Evaluation of M1-microglial activation by neurotoxic metals using optimized organotypic cerebral slice cultures

Abstract

-M1-microglia (neurotoxic microglia) regulate neuronal development and cell death and are involved in many pathologies in the brain. Although organotypic brain slice cultures are widely used to study the crosstalk between neurons and microglia, little is known about the properties of microglia in the mouse cerebral cortex slices. Here, we aimed to optimize the mouse cerebral slice cultures that reflect microglial functions and evaluate the effects of neurotoxic metals on M1-microglial activation. Most microglia in the cerebral slices prepared from postnatal day (P) 7 mice were similar to mature microglia in adult mice brains, but those in the slices prepared from P2 mice were immature, which is a conventional preparation condition. The degree of expression of M1-microglial markers (CD16 and CD32) and inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) by lipopolysaccharide, a representative microglia activator, in the cerebral slices of P7 mice were higher than that in the slices of P2 mice. These results indicate that M1-microglial activation can be evaluated more accurately in the cerebral slices of P7 mice than in those of P2 mice. Therefore, we next examined the effects of various neurotoxic metals on M1-microglial activation using the cerebral slices of P7 mice and found that methylmercury stimulated the activation to M1-microglia, but arsenite, lead, and tributyltin did not induce such activation. Altogether, the optimized mouse cerebral slice cultures used in this study can be a helpful tool to study the influence of various chemicals on the central nervous system in the presence of functionally mature microglia.

Methylmercury causes neuronal cell death via M1-microglial activation in organotypic slices prepared from mouse cerebral cortex

“…M0-microglia are ramified with extended processes, and change to the spherical amoeboid-type when activated to M1-microglia (Cho and Choi, 2017). We recently found that microglia morphology could be changed from the ramified-type to the amoeboid-type by treatment with methylmercury in mouse cerebral slice cultures (Hoshi et al, 2019). Therefore, we first examined the effects of minocycline on the change in microglial morphology by methylmercury, and the pretreatment of 10 μM minocycline inhibited the reduction of the microglial process-es by methylmercury (Fig.…”

“…We prepared organotypic cerebral slices as we recently reported (Hoshi et al, 2019). Briefly, the cerebral cortices prepared from P7 mice were dissected and placed in ice-cold Hank's balanced salt solution (HBSS) containing 6 mg/mL glucose and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for 5 min.…”

“…Organotypic brain slices can be cultured while maintaining the cellular composition and structure of the brain, and has been used as a useful culture system for examining the intercellular communication networks in the brain. We recently improved the conventional preparation method for mouse brain slices, and produced mouse cerebral slice cultures in which most microglia exist in the mature state, as in the adult mouse brain (Hoshi et al, 2019). We also examined the effects of methylmercury, lead, tin and arsenic, which are known to exhibit neurotoxicity on M1-microglial activation using slice cultures, revealing that only methylmercury activated M0-microglia to M1-microglia (Hoshi et al, 2019).…”

“…We recently improved the conventional preparation method for mouse brain slices, and produced mouse cerebral slice cultures in which most microglia exist in the mature state, as in the adult mouse brain (Hoshi et al, 2019). We also examined the effects of methylmercury, lead, tin and arsenic, which are known to exhibit neurotoxicity on M1-microglial activation using slice cultures, revealing that only methylmercury activated M0-microglia to M1-microglia (Hoshi et al, 2019). However, the effects of activation to M1-microglia by methylmercury on neuronal cell death is unknown.…”

Cellular and Molecular Mechanisms Mediating Methylmercury Neurotoxicity and Neuroinflammation

“…In the presence of MeHg, microglia display an activated morphology characterized by a round shape with shorter, thicker, and less numerous processes in microglial primary cultures, organotypic cerebral cortex slices, hippocampal sandwich cultures, and animal models [122,123,[134][135][136][137]. Sholl analysis and time-lapse recordings show a reduction in morphological complexity and process extension and retraction activities [134].…”