Fig. 1.

Neo-self-antigen–specific Th1 cells trigger focal hypothalamic inflammation but no loss of orexinergic neurons. (A–D) Immunohistochemistry staining for CD3 (violet) and orexin-A (brown) in Orex-HA mice (A and C) and WT littermate control mice (B and D) 8 d after transfer of 3 × 10⁷ neo-self-antigen–specific Th1 cells. (E and F) Immunohistochemistry staining for Iba-1 (violet) and orexin⁺ neurons (brown) in Orex-HA (E) and WT (F) mice. Representative results from four or five mice per group are shown. Arrowheads in C point to infiltrating CD3⁺ cells. [Scale bars: 200 μm (A and B) or 50 μm (C–F).] (G) Quantification of CD3⁺ T cells in the hypothalamus of WT or Orex-HA mice at different time points after Th1 injection. Each symbol represents an individual mouse. Results are expressed as mean ± SEM of four or five mice per group for each time point. (H) Representative FACS plots of brain-infiltrating cells from WT or Orex-HA animals at day 8 after Th1 transfer. Representative results from three independent experiments are shown. (I and J) Frequency of CD11c⁺ among CD45^high CD11b⁺ cells was assessed by flow cytometry (I) and MHC class II expression on CD45^dim CD11b⁺ Thy1.2⁻ microglia (J). Results are expressed as mean ± SEM of 12 mice per group from three independent experiments. (K and L) At 60 d after Th1 cell transfer, double immunohistochemistry, staining for orexin⁺ neurons (black) and MCH⁺ neurons (brown) in Orex-HA mice (K) and WT controls (L). Representative results from seven or eight mice per group are shown. [Scale bars: 125 μm (K and L).] (M and N) The density of orexin⁺ neurons (M) and MCH⁺ neurons (N) in Orex-HA and WT mice is shown at day 60 after Th1 injection. Results are expressed as mean ± SEM of seven or eight mice per group from two independent experiments. Statistical analysis was performed by using the Mann–Whitney u test. ns, not significant. *P < 0.05; ***P < 0.001.