The Endocrine Program, The State University of New Jersey, Rutgers, New Brunswick, NJ, USA.
Department of Animal Sciences, State University of New Jersey, Rutgers, New Brunswick, NJ, USA.
J Neuroinflammation. 2024 Oct 30;21(1):279. doi: 10.1186/s12974-024-03274-6.
Microglia, a type of resident immune cells within the central nervous system, have been implicated in ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing β-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined if microglial extracellular vesicles (exosomes) are involved in the ethanol-induced neuronal death of the β-endorphin neuron via secreting elevated levels of the chemokine monocyte chemoattractant protein 1 (MCP1), a key regulator of neuroinflammation.
We employed an in vitro model, consisting of primary culture of hypothalamic microglia prepared from postnatal day 2 (PND2) rat hypothalami and treated with or without 50 mM ethanol for 24 h, and an in vivo animal model in which microglia were obtained from hypothalami of PND6 rats fed daily with 2.5 mg/kg ethanol or control milk formula for five days prior to use. Exosomes were extracted and characterized with nanosight tracking analysis (NTA), transmission electron microscopy and western blot. Chemokine multiplex immunoassay and ELISA were used for quantitative estimation of MCP1 level. Neurotoxic ability of exosome was tested using primary cultures of β-endorphin neurons and employing nucleosome assay and immunocytochemistry. Elevated plus maze, open field and restraint tests were used to assess anxiety-related behaviors.
Ethanol elevated MCP1 levels in microglial exosomes both in vitro and in vivo models. Ethanol-activated microglial exosomes when introduced into primary cultures of β-endorphin neurons, increased cellular levels of MCP1 and the chemokine receptor CCR2 related signaling molecules including inflammatory cytokines and apoptotic genes as well as apoptotic death of β-endorphin neurons. These effects of microglial exosomes on β-endorphin neurons were suppressed by a CCR2 antagonist RS504393. Furthermore, RS504393 when injected in postnatal rats prior to feeding with ethanol it reduced alcohol-induced β-endorphin neuronal death in the hypothalamus. RS504393 also suppressed corticosterone response to stress and anxiety-like behaviors in postnatally alcohol-fed rats during adult period.
These data suggest that alcohol exposures during the developmental period elevates MCP1 levels in microglial exosomes that promote MCP1/CCR2 signaling to increase the apoptosis of β-endorphin neurons and resulting in hormonal and behavioral stress responses.
小胶质细胞是中枢神经系统中固有免疫细胞的一种，在内脏酒精谱系障碍的产后大鼠模型中，已被牵连到乙醇激活的应激调节前阿黑皮素原（POMC）神经元中β-内啡肽肽的神经元死亡，该神经元产生β-内啡肽。我们确定小胶质细胞细胞外囊泡（外泌体）是否通过分泌升高水平的趋化因子单核细胞趋化蛋白 1（MCP1）而参与乙醇诱导的β-内啡肽神经元的神经元死亡，MCP1 是神经炎症的关键调节剂。
我们采用了一种体外模型，该模型由来自产后第 2 天（PND2）大鼠下丘脑的原代培养的下丘脑小胶质细胞组成，并进行了或不进行 50 mM 乙醇处理 24 小时的处理，以及一种体内动物模型，其中小胶质细胞来自 PND6 大鼠的下丘脑，在使用前五天每天给予 2.5mg/kg 乙醇或对照奶配方。使用纳米粒子跟踪分析（NTA）、透射电子显微镜和western blot 提取并表征外泌体。使用趋化因子多重免疫测定法和 ELISA 定量估计 MCP1 水平。使用β-内啡肽神经元的原代培养物并使用核小体测定法和免疫细胞化学法测试外泌体的神经毒性能力。高架十字迷宫、旷场和束缚试验用于评估焦虑相关行为。
乙醇在体外和体内模型中均升高了小胶质细胞外泌体中的 MCP1 水平。当将乙醇激活的小胶质细胞外泌体引入β-内啡肽神经元的原代培养物中时，会增加细胞内 MCP1 水平以及趋化因子受体 CCR2 相关信号分子，包括炎症细胞因子和凋亡基因以及β-内啡肽神经元的凋亡死亡。小胶质细胞外泌体对β-内啡肽神经元的这些作用可被 CCR2 拮抗剂 RS504393 抑制。此外，在给新生大鼠注射 RS504393 之前，在给它们喂食乙醇之前，它可以减少乙醇诱导的下丘脑β-内啡肽神经元死亡。RS504393 还抑制了发育期酒精喂养大鼠在成年期对皮质酮应激和焦虑样行为的反应。
这些数据表明，发育期间的酒精暴露会升高小胶质细胞外泌体中的 MCP1 水平，促进 MCP1/CCR2 信号传导，增加β-内啡肽神经元的凋亡，并导致激素和行为应激反应。
