Rat primary cortical cell tri-culture to study effects of amyloid-beta on microglia function

IntroductionThe etiology and progression of sporadic Alzheimers Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (A{beta}) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in A{beta} clearance, and alterations in microglial function due to aging or disease may result in A{beta} accumulation and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and A{beta} clearance) and proteomic response to exogenous A{beta}. MethodsWe established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days in vitro (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled A{beta} (FITC-A{beta}) particles for varying durations. Images were analyzed to determine the number of FITC-A{beta} particles after specific lengths of exposure. A group of cells were stained for {beta}III-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio). Results and discussionFITC-A{beta} particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-A{beta} particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-A{beta} significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-A{beta} addition revealed a significant increase in proinflammatory cytokines (TNF-, IL-1, IL-1{beta}, IL-6) in tri-culture, but not co-culture. In addition, A{beta} addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and A{beta} clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.