Animals and study design
Male Wistar rats (Bantham and Kingman, UK; n = 5-7) aged 3 months (250-350 g) or 22 months (550-650 g), housed in pairs or groups of 4 respectively. All these experiments were performed under license (Department of Health, Ireland) and in accordance with local ethical guidelines.
In the first study, young and aged rats were anaesthetized with urethane (1.5 g/kg intraperitoneally) and transcardially perfused with 200-300 ml saline before tissue was harvested. In the second study, young and aged rats (n = 5-7) were divided into a control group and a group that received atorvastatin orally (5 mg/kg per day) for 8 weeks. Mean body weights of young animals (g) before and after treatment were 222 ± 9 and 356 ± 15 in the control group and 219 ± 7 and 340 ± 13 in the atorvastatin-treated group; the corresponding weights in aged rats were 534 ± 17 and 555 ± 22 (controls) and 527 ± 16 and 577 ± 22 (atorvastatin). In the third study, young rats, assigned to a control- or an Aβ-treated group, were subdivided into rats which did/did not receive atorvastatin (5 mg/kg per day) for 3 weeks; mean body weights (g) before and after treatment were 291 ± 12 and 337 ± 9 in the control group and 277 ± 13 and 336 ± 15 in the atorvastatin-treated group.
Ground atorvastatin calcium tablets (Lipitor™; Pfizer-Parke Davis, Ireland) were dissolved in vegetable oil (5 ml/150 g chow) and added to laboratory chow (Red Mills, Ireland); control animals received laboratory chow with added vegetable oil. The dose of atorvastatin was chosen on the basis of previous findings [3, 8]. Food intake (18 g and 25 g per rats per day for young and aged rats respectively) was assessed before and during the study to ensure animals received their full atorvastatin dose and fresh food was prepared every 2 days. Mean atorvastatin concentrations (± SEM; ng ATV/mg protein) in plasma and liver respectively were 0.008 ± 0.002 in young rats and 0.005 ± 0.002 in aged rats, and 0.026 ± 0.05 in young rats and 0.020 ± 0.05 in aged rats.
Aβ1-42 (5 μl; 200 μM; aggregated for 48 hours at 37°C; Biosource International Inc. US) or vehicle was delivered intracerebroventricularly (2.5 mm posterior and 0.5 mm lateral to bregma; depth 3.5 mm) to urethane-anaesthetized rats , on the last day of atorvastatin treatment. [We have previously established that treatment of animals with reverse peptide, Aβ40-1 exerted no effect on hippocampal expression of MHCII, CD40 or CD11b mRNA, or on hippocampal IL-1β at mRNA or protein level]. Rats were killed 4 hours after Aβ injection , the hippocampus was dissected and used for preparation of dissociated cells by flow cytometry or for analysis of mRNA or for western immunoblot.
Assessment of NK cells by flow cytometry
Following transcardial perfusion hippocampal tissue was harvested and passed through a cell strainer (70 μm) and centrifuged (170 × g, 10 min). The pellet was resuspended in PBS containing collagenase D (1 mg/ml) and DNase I (200 μg/ml), incubated at 37°C for 30 min and centrifuged (170 × g, 5 min). Pellets were resuspended in 1.088 g/ml Percoll (9 ml), underlaid with 1.122 g/ml Percoll (5 ml), and overlaid with 1.072 g/ml and 1.030 g/ml (9 ml each) Percoll and PBS (9 ml) and centrifuged (1250 × g, 45 min). The mononuclear cells (between the 1.088:1.072 and 1.072:1.030) were centrifuged and the pellets were washed, and blocked for 15 min at room temperature in FACS block (Mouse BD Fc Block (BD Pharmingen, UK); 1:500 in FACS buffer). Cells were incubated with CD161a-FITC (1:100 in FACS buffer; BD Biosciences, UK) and NKp30-PE (1:30 in FACS buffer; Santa Cruz Biotechnology Inc, USA). Immunofluorescence analysis was performed on a DAKO Cyan ADP 7 colour flow cytometer (DAKO Cytomation, UK) with Summit v4.3 software.
Isolation of NK cells
IgG-precoated magnetic beads (100 μl; 4 × 106 beads; monoclonal human anti-mouse IgG; Invitrogen, UK) were incubated overnight at 4°C with mouse anti-rat CD161 primary antibody (1 mg/ml: Serotec, UK) or mouse IgG (0.4 mg/ml; Santa Cruz, USA). Perfused hippocampal or cortical tissue was triturated in pre-warmed DMEM (Gibco BRL, Scotland), centrifuged (2000 × g, 4 min) and trypsinized (Tryple Express; Invitrogen, UK; 2 min, RT). Cells were centrifuged (2000 × g, 4 min), resuspended in DMEM and filtered 3 times, incubated with CD161-coated or IgG-coated beads (2 hours, 4°C) and collected by magnetic isolation. Cell isolation was checked by gel electrophoresis, and visualized following western immunoblot using a monoclonal anti-CD161 antibody (Serotec, UK) and a HRP-linked secondary antibody. These CD161+ cells were incubated with/without IL-2 (100 ng/ml; Biosource, USA) for 24 hours and IFNγ was assessed in supernatants.
In another experiment, a human NK cell line was grown in RPMI with glutamax (Gibco, UK) supplemented with foetal calf serum (10%; Gibco, UK), penicillin (100 U/ml; Gibco, UK), streptomycin (100 μg/mg; Gibco, UK) and IL-2 (20 ng/ml; R&D Systems, UK). Cells were washed (1200 g × 10 min) 3 times, resuspended in warmed media and incubated for 24 hours in the presence/absence of IL-2 (20 ng/ml). Supernatants were assessed for IFNγ by ELISA.
Preparation of mixed glial cultures
Primary cortical mixed glia were prepared from 1 day old male Wistar rats as previously described . Briefly cortical tissue was chopped, incubated for 5 minutes in 5% CO2 at 37°C in warm filter-sterilized Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, UK) supplemented with penicillin (100 μl/ml; Gibco, UK), streptomycin (100 μl/ml; Gibco, UK) and FBS (10% w/v; Gibco, UK). A single cell suspension was prepared by triturating tissue, filtering though a 40 μm sterile nylon mesh filter (BD Biosciences, USA), centrifuging at 2000 g for 3 minutes at 20°C, and resuspending the pellet 1 ml warm filter-sterilized culture media. Tissue was triturated and resuspended glia were counted, equal numbers of cells were plated onto 6 well plates and incubated for 2 hours in 5% CO2 at 37°C to allow the cells to adhere. After 2 hours the cells were flooded with 1 ml warm culture media and incubated for 3 days in 5% CO2 at 37°C. Culture media was replaced with fresh culture media every 3 days for 12 days until cells were ready for treatments. After 12 days in culture, cells were incubated in the presence or absence of IFNγ (50 ng/ml; 24 hours at 37°C; R&D Systems, UK). This concentration has been shown to robustly increase MHCII mRNA . Cells were harvested for analysis of MHCII and CD11b mRNA.
Total RNA was extracted from snap-frozen hippocampal tissue or cultured glial cells using a NucleoSpin® RNAII isolation kit (Macherey-Nagel Inc., Germany). Total RNA concentrations were determined by spectrophotometry, samples were equalised and cDNA synthesis was performed on 1 μg total RNA using a High Capacity cDNA RT kit (Applied Biosystems, Germany). Real-time PCR was performed using Taqman Gene Expression Assays (Applied Biosystems, Germany); the assay IDs for MHCII, CD11b, MCP-1 and IP-10 were Rn01768597_m1, Rn00709342_m1, Rn00580555_m1 and Rn00594648_m1, respectively. Real-time PCR was conducted using an ABI Prism 7300 instrument (Applied Biosystems, Germany). A 20 μl volume was added to each well (9 μl of diluted cDNA, 1 μl of primer and 10 μl of Taqman® Universal PCR Master Mix). Samples were assayed in duplicate (40 cycles; 95°C for 10 min, 95°C for 15 sec for each cycle (denaturation) and the transcription step at 60°C for 1 min). β-actin was the endogenous control (VIC-labeled MGB Taqman probe, Applied Biosystems, Germany; Assay ID: 4352341E). Gene expression was calculated relative to the endogenous control samples and to the control sample giving an RQ value (2-
DDCt, where CT is the threshold cycle)
Analysis of CD161 and NKp46 by western blot
Hippocampal tissue was equalized for protein, aliquots (10 μl) were loaded onto 10% gels, separated (30 mA; 60 min) and transferred onto nitrocellulose (225 mA; 90 min). Proteins were immunoblotted over night hours at 4°C with anti-CD161 antibody (1:500 in TBS containing 0.05% Tween-20/2% BSA; Serotec, UK) or anti-NKp46 antibody (1:500 in TBS containing 0.05% Tween-20/2% BSA; R&D Systems, UK). Membranes were washed, incubated with the HRP-linked secondary antibody (1:1000; anti-mouse IgG; Sigma, UK) Immunoreactive bands were detected with ECL (GE Healthcare, UK) chemiluminescence. Membranes were then stripped with Re-blot (Re-blot Plus Strong Solution; Chemicon, UK) for 10 min at room temperature (1:10 dilution; Chemicon International, US) and probed for actin expression (Sigma, UK). Bands were quantified relative to the endogenous control using densitometry (Labworks, UVP BioImaging Systems, UK).
Analysis of IFNγ and IL-2
Cytokines were assessed by ELISA in hippocampal tissue prepared from rats and, in the case of IFNγ, also in a human NK cell line. For IFNγ, 96-well plates were coated with monoclonal mouse anti-rat or anti-human antibody. Pre-coated 96-well plates (BioSource, US) were used for analysis of IL-2. Samples or standards, and subsequently detection antibody (biotinylated goat anti-rat or anti-human antibody), were added and after incubation in the presence of streptavidin-horseredish peroxidase, substrate solution was added . Cytokine concentrations were evaluated with reference to standard curves.
Data were analyzed using either Student's t-test for independent means, or a 1 way ANOVA followed by post hoc Student Newman-Keuls test. Data are expressed as means of between 5 and 7 replicates (± SEM).