BACKGROUND Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or "primed" by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo. METHODS OHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated. RESULTS Microglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1β in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1β that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol. CONCLUSIONS Microglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.

中文翻译：

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脑切片培养物中的小胶质细胞耗竭和重新分布可正常化敏化的促炎信号。

背景小胶质细胞是跨多种神经系统疾病的神经免疫病理学的关键介质。小胶质细胞可以被Toll样受体（TLR）激活，乙醇，压力和其他伤害持续激活或“引发”。因此，对于涉及逐渐增加的小胶质细胞活化的疾病，预防或逆转小胶质细胞引发的策略可能是有益的。小胶质细胞减少和再填充正在成为使慢性免疫激活正常化的潜在疗法。主要的器官型海马切片培养物（OHSC）可以用于神经免疫激活以及小胶质细胞耗竭和再填充的研究，而无需涉及外周免疫激活。OHSC经历功能成熟并保留类似于体内的细胞结构。方法OHSC接受CSF1R拮抗剂PLX3397进行小胶质细胞清除，去除PLX3397后可重新填充或不重新填充。在小胶质细胞耗竭和繁殖的背景下，评估了对TLR 2、3、4、7和9激动剂以及乙醇产生的免疫，营养和突触基因变化。小胶质细胞的Gi-DREADD抑制作用被用于确认选择观察到的耗竭现象。还研究了小胶质细胞再填充防止由慢性乙醇引起的进行性促炎基因诱导的能力。结果PLX3397在OHSC中消耗了小胶质细胞（> 90％）。小胶质细胞耗竭减弱了对几种TLR激动剂以及乙醇的促炎反应，而Gi-DREADD对OHSC小胶质细胞的抑制作用模仿了乙醇。除去PLX3397，然后完全重填小胶质细胞。带有小胶质细胞繁殖的OHSC显示出抗炎细胞因子（例如IL-10），小胶质细胞抑制信号（例如CX3CL1）和生长因子（例如BDNF）的基线表达增加。这与对TLR4和TLR7激动剂的应答导致TNFα和IL-1β的诱导减弱（约50％）有关。此外，从体外4天（DIV）到16DIV的慢性循环乙醇导致TNFα和IL-1β的2倍速诱导，而40DIV诱导的年龄增长了约4倍。慢性乙醇后，小胶质细胞的耗竭和再填充完全消除了这种持续的炎症基因表达。结论OHSC中的小胶质细胞介导对TLR激动剂和乙醇的促炎反应。小胶质细胞的繁殖促进了抗炎，营养神经环境和正常化的促炎基因表达。