Figure 3

Neutralization of Toll-like receptor 4 (TLR4) with its antibody attenuated the hypoxia-induced expression of TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS) in primary cultured microglia. TLR4 was neutralized with its antibody to assess its role in production of inflammatory factors. (A) Confocal images showing the expression of TNF-α (d-f), IL-1β (m-o) and iNOS (v-x) in primary microglia labeled with lectin (a-c, j-l, s-u; green) in different groups. Very weak TNF-α, or almost undetectable IL-1β and iNOS immunoexpression is detected in the control microglia (g, p, y). The immunoflurescence intensity is enhanced in microglia subjected to hypoxia exposure (h, q, z). This increased immunofluorescence intensity is attenuated in microglia exposed to hypoxia but pretreated with TLR4 Ab (i, r, za). TNF-α (B) and IL-1β (C) concentration in the supernatant of microglia in different groups was investigated with ELISA. Release of TNF-α and IL-1β is significantly increased after hypoxia in comparison to the control levels. The hypoxia-induced increase in TNF-α and IL-1β release was suppressed when the microglia were neutralized with TLR4 antibody. Significant differences in protein levels between different groups are indicated as *P <0.05 and **P <0.01. The values represent the mean ± SD in triplicate. Scale bars in A = 20 μm.