Abstract
BackgroundThere is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved.MethodsThe number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation.ResultsThe percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia.ConclusionsBecause contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.
Highlights
There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation
By comparing the gene expression profiles of microglia-eliminated and microglia-contaminated primary astrocyte cultures after interleukin-6 (IL-6) stimulation, we found that contaminating microglia induced deceptive up-regulation of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β genes in conventional primary astrocyte cultures through the activation of the Janus kinase/signal transducer and activator of the transcription (JAK/Signal transducer and activator of transcription (STAT)) pathway, highlighting the importance of microglial elimination from primary astrocyte cultures
Even after utilizing the conventional shake-off method, many microglia were observed in primary astrocyte cultures and these remaining amoeboid microglia had multiple short spinous processes (Figure 1B), suggesting strong adhesion of these cells to culture dishes
Summary
There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. Astrocytes were originally defined as gap fillers for neuronal networks, they have been found to play prominent roles in normal CNS functions, such as energy metabolism, neurotransmission, maintenance of blood–brain barrier, extracellular ion homeostasis and cerebrovascular regulation [1,2]. In pathological conditions, they participate in neuroinflammation and tissue repair [2,3,4,5]. These modifications result in cultures in which astrocytes are the predominant cells, but never comprise 100% of the cells in these preparations (approximately 90% to 95%) [6]
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