Mice and diets
C57BL/6 male mice (8-week-old; Harlan UK Limited, UK) were randomly assigned a high-fat diet (60 % energy from fat, 35 % fat content by weight, 13 % saturated fatty acids, 58G9, Test Diets®, supplied by IPS Product Supplies Ltd, UK) or control diet (12 % energy from fat, 5 % fat content by weight, 0.78 % saturated fatty acids, 58G7) and housed in groups of 4–5. Separate groups of mice were then maintained on their respective diets for 2, 3, 4 or 6 months. Each duration of feeding diet (i.e. time on diet) was performed as a separate experiment, and randomisation to diet was therefore done independently for each experiment. All mice were given ad libitum access to their respective diets and water and were housed at a constant ambient temperature of 21 ± 2°C on a 12-h light, 12-h dark cycle (lights on at 0800 h). All experimental procedures using animals were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986 and approved by the Home Office and the local Animal Ethical Review Group, University of Manchester.
Measurement of physiological and haematological parameters
Body weight was monitored in all mice. In tail vein blood samples, blood glucose was measured using a hand-held glucose monitor (Accu-Check Aviva, Roche, UK) and blood cell counts were analysed using a haematometer (Pentra ES 60, Horiba Ltd, UK). Blood pressure was measured in conscious mice using a tail cuff system (BP-2000, Visitech Systems, USA).
Focal cerebral ischaemia
Focal ischaemia was induced by transient middle cerebral artery occlusion (MCAο). Briefly, under isoflurane anaesthesia (in a mixture of 30 % oxygen and 70 % nitrous oxide), the carotid arteries were exposed and a 6–0 silicon rubber-coated monofilament (Doccol, USA) with a 2-mm tip (210 μm diameter, coating length 405 mm) was introduced into the external carotid artery and advanced along the internal carotid artery until occluding the origin of the MCA. Cerebral blood flow was monitored in all mice by laser-Doppler (Moor Instruments, UK), and middle cerebral artery occlusion (MCAo) was confirmed by a drop in cerebral blood flow of at least 2 0% of baseline. If this drop in blood flow was not attained, animals were excluded from the analysis. After 20 or 30 min (see below), the filament was withdrawn to establish reperfusion and the wound sutured. During surgery, core body temperature was monitored using a rectal probe and maintained at 37 ± 0.5 °C, using a homeothermic blanket (Harvard Apparatus, Kent, UK) and all mice were kept anaesthetised throughout the whole surgical procedure. During recovery all mice were given saline (0.5 ml, subcutaneously). In some groups, tail vein blood was taken immediately before MCAo (time 0), and at 4 and 24 h after reperfusion, and plasma obtained after centrifugation (13,000 × g, 10 min) was stored at −80°C until further use.
In separate experiments mice fed a control or high-fat diet for 2 (n = 9/group), 3 (n = 7–8/group), and 4 (n = 9–12/group) months, the MCA was occluded for 30 min. In the 6-month group, MCAo was induced for either 20 (n = 5–8/group) or 30 (n = 5-6/group) min. In a separate study, MCAo was induced for 30 min in 6-month high-fat- or control-fed mice (n = 5–6/group), and sham-operated animals (n = 5–6/group) were also prepared where the filament was advanced along the internal carotid artery and was retracted immediately. For all groups of animals, mice within a given cage were randomly assigned to undergo either MCAo or sham surgery or no surgical intervention (naïve). In total, 10 % of mice died during or after the surgical procedure and 8 % were excluded due to lack of drop in cerebral blood flow as described above. A reduction in sample size for some groups is therefore shown in the results section.
Twenty-four hours after MCAo, mice were terminally anaesthetised with isoflurane and perfused transcardially with 0.9 % saline, and samples of the liver, spleen and lung were taken, frozen and stored at −80 °C until analysis. In a separate study, the brain was also taken after saline perfusion, and from the ipsilateral and contralateral hemisphere, the striatum and cortex were dissected and frozen. Following perfusion with saline, animals were then perfuse-fixed with 4 % paraformaldehyde (PFA; in 0.1 M phosphate buffer, PB). Brains were removed and post-fixed (in 4 % PFA), cryoprotected (30 % sucrose in 0.1 M PB) and frozen in isopentane on dry ice. Coronal brain sections (30 μm) were cut on a freezing sledge microtome (Bright 8000–001, Bright Instrument Co Ltd, UK). The liver was also removed and post-fixed (in 4 % PFA) before embedding in paraffin wax. Sections of liver were cut at 5 μm with a rotary microtome and mounted onto slides.
Measurement of ischaemic damage
Brain sections were stained with either cresyl violet or haematoxylin and eosin (H&E). The volume of ischaemic damage was calculated as described previously . Briefly, areas of damage on cresyl violet-stained sections were directly transcribed onto brain maps at eight anatomically defined coronal levels (bregma levels; 2.22, 1.54, 0.98, 0.14, −0.58, −1.22, −1.82 and −2.54 mm as defined by ) and are therefore corrected for oedema. The area of damage at each level was then measured using ImageJ (NIH, Bethesda, MD, USA) and the volume calculated. The volume of damage was expressed as the total amount of ischaemic damage, which was the sum of the damage in the striatum, cortex and hippocampus and thalamus combined (‘other’).
Immunoperoxidase labelling for neutrophils was performed on PFA-fixed brain or paraffin-embedded liver sections mounted onto slides. For liver, sections were deparaffinised, rehydrated and antigen retrieval performed by incubation in heated citrate buffer (10 mM sodium citrate, 0.05 % Tween 20). All sections were initially incubated in 0.3 % H2O2 in 0.1 M PB for 10 min to quench endogenous peroxidase activity. Non-specific binding of antibodies was blocked for 1 h with 2 % normal serum (Vector Laboratories, UK) from species in which the secondary antibody was raised. Sections were incubated with a rabbit anti-neutrophil antibody (SJC for neutrophils, 1:1000, kindly provided by Drs. Daniel Anthony and Sandra Campbell, University of Oxford, UK) before being incubated in an anti-rabbit biotinylated (for brain: 1:500, Vector Labs) or HRP-labelled polymer (for liver: EnVision Plus, Dako) secondary antibody for 30 min–2 h at room temperature. For the brain, signal amplification was then performed by incubating sections in avidin-biotin complex (Vectastain ABC Elite, Vector Labs). For peroxidase visualisation, sections were incubated in 3, 3′-diaminobenzidine solution (DAB; for the liver: EnVision Plus, or for the brain: SigmaFast DAB with metal enhancer, Sigma-Aldrich, UK). All tissues stained for neutrophils were then counterstained with Papanicoloau haematoxylin and coverslipped. For visualisation of microglia/macrophages in the brain, PFA-fixed brain sections were treated as above but incubated in a rabbit anti-Iba1 antibody (1:1000, Wako Chemicals) overnight at 4 °C. For assessment of BBB disruption, primary antibody was omitted and a biotinylated anti-mouse IgG secondary antibody used (1:500, Vector Labs) before incubation in DAB (no metal enhancer).
SJC-positive neutrophils were counted in a defined area of the liver and results expressed as number of neutrophils/area. For the brain, the number of neutrophils per section (3–7 sections depending on the region analysed) was counted in the ipsilateral cortex (1.54 to −2.7 mm), striatum (1.54 to −1.22 mm) and hippocampus (−1.22 to −2.7 mm). The average number of cells per section was then calculated and the group mean determined for each brain region. For Iba1-positive cells, three separate images were taken in the striatum and cortex (for the ipsilateral and contralateral hemisphere). The area of Iba1 staining was assessed in each image using the threshold function of ImageJ (NIH, Bethesda, USA), with the threshold value kept constant and verified on all images so that only the area of positive Iba1 staining was measured. The area of Iba1 staining was expressed as percentage increase from the contralateral hemisphere. For BBB disruption, the intensity of IgG staining was analysed (using ImageJ) in the contralateral and ipsilateral cortex, striatum and hippocampus (as defined above) and results expressed as percentage increase from the contralateral hemisphere.
Chemokine protein analysis
Saline-perfused liver, spleen, lung and brain samples were homogenised in buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl2 and 0.02 % NaN3) containing 1 % Triton-X 100 and a protease inhibitor cocktail (Set I; Calbiochem, Merck Chemicals Ltd). All homogenates were centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant from the liver samples was further ultra-centrifuged at 100,000 × g for 1 h at 4 °C. All supernatants were then stored at −20 °C until analysis. CXCL-1 and CCL3 were assessed due to their chemoattractant properties for neutrophils. Mouse CXCL-1 (KC) was analysed by cytometric bead array (BD Biosciences, UK) and mouse CCL3 (MIP-1α) by ELISA (Duoset; R&D Systems, UK), according to the manufacturer’s instructions. Cytokine concentrations were determined by reference to the relevant standard curves. For liver, spleen, lung and brain supernatant, protein concentration was assessed by a bicinchonic protein assay (BCA; Pierce Biotechnology, USA) and results expressed as picogram per milligram protein for the liver, spleen and lung and the ratio of ipsilateral/contralateral for the brain. For the liver, spleen and lung, a separate group of 6-month high-fat- or control-fed mice (naïve) were treated as above but in the absence of any intervention (sham or MCAo).
Data and statistical analyses
For all analyses, data are represented as mean ± standard error of the mean (SEM). Sample sizes were determined by power calculation (α = 0.05, β = 0.2) of our previous data at http://www.stattools.net/SSizAOV_Pgm.php. For all ex vivo analyses, the investigator was blinded to diet (control or high-fat) and treatment (e.g. length of occlusion of MCA, sham or naive).
For two groups, parametric data were analysed using Student’s t-test and for data with unequal variances, a Welch’s correction was applied. All other data was analysed using a two-way ANOVA with diet and treatment (sham or MCAo) or duration of MCAo as the fixed factors followed by a Bonferroni test for multiple comparisons. P < 0.05 was considered significant.