Fig. 6

Higher Il-1β expression occurs mainly in aged SAMP8 CD45⁺ brain parenchymal cells. qRT-PCR analysis of Il-1β mRNA expression. (a) (i) Scheme of BP CD45 magnetic cell separation used for CD45⁺ isolation, and histograms (bottom) showing the CD45 staining by cytometry from CD45⁺ and CD45⁻ fraction. Quantification of BP CD45⁺ obtained in positive fraction that was of 97 ± 2% (arithmetic mean ± SEM (n = 5) of CD45⁺ cells from alive cells, determined as in additional Fig 2s. (ii) Il-1β mRNA expression from BP CD45⁺ isolated cells from young (2 months) and aged (10 months) SAMR1 (green) and SAMP8 (red) animals. Data in the box and whisker plot (as in Fig. 5a iii) are presented as dCt (Ct _il1β-Ct _36b4). Lower values of dCt means higher Il-1β transcript expression in the sample. (iii) Relative quantification of Il-1β mRNA expression was referred to average values of 2 months old SAMR1 as control animals that were given a value of 1. Plot represents individual values are mean ± SEM of n = 3 to 6 male brains. SAMR1 is shown in green and SAMP8 in red. Two months data are represented with triangles and 10 months data with circles. (b) (i) Dissected choroid plexus and meningeal membranes (m/Ch) were pooled into an Eppendorf tube and total RNA was obtained as indicated in material and methods. Specific brain regions were stained with anti CD31 for brain vessels (red), Iba1 (green) and DAPI for nuclei (blue), and representative images of these areas were taken with a SP5 Leica TCS confocal fluorescent microscope, show abundance of Iba1⁺ cells (green) by immunofluorescence in both brain localizations. Scale bars are included in the images. (ii) Il-1β mRNA expression from m/Ch preparations from young (2 months) and aged (10 months) SAMR1 (green) and SAMP8 (red) animals. Specific mRNAs were amplified from total mRNA qRT-PCR using 36b4 as reference gene. Data are presented as dCt (Ct _il1β-Ct _36b4) as in panel A (ii). Lower values of dCt means higher Il-1β transcript expression in the sample. (iii) As before, relative quantification of Il-1β mRNA expression was referred to average values of 2 months old SAMR1 m/Ch values as control animals that were given a value of 1. Two months data are represented with triangles and 10 months data with circles. SAMR1 is shown in green and SAMP8 in red. Plot represents individual values are mean ± SEM of n=3 to 6 male brains. **p < 0.01 and ***p < 0.001: (ns) means non-significant differences, n ≥ 3 male