injury group. Moreover, more apoptotic neurons were observed in the cerebral cortex and periventricular parenchyma of the brain in the burn mice relative to the sham injury mice. Conclusion As soon as 24 hours after moderate burn,infiltration of peripheral T cells and increasing number of apoptotic neurons can be observed in the brain, which may contribute to the following neuropsychiatric disorders.