Figure 4.

Il33^−/− mice fail to initiate the repair of rapidly increasing DNA double-strand breaks (DSBs) in the cortical and hippocampal neurons after 40 weeks. (a) Terminal-deoxynucleotidyl-transferase dUTP nick-end labeling (TUNEL) reveals the accumulation of DSBs (green) in neurons in the cortex and hippocampus (CA) of a 40-week Il33^−/− mouse, in comparison to a 40-week wild-type (WT) mouse. (b) Co-staining of TUNEL (green) with tubulin β3 (red) for numbered boxes in a shows that neurons are TUNEL⁺, but glial cells (arrowheads) are TUNEL negative. (c) Time course of TUNEL density in brains reveals a rapid increase in TUNEL⁺ neurons in Il33^−/− mice between 35 and 40 weeks. TUNEL⁺ density in each brain is quantitated as total fluorescent intensity/brain section area. Liner progression was used for comparison between WT and Il33^−/− group, and four-parameter progression for construction of curves. (d) Immunofluorescence demonstrates incorporated BrdU (red) in the nuclei of WT (left) but not in Il33^−/− (right) neurons in cortex and hippocampus at 40 weeks (n=4). (e) Enlarged numbered boxed areas in d showed nuclear BrdU (red) only in WT neuronal nuclei. (f) Quantitation of BrdU incorporation in hippocampal dentate gyrus (n=4 per age). Each dot represents BrdU incorporation in one neuron (fluorescent intensity/nuclear area). (g) Immunofluorescence reveals PγH2AX (red) in the cortex and hippocampus (CA) of WT mice, but not in Il33^−/− mice, at 40 weeks (n=4). Note heavy loss of neurons/neurites in Il33^−/− mice as revealed by co-staining for tubulin β3 (green). (h) Enlarged-numbered-boxed areas in g show PγH2AX (red) only in WT neuronal nuclei (green). (i) Summary of PγH2AX⁺ neuron density (%) in dentate gyrus at various ages (n=3 per group). (j) Western blot on cortical proteins shows reduced PγH2AX in Il33^−/− mice at 40 weeks. (k) Distribution of PγH2AX⁺ cells in the mouse brains at 40 weeks (n=3). Cell densities (%) are indicated by color scale.