脂多糖诱导小胶质细胞依赖的早产鼠脑白质损伤机制研究

目的研究中枢神经系统小胶质细胞（MG）正常发育尤其是少突胶质细胞前体细胞（OPCs）最易受损阶段的发育,探讨宫内感染早产鼠MG依赖的OPCs损伤机制。方法①观察正常C57B/L鼠不同胎龄（孕10、15d）和生后（0、5、10d）MG和OPCs在脑白质的发育分布情况,明确两者在发育和分布上的关联。②建立脂多糖（LPS）宫内感染新生鼠模型（宫内分别接种LPS5、10和20μg·mL-1为感染A-C组）,以PBS溶液接种为对照组。以Tomato lectin作为静息状态MG标志,CD68作为活化MG的特殊抗体,O4＋作为OPCs抗体,抗体浮片法进行免疫组化染色并计数分析。③Western blot法检测各组脑室周围白质组织Toll样受体-4（TLR-4）蛋白表达。④采用ELISA法检测各组MG活化后IL-2、TNF-α和SOD水平变化。结果①MG在孕10d胎鼠Tomato lectin表达低下,孕15d胎鼠表达显著增高,MG主要分布在脑室周围白质区域,灰质皮质几乎不表达。出生后,脑室周围白质区域MG的表达有所下降,灰质皮质的表达逐渐增高。②感染A-C组CD68＋细胞数量均显著增加,与对照组差异有统计学意义（P〈0.01）,但感染C组与B组CD68＋细胞数量差异无统计学意义（P〉0.05）。与对照组比较,感染A-C组均可见O4＋细胞数量显著性下降（P〈0.01）,其中以感染C组下降最为明显。③对照组未检测到TLR-4蛋白表达,感染A-C组均可见LPS剂量依赖的TLR-4蛋白表达增加,与对照组差异有统计学意义（P〈0.05）。④随接种LPS剂量增大,IL-2和TNF-α水平较对照组呈显著增加趋势,SOD水平较对照组呈显著降低趋势。结论新生鼠发育依赖的MG在脑白质受损区域过度表达,表明活化MG起到本底激活效应,是早产儿脑白质损伤的物质基础。

Mechanisms of activated microglia dependent white matter injury following Lipopolysaccharide-induced intrauterine infection

Objectives To examine the normative baseline microglia （MG） development in C57B/L fetus and early infants especially the most vulnerable period of oligodendrocyte progenitor cells （OPCs） injury, and to explore the mechanisms of activated microglia dependent white matter injury following LPS -induced intrauterine infection. Methods ① The developmental profile of MG in mouse cerebral white matter was examined, immunocytochemistry with MG and OPCs specific markers （Tomato lectin and O4＋） were used to identify the normal distribution of MG and its relationship with OPCs. ② Intrauterine infection brain injury model was established by intrauterine injection of LPS（5, 10 and 20 μg·mL-1 as group A, B and C）and compared with PBS control group to further verify the causal relationship of MG activation to OPCs damage using special antibodies to MG （CD68） and OPCs （O4＋）. ③ Western blotting was used to test the Toll like receptor-4 （TLR-4） protein expression in peri-ventricular white matter tissue. ④ Cytokine concentrations released by activated MG were measured by ELISA and the relationship to brain injury was analyzed. Results ① MG （Tomato lectin ＋） expression was low in the middle gestational age （GA） and the expression peaked at late GA and earlier after birth which was mainly distributed to the periventricular white matter and correlated closely to the development of OPCs between 10 gestational days and 0 day after birth （P0）. ② Quantitative analysis with CD68＋ and O4＋ showed increased density of activated MG and OPCs proportionally in control group. There was a significant increase of CD68＋ cells following increasing dose of LPS, but there was no significant difference between group B and C. Contrary to the significant increase of CD68＋ cells, there was a significant decrease of O4＋ cells in group A, B and C. ③ No TLR-4 protein expression was found in control group, dose dependent significant increase of TLR-4 protein was found in group B and C. ④ Pro-cytokines IL-2 and TNF-α levels released by MG significantly increased with LPS dose increased, which were higher than control group, whereas the SOD levels were significantly lower than control group. Conclusions The primary finding of a transient, developmental dependent overabundance of CD68-activated MG in the cerebral white matter of the C57B/L pups suggested a potential ＂priming＂ effect of this area on infectious brain insults characterized by activation of MG, particularly PVL.