Effect of Repetitive Lipopolysaccharide Exposure on Oligodendrocyte Differentiation at Different Developmental Stages: an In Vitro Study

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 an oligodendrocyte and microglial cell co-culture 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 induced microglia activation while did not have excessive cytotoxicity to OPCs. The co-culture was treated with 0.01 μg/mL LPS at the developmental stages D1 (early LPS group), D3 (late LPS group), or D1 and D3 (repetitive 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 OPCs. 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 repetitive LPS group 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.

본 연구는 희소돌기아교세포 (oligodendrocyte)와 미세아교세포 (microglia cell)의 공동 배양 환경에서 발달 단계에 따른 희소돌기아교세포의 분화에 대한 지질다당질 (Lipopolysaccharide; LPS)-유발 감염 및 염증의 영향과 반복 자극(repetitive-hit) 모델에 대한 연구로서 진행되었다. 생후 2일된 쥐의 뇌에 있는 희소돌기아교세포와 미세아교세포를 추출하여 통제 집단, 1일차 LPS노출 집단(초기), 3일차 LPS 노출 집단(후기), 1일 및 3일차 LPS 노출 집단(초기 및 후기; repetitive)의 네 가지 조건을 만들어 MBP와 NG2 수준을 분석하였다. 그 결과 통제 집단과 비교했을 때, 초기 LPS 노출은 MBP 수준을 낮추고 희소돌기아교세포 분화에 영향을 미쳐 뇌세포의 발달을 저해한 반면, 후기 LPS 노출만으로는 뇌세포 발달에 영향을 미치지 않은 것을 확인하였다. 또한 초기 및 후기 모두 LPS 자극을 받은 경우, NG2 세포의 비율이 크게 증가하여 희소돌기아교세포 계통의 발달이 더 심하게 저해된 것을 알 수 있었다. 이를 통해 희소돌기아교세포 초기 발달 단계에서의 LPS 노출이 뇌의 염증 취약성을 유발시킬 수 있으며, 반복되는 LPS 자극에 의해 염증 자극에 대한 감수성이 증가될 수 있음을 추측할 수 있다.