Alleviation of the complement activation, inflammation, and neurodegeneration in double knockout (DKO) mice by disruption of complement C3 gene. (A) To clarify the involvement of complement systems in the inflammatory reactions and neurodegenerations in DKO mice, DKO- and C3-deficient mutant mice were mated, generating triple knockout (TKO) mice. (B) Expression levels of complement mRNAs from the cerebellum of 40-week-old mice were analyzed for C1qα, C3, IL-1β, and TNFα genes with real-time reverse transcription-polymerase chain reaction (RT-PCR). Relative expression levels are presented after correction by the GAPDH gene. The numbers of mice examined were as follows: wild-type (WT) (n = 5), DKO (n = 5), TKO (n = 5), and C3KO (n = 3); data are presented as mean ± standard deviation (SD) and were analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc test (*P <0.05; **P <0.01; ***P <0.001). (C) Immunohistostaining of F4/80 (microglia marker) in the spinal cord of 40-week-old WT and DKO mice by using monoclonal anti-mouse F4/80 and Alexa Fluor 488-conjugated anti-rat IgG2b. The number of microglia was quantified as an average in a visual field of the fluorescence microscope (×400). Numbers of section area examined were as follows: WT (n = 3), DKO (n = 3), TKO (n = 3), and C3KO (n = 3); data are presented as mean ± SD and were analyzed by one-way ANOVA with Tukey-Kramer post hoc test (**P <0.01; ***P <0.001). The thickness of sections was 7 μm (scale bar, 50 μm).