Figure 5
Reduction of astrocytes in the Xpnpep1−/− hippocampus. (a) Astrocytes in the hippocampal CA3 regions from 4- to 5-week-old WT (top) and Xpnpep1−/− (bottom) mice were stained with GFAP and S100β antibodies. DAPI was used to visualize cell nuclei, and arrows indicate vacuoles. Immunofluorescence images of hippocampal CA3 regions show fewer astrocytes in Xpnpep1−/− mice than in WT mice. (b,c) Astrocytes in the CA1 (b) and DG (c) subfields of Xpnpep1+/+ (top) and Xpnpep1−/− (bottom) mice are visualized. Scale bars, 50 µm (a–c). SP, stratum pyramidale; SR, stratum radiatum; SO, stratum oriens; ML, molecular layer; GL, granule cell layer. (d–f) The density of GFAP- and S100β-positive cells in the CA3, CA1, and DG regions were lower in Xpnpep1−/− mice than in WT mice. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t-test, n = 6 slices from 3 mice for each genotype. (g) The morphology of astrocytes and cell nuclei in the hippocampal CA3 subfield were visualized by GFAP (green) and DAPI (blue), respectively. The morphological features of reactive astrocytes, such as hypertrophy and extension of processes, were not detected in Xpnpep1−/− astrocytes. Arrow indicates vacuole. Scale bars, 10 µm. (h) Sholl analysis of astrocyte complexity in the hippocampal CA3 subfield. n = 28 cells from 3 mice per genotype. p > 0.05 by Student’s t-test and Mann–Whitney test. (i) Representative western blots showing the expression levels of aminopeptidase P1 and GFAP in the hippocampal extracts. Western blotting using anti-α-tubulin antibody was performed to ensure equal protein loading and transfer, and quantification of protein levels in each sample. (j) Quantification of GFAP protein levels (right) in the Xpnpep1+/+ (n = 4) and Xpnpep1−/− (n = 4) hippocampus. t(6) = 4.62, **p = 0.0036 by Student’s t-test.
