J Korean Med Sci.  2021 Dec;36(49):e332. 10.3346/jkms.2021.36.e332.

Effects of Lipopolysaccharide on Oligodendrocyte Differentiation at Different Developmental Stages: an In Vitro Study

• Ahn JH
• Lee HJ
• Lee K
• Lim J
• Hwang JK
• Kim CR
• Kim HA
• Kim HS
• Park HK

• KMID: 2523552
• DOI: http://doi.org/10.3346/jkms.2021.36.e332

Abstract

Background
Lipopolysaccharide (LPS) exerts cytotoxic effects on brain cells, especially on those belonging to the oligodendrocyte lineage, in preterm infants. The susceptibility of oligodendrocyte lineage cells to LPS-induced inflammation is dependent on the developmental stage. This study aimed to investigate the effect of LPS on oligodendrocyte lineage cells at different developmental stages in a microglial cell and oligodendrocyte coculture model.
Methods
The primary cultures of oligodendrocytes and microglia cells were prepared from the forebrains of 2-day-old Sprague–Dawley rats. The oligodendrocyte progenitor cells (OPCs) co-cultured with microglial cells were treated with 0 (control), 0.01, 0.1, and 1 µg/mL LPS at the D3 stage to determine the dose of LPS that impairs oligodendrocyte differentiation. The co-culture was treated with 0.01 µg/mL LPS, which was the lowest dose that did not impair oligodendrocyte differentiation, at the developmental stages D1 (early LPS group), D3 (late LPS group), or D1 and D3 (double LPS group). On day 7 of differentiation, oligodendrocytes were subjected to neural glial antigen 2 (NG2) and myelin basic protein (MBP) immunostaining to examine the number of OPCs and mature oligodendrocytes, respectively.
Results
LPS dose-dependently decreased the proportion of mature oligodendrocytes (MBP+ cells) relative to the total number of cells. The number of MBP+ cells in the early LPS group was significantly lower than that in the late LPS group. Compared with those in the control group, the MBP+ cell numbers were significantly lower and the NG2+ cell numbers were significantly higher in the double LPS group, which exhibited impaired oligodendrocyte lineage cell development, on day 7 of differentiation.
Conclusion
Repetitive LPS stimulation during development significantly inhibited brain cell development by impairing oligodendrocyte differentiation. In contrast, brain cell development was not affected in the late LPS group. These findings suggest that inflammation at the early developmental stage of oligodendrocytes increases the susceptibility of the preterm brain to inflammation-induced injury.

Keyword

Lipopolysaccharide; Preterm; Periventricular Leukomalacia; Oligodendrocyte; Microglia

Figure

• 

  Fig. 1 Schematic outline of the experimental protocol. The oligodendrocyte-microglial cell co-culture was treated with the lowest dose of LPS (0.01 µg/mL), which does not inhibit oligodendrocyte differentiation, at the developmental stages D1 (early LPS group), D3 (late LPS group), or D1 and D3 (double LPS group).LPS = lipopolysaccharide, MBP = myelin basic protein.

• 

  Fig. 2 Effect of various doses of LPS on oligodendrocyte differentiation. On day 3, the co-culture was treated with 0.01, 0.1, and 1 µg/mL LPS. On day 7 of differentiation, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). The oligodendrocyte-microglia co-culture was subjected to MBP (green; mature oligodendrocyte marker) and IBA1 (red; microglia marker) immunostaining. The co-culture was treated with vehicle (control) (A) and 0.01 (B), 0.1 (C), and 1 µg/mL LPS (D). Scale bar = 100 µm (200 × magnification).LPS = lipopolysaccharide, MBP = myelin basic protein.

• 

  Fig. 3 Representative images of oligodendrocytes treated with various doses of LPS on day 7 of differentiation (n = 4/group).LPS = lipopolysaccharide, OPC = oligodendrocyte progenitor cell, MBP = myelin basic protein, MG = microglia.P values from Mann-Whitney U test: a0.007 vs. control; b0.009 vs. OPC + MG + LPS 0.1. P values from one-way analysis of variance analysis of variance with Bonferroni: c< 0.001 vs. control.

• 

  Fig. 4 Representative images of oligodendrocyte-microglia co-culture following different schedules of LPS treatment. Representative images of the oligodendrocyte-microglia co-culture subjected to immunostaining after in vitro treatment with vehicle (control) (A) or 0.01 µg/mL LPS at D1 (early LPS group) (B), D3 (late LPS group) (C), or D1 and D3 (double LPS group) stages. Scale bar = 100 µm (200 × magnification).LPS = lipopolysaccharide, MBP = myelin basic protein.

• 

  Fig. 5 Quantification of the relative numbers of NG2+ and MBP+ cells. Relative numbers of NG2+ and MBP+ cells were quantified after treatment with vehicle (control) or 0.01 µg/mL LPS at D1 (early LPS group), D3 (late LPS group), or D1 and D3 (double LPS group) stages. The number of MBP+ cells in the early LPS group was significantly lower than that in the control group. Compared with those in the control group, the MBP+ cell numbers were significantly lower and the NG2+ cell numbers were significantly higher in the double LPS group at day 7 of differentiation (n = 5/group).LPS = lipopolysaccharide, MBP = myelin basic protein.All P values from one-way analysis of variance analysis of variance with Bonferroni; a< 0.001 vs. control; b< 0.001 vs. early LPS; c0.004 vs. early LPS; d< 0.001 vs. late LPS.

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