Department of Biomedical Engineering, University of California - Davis, Davis, CA, 95616, USA.
Department of Molecular Biosciences, University of California - Davis, Davis, CA, 95616, USA.
J Neuroinflammation. 2020 May 11;17(1):155. doi: 10.1186/s12974-020-01819-z.
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.
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.
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.
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.
神经元、星形胶质细胞和小胶质细胞之间的相互作用对中枢神经系统损伤后的神经炎症反应有重要影响。体外星形胶质细胞和小胶质细胞培养是研究神经炎症中涉及的特定分子途径的有力工具；然而，为了更好地理解细胞串扰对神经炎症的影响，需要新的多细胞培养模型。
从新生大鼠中分离出的原代皮质细胞在无血清的“三培养”培养基中培养，该培养基旨在支持神经元、星形胶质细胞和小胶质细胞，或在仅支持神经元和星形胶质细胞的“共培养”培养基中培养。使用半胱天冬酶 3/7 活性和形态变化来量化两种培养物类型对模拟无菌细菌感染（脂多糖（LPS）暴露）、机械损伤（划痕）和癫痫发作活动（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 LPS 暴露的共培养和三培养的分泌细胞因子谱。
三培养在体外保持了 14 天具有生理相关性的神经元、星形胶质细胞和小胶质细胞，而共培养保持了相似的神经元和星形胶质细胞群体，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的神经元整体健康产生负面影响，三培养中的神经元的半胱天冬酶 3/7 活性降低，与共培养相似的轴突生长，以及小胶质细胞分泌的神经营养因子 IGF-1 增加，条件培养基中 CX3CL1 的浓度显著降低。LPS 暴露的三培养显示出明显的星形胶质细胞肥大、半胱天冬酶 3/7 活性增加以及许多促炎细胞因子（例如 TNF、IL-1α、IL-1β 和 IL-6）的分泌，而这些都没有在 LPS 暴露的共培养中观察到。机械损伤后，与共培养相比，三培养中的半胱天冬酶 3/7 活性增加，同时星形胶质细胞向损伤源迁移增加。最后，谷氨酸诱导的兴奋性毒性过程中小胶质细胞在三培养中发挥了显著的神经保护作用，三培养中的神经元丢失和星形胶质细胞肥大明显减少。
由神经元、星形胶质细胞和小胶质细胞组成的三培养比标准的单培养和共培养更能真实地模拟体内神经炎症反应。这种三培养可以成为一种有用的体外研究神经炎症的工具，在预测体内神经炎症现象方面具有更高的准确性。
