Materials and Methods
15- and 135-week-old Wild-type (WT) and GRN-deficient (KO) C57BL/6J male mice were used for investigating the difference between the genotypes in young and old mice. The animals were housed at constant ambient temperature of 24 ?C with controlled lighting (lights on, 0700-1900 hr), and given free access to food and water. All animal handling and experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, College of Animal Science and Technology, Henan University of Science and Technology.
Mice were decapitated, the brain was quickly removed from the skull, and the hippocampus was excised, fresh-frozen in liquid nitrogen, and stored at -80 °C until gene expression analysis by real-time reverse transcription (RT)-PCR. In addition, in order to determine the specific contribution of PGRN in mediating the influence of aging on neuronal activity, WT and KO mice were deeply anesthetized with pentobarbital (Kyoritsu Seiyaku, Tokyo, Japan; i.p.) and then transcardially perfused with chilled saline followed by 4% paraformaldehyde in 0.02 M phosphate buffered saline (PBS, pH 7.2), and then the whole brain was quickly removed. Brains were postfixed in the same fixative overnight and then submerged in 10% sucrose solution overnight, 20% sucrose solution for 24 h, and 30% sucrose until the brain completely sank.
Total RNA was isolated from the hypothalamus using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. RT reactions were performed as previously described (42). Briefly, 2 ?g total RNA and 1 ?l Oligod (T)16 (50 ?M, Applied Biosystems, Branchburg, NJ, USA) were heated at 65 °C for 15 min. Subsequently, 1 ?l SuperScript II reverse transcriptase (200 U/?l, Invitrogen Life Technologies, Carlsbad, CA, USA), 4 ?l 5× First-Strand buffer (Invitrogen), 5 ?l (10 mM) dNTP Mix with dTTP (2.5 mM each, Applied Biosystems, Warrington, UK), 2 ?l DTT (0.1 M), and 2 ?l RNaseOUT (40 U/?l, Invitrogen) were added, and the mixture was incubated at 42 °C for 60 min. Finally, the reaction was inactivated by heating at 94 °C for 10 min. Target-specific gene levels were quantified by real-time RT-PCR using the LightCycler 2.0 system according to the manufacturer’s protocols (Roche Diagnostics GmbH, Mannheim, Germany). Each reaction mixture contained 5 ?l cDNA synthesized from total RNA, 10 ?l THUNDERBIRD SYBR qPCR Mix (Toyobo, Co., LTD., Osaka, Japan), 3 ?l diethyl pyrocarbonate treated water, and 1 ?l (10 µM) of each forward and reverse primer. The following primer sets were used: ionized calcium-binding adapter molecule 1 (Iba-1) forward: 5?-GGATTTGACGGGAGGAAAA-3? and reverse: 5?-TGGGATCATCGAGGAATTG-3?; lysozyme M (Lyz2) forward: 5?-GAATGGAATGGCTGGCTACT-3? and reverse: 5?-CGTGCTGAGCTAAACACACC-3?; macrophage expressed gene 1 (Mpeg1) forward: 5?-ACTGTCAGGTGATGTTCTTTGC-3? and reverse: 5?-AAATCACCTGTGAGCAGGTTC-3?; cathepsin Z (Ctsz) forward: 5?-AACCAGCACATCCCACAGTA-3? and reverse: 5?-TTGATGTTGATTCGGTCTGC-3?; IL-1? forward: 5?- AGTTGACGGACCCCAAAAG-3? and reverse: 5?-AGCTGGATGCTCTCATCAGG-3?; IL-6 forward: 5?- GCTACCAAACTGGATATAATCAGGA-3? and reverse: 5?- CCAGGTAGCTATGGTACTCCAGAA-3?; TNF-? forward: 5?-CTGTAGCCCACGTCGTAGC-3? and reverse: 5?- TTGAGATCCATGCCGTTG-3?. For standardization, the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) was evaluated as an internal control: HPRT forward: 5?-AGTCCCAGCGTCGTGATTAGCCAT-3? and reverse: 5?-CTTGAGCACACAGAGGGCCACAAT-3?. PCR amplification was run for 45 cycles with three repeating steps that consisted of denaturation at 95 ?C for 5 s, annealing of primers at optimal temperature for 30 s, and extension at 72 ?C for 60 s. Relative mRNA levels of each target gene were normalized to the housekeeping gene HPRT (41).
Immunohistochemistry for c-Fos
Brain sections of 30 ?m thickness were cut using a cryostat (Microm HM550, Thermo Scientific, Waltham, MA, USA) and collected serially in a 24-well plates containing PBS (0.02 M, pH 7.2). One out of every six slices was used for immunostaining for c-Fos protein. Free-floating sections were rinsed with PBS for 10 min three times. Sections were then incubated with 0.3% H2O2 in PBS for 30 min at room temperature and rinsed with PBS for 10 min three times. Thereafter, sections were incubated in blocking solution (Block Ace, Snow Brand Milk Products Co., Sapporo, Japan) for 2 hr. Tissue sections were incubated in rabbit anti-Fos primary antibody (1:5000 dilution with 0.02 M PBS containing 1% BSA, 0.3% Triton X-100; Ab-5, Oncogene Research Product, Calbiochem, MA, USA) at 4 ?C for 60 hr, washed three times with 0.02 M PBS containing 0.03% Triton X-100 (0.03% PBST), and then the sections were incubated with the biotinylated goat-anti-rabbit secondary antibody IgG (1:800 dilution with 0.02 M PBS containing 0.3% Triton X-100; Vector Laboratories, Burlingame, CA, USA) at room temperature for 2 hr. Sections were then rinsed three times with 0.03% PBST and incubated at room temperature for a further 2 hr with an avidin-biotin-horseradish peroxidase complex solution (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA). Finally, sections were treated for approximately 1.5 min in 0.5 mg/ml diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA; dissolved in 0.02 M PBS, with 0.01% hydrogen peroxide and 0.25% nickel chloride). The sections were mounted on slides, air dried, dehydrated in ethanol solutions and xylene, and coverslipped.
Quantification of c-Fos immunoreactive cells
Brain regions were identified using the mouse brain in stereotaxic coordinates (Paxinos and Franklin, 2008). The number of c-Fos-immunoreactive (IR) cells in the paraventricular nucleus (PVN) of hypothalamus was counted using an Olympus Optical System (BX-51, Olympus Optical Co., Tokyo, Japan) and the digitized images were analyzed with IPLab image analyzing software (Scanalytics Corp., Fairfax, VA, USA). A threshold was set to delineate c-Fos-positive stained nuclei from background, and only cells above the threshold were included. Up to four sections per animal for each region were quantified bilaterally and then averaged.
All statistical analyses were performed with SPSS 20.0 Version (SPSS Inc., Chicago, IL, USA). Data were analyzed by analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test. Differences at p ; 0.05 were considered statistically significant. Data are presented as the mean ± SEM.