2.1. Animals
Wild type (WT) C57Bl6J and transgenic mice overexpressing normal human SOD1 were used, derived from a total cohort of 240 male mice. Both types of animals were assigned to receive either a control diet (CD) or a calorie restricted diet. Details on the generation and characterization of the cohort, preparation of the diets, and weight and survival curves of the animals have been described previously (
Rutten et al., 2010;
Weindruch et al., 1986). Briefly, the cohort was generated from 4 breeder pairs of female WT C57Bl6J and male transgenic hemizygous for SOD1 on a C57Bl6J background (carrying 7 copies of the entire human SOD1 sequence, in order to achieve increased expression and enzyme activity in brain and other tissues;
Epstein et al., 1987;
Przedborski et al., 1992). The animals were assigned to the 2 diet groups after weaning and for their entire life. The reduction of caloric intake in the CR groups was approximately 50%, while the CD was approximately 85% of the ad libitum consumption, in order to monitor the caloric intake and ensure that all the food pellets would be consumed. The detailed compositions, feeding patterns, and the weight and survival curves have been reported by (
Rutten et al., 2010). All experiments were carried on the Central Animal Facilities, Maastricht University, Maastricht, The Netherlands, and the animals were maintained under standard temperature and humidity conditions on a 12-hour light-dark circle, housed individually, with water available ad libitum, under specified pathogen-free conditions. All experiments were approved by the Animals Ethics Board of Maastricht University.
2.2. Experimental design
Four groups were generated, based on the genotype and diet of the animals: (1) WT mice on control diet (WT-CD), (2) SOD1 mice on control diet (SOD1-CD), (3) WT mice on caloric restriction (WT-CR), and (4) SOD1 mice on caloric restriction (SOD1-CR). Six animals from each group were euthanized at the age of 12 months and another 6 animals per group at 24 months of age for histological analyses.
2.3. Tissue processing
The animals were deeply anesthetized and transcardially perfused with 20 mL tyrode solution and 20 mL of fixative solution (4% parafolmaldehyde, 0.9% NaCl) followed by 30 mL of a second fixative solution (8% parafolmaldehyde, 0.9% NaCl, 1% acetic acid). After removal, the brains were post-fixed at 4 °C for 24 hours in the 8% parafolmaldehyde, without the acetic acid. Consequently, the brains were hemisected in the midsagittal line, cryoprotected in sucrose solution (10%, 20%, and finally 30% sucrose in Tris-HCl buffer, 2 × 12 hours per solution at 4 °C) and embedded in Tissue Tek® (Sakura Finetec Europe, Zoeterwoude, The Netherlands). Then, the left brain halves were quickly frozen and stored at −80 °C, until they were cut serially in 30-µm thick free-floating coronal sections using a cryostat (type HM 500 OMV, Microm, Walldorf, Germany), yielding 10 subseries of every tenth section, and stored until further histological processing. The right brain halves were not used in the present study.
2.4. Immunohistochemical detection of 5-mC
Using standard immunohistochemical procedures that were previously described (
Chouliaras et al., 2011), with a mouse monoclonal anti-5-mC (dilution 1:500; GenWay Biotech, San Diego, CA, USA) as primary antibody and a donkey antimouse biotine (dilution 1:200; Jackson, Westgrove, PA, USA) as a secondary antibody, a series of sections containing the hippocampus were stained. In order to visualize the reaction product, the sections were incubated in 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution (1:1 3,3′-diaminobenzidine tetrahydrochloride: Tris-HCl, 0.3% H
2O
2) (Sigma, Uithoorn, The Netherlands) for 10 minutes. Control experiments, omitting the primary antibody or using an anti-5-mC primary antibody from another company (Santa Cruz, CA, USA) confirmed the specificity of immunoreactivity (data not shown).
2.5. Immunofluorescent labeling of 5-mC and DAPI
After incubation with the primary and secondary antibodies (see above), the sections were incubated with streptavidin Alexa Fluor 594 conjugate (Invitrogen, Eugene, OR, USA) and counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich, Zwijndrecht, The Netherlands).
2.6. Analysis of 5-mC IR
2.6.1. Mean intensity and surface area of 5-mC IR Gray value measurements and percentage of surface area below (i.e., darker as compared with) the calculated threshold (see below) were used for the assessment of 5-mC IR. As illustrated using the white boxes in , 4 images from the granule cell layer of the dentate gyrus (DG), 2 images from the CA3 cell layer, and 2 images from the CA1–2 regions were taken in 4 selected sections per series, at 4 bregma levels (−1.58, −1.82, −3.40, and −3.52) according to
Franklin and Paxinos (1997). Thus, a total of 32 images per animal were analyzed. The images were taken using the 40× objective, with a digital camera (f-view; Olympus, Tokyo, Japan) connected to an Olympus AX70 brightfield microscope (analySIS; Imaging System, Münster, Germany). The pattern of the staining revealed densely stained small particles, reflecting hypermethylated DNA fragments (
Brown et al., 2008;
Hernandez-Blazquez et al., 2000). Threshold values were set in order to correct for background signal (
Strackx et al., 2008). Surface area was defined as the percentage of the area delineated with a gray value below the background threshold. Further, the surface area measurements were corrected for total hippocampal volume differences (
Rutten et al., 2010), simply by multiplying the surface area by the volume. However, the surface area measurements, despite the performed corrections, might still be hampered by hippocampal volume differences. Therefore, in addition, mean gray values were calculated in order to further replicate the results. The mean gray value was defined as the sum of gray values of all pixels below the calculated threshold in the selected area divided by the number of all pixels, and such measurement is independent of volume or even cell number differences. Results on gray values are represented as mean intensity, i.e., the inverse mean gray value, with higher intensities representing increased IR.
2.6.2. Imaging of 5-mC and DAPI Colocalization of 5-mC and DAPI was demonstrated after double immunofluorescence (). Image stacks of 16-µm thick and consisting of 80 confocal images (0.2 µm apart) of double-labeled cell particles were made with a 100× objective (Olympus UPlanSApo; numerical aperture [NA] = 1.40) and the SI-SD system (MBF Bioscience, Williston, VT, USA). The system consisted of a modified Olympus BX51 fluorescence microscope with customized spinning disk unit (DSU; Olympus), computer-controlled excitation and emission filter wheels (Olympus), 3-axis high-accuracy computer-controlled stepping motor specimen stage (4 × 4 Grid Encoded Stage, Ludl Electronic Products, Hawthorne, NY, USA), linear z-axis position encoder (Ludl), ultra-high sensitivity monochrome electron multiplier CCD camera (1000 × 1000 pixels, C9100-02, Hamamatsu Photonics, Hamamatsu City, Japan) and controlling software (StereoInvestigator; MBF BioScience, Williston, VT, USA).
2.7. Correlation analysis
Dnmt3a IR was assessed previously in sections from the same mouse brains that were used in the present study. Pearson’s correlation coefficient (rp) was calculated for gray values when correlating Dnmt3a IR with 5-mC IR per hippocampal subregion per animal.
2.8. Statistical analysis
All data are presented as mean and standard error of the mean. The general linear model univariate analysis of variance was used for comparisons between groups, accounting for the main and interactive effects of age, genotype, and diet. Statistical significance was set at an α level of 0.05. Pair-wise comparisons were performed with a Bonferroni post hoc test. All statistical calculations were performed using the Statistical Package for the Social Sciences, (SPSS 16, SPSS, Inc., Chicago, IL, USA). Graphs were built in GraphPad Prism (Version 4, GraphPad Software, San Diego, CA, USA).
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