Effects of lipoic acid on primary murine microglial cells

Abstract

The anti-oxidant lipoic acid (LA) is beneficial in murine models of multiple sclerosis (MS) and has recently been shown to slow brain atrophy in secondary progressive MS. The mechanism of these effects by LA is incompletely understood but may involve effects on microglia. The objective of this study is to understand how LA affects microglial cells. We cultured primary microglial cells from C57BL/6 adult mice brains and stimulated the cells with lipopolysaccharide (LPS) and interferon gamma (IFN-γ) in the presence or absence of LA. We demonstrate the inhibition of phagocytosis, rearrangement of actin, and formation of membrane blebs in stimulated microglia in the presence of LA. These experiments suggest that LA causes changes in microglial actin, which may lead to alterations in phagocytosis, mobility, and migration.

Keywords: Actin; Blebs; Cytoskeleton; Microglia; Phagocytosis.

Fig. 1.

Fluorescent beads engulfed by an activated microglial cell. The z stack image taken on a Zeiss confocal microscope. Ortho image assembled from a Z-stack. The image shows XZ plane (green line) and YZ plane (red line) through the stack of images. Section of the XY plane (blue) is the slice plane of the stack. The result of an orthogonal section is visible at the image margins (above and right). Scale bar 5 μm.

Fig. 2.

LA decreases phagocytosis. a. LPS/IFN-γ stimulated cells b. LPS/IFN-γ stimulated cells in presence of LA 25 μg/ml. Beads are red and microglial cells stained with anti-CD11b are green. Scale bar 50 μm.

Fig. 3.

LA causes disorganization of actin. After 24 hours of stimulation, microglial cells were stained with phalloidin. a. LPS/IFN-γ stimulated cells shows normal fibrillary pattern of staining for actin b. LPS/IFN-γ stimulated cells co-cultured with LA 25 μg/ml shows loss of normal pattern and clumping of actin. Scale bar 10 μm.

Fig. 4.

Morphological alterations were seen in stimulated cells in absence and presence of LA. The cells are outlined in blue to highlight difference in cell size. a. LPS/IFN-γ stimulated cells b. LPS/IFN-γ stimulated cells that were also treated with LA 25 μg/ml. Microglial cells were fixed with paraformaldehyde and stained with WGA after 24 hrs. Arrows indicate blebs. Scale bar 10 μm.

Fig. 5.

LA induces the formation of blebs. After 6 hours of stimulation, live cells were stained with WGA and images were acquired on a confocal using z stack (20X, 2 zoom). Arrows show blebs. a. LPS/IFN-γ stimulated cells b. LPS/IFN-γ stimulated cells co-cultured with LA 25 μg/ml. Scale bar 20 μm.