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A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.
Journal of Neuroinflammation ( IF 9.3 ) Pub Date : 2020-05-11 , DOI: 10.1186/s12974-020-01819-z
Noah Goshi 1 , Rhianna K Morgan 2 , Pamela J Lein 2 , Erkin Seker 3
Affiliation  

BACKGROUND Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required. METHODS Primary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared. RESULTS The tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture. CONCLUSIONS The tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena.

中文翻译:

一种初级神经细胞培养模型,用于研究神经炎症中的神经元、星形胶质细胞和小胶质细胞的相互作用。

背景神经元、星形胶质细胞和小胶质细胞之间的相互作用严重影响对中枢神经系统损伤的神经炎症反应。体外星形胶质细胞和小胶质细胞培养是研究参与神经炎症的特定分子途径的有力工具;然而,为了更好地了解细胞串扰对神经炎症的影响,需要新的多细胞培养模型。方法 取自新生大鼠的原代皮层细胞在配制为支持神经元、星形胶质细胞和小胶质细胞的无血清“三培养”培养基或配制为仅支持神经元和星形胶质细胞的“共培养”培养基中培养。Caspase 3/7 活性和形态变化用于量化两种培养类型对模拟无菌细菌感染(脂多糖 (LPS) 暴露)、机械损伤(划痕)和癫痫发作(谷氨酸诱导的兴奋性毒性)的不同神经炎症刺激的反应. 还比较了对照和暴露于 LPS 的共培养和三培养的分泌细胞因子谱。结果 三培养在体外维持了 14 天神经元、星形胶质细胞和小胶质细胞的生理相关表现,而共培养物维持了相似的神经元和星形胶质细胞群,但缺乏小胶质细胞。小胶质细胞的持续存在并没有对三培养中神经元的整体健康产生负面影响,这表明 caspase 3/7 活性降低,并且与共培养物相似的神经突生长,随着小胶质细胞分泌的神经营养因子 IGF-1 的增加和条件培养基中 CX3CL1 的浓度显着降低。暴露于 LPS 的三培养物显示出显着的星形胶质细胞肥大、半胱天冬酶 3/7 活性增加以及大量促炎细胞因子(例如 TNF、IL-1α、IL-1β 和 IL-6)的分泌,无其中在暴露于 LPS 的共培养物中观察到。机械损伤后,与共培养相比,三培养显示 caspase 3/7 活性增加,同时星形胶质细胞向损伤源的迁移增加。最后,三培养中的小胶质细胞在谷氨酸诱导的兴奋性毒性过程中发挥了重要的神经保护作用,显着减少了三培养中的神经元损失和星形胶质细胞肥大。结论 由神经元组成的三文化,星形胶质细胞和小胶质细胞比标准的单一和共培养物更忠实地模拟体内神经炎症反应。这种三培养可以成为研究体外神经炎症的有用工具,提高了预测体内神经炎症现象的准确性。
更新日期:2020-05-11
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