Mice and chemicals

We used 5-week old C57BL/6 male mice purchased from Charles River for all experiments. Animals were housed in groups of maximum 6 mice per cage under specific-pathogen-free conditions and with free access to food and water. Mice were housed for 7 days prior to testing, making them 6 weeks old when the massage treatment began. All tests were conducted in a blinded fashion and according to the UK Animals (Scientific Procedures) Act, 1986. All experimental protocols were approved by the local biological service unit at Queen Mary University of London. Mice treated with hydrocortisone received a suspension of 2.5 mg of hydrocortisone hemisuccinate (Sigma-Aldrich) in 200 μl of PBS.

Massage-like stroking therapy

Tests were performed double-blind during the light phase of the light-dark cycle. All efforts were made to minimize mouse discomfort in these behavioural experiments. Mice were brought to the testing room at least 30 minutes before the start of the test session to allow habituation to the testing environment. Unless otherwise specified, standard lighting (~50 lux) and quiet conditions were maintained throughout each experiment.

Mice were divided into three groups: 1) control (non-stroked) mice; 2) brush-stroked mice; 3) hand-stroked mice, each involving 6 mice per group, per experiment. Treatments were administered by placing a single mouse into a new cage every day as previously described20. These cages were identical to the home cage, however the lid, shredded paper, food and water were removed. New cages were used for each mouse to reduce odour from the previous mouse. Brush and hand stroked mice were stroked at a pressure of 100-150 mmH 2 O (or 7-11 mmHg) and at a speed of about 3 cm/sec according to a previously described protocol 21 whereas control mice were not touched (by hand or brush) throughout the 60-minute treatment. Brush stroking was applied using a No.5 da Vinci paintbrush while hand stroking was applied using three fingers of the preferred hand of the investigator as previously described22. For the hand-stroked treatment, the experimenter wore Bizzybee disposable vinyl gloves (Amazon, UK). These odourless gloves reduced human smell but maintained human contact i.e. warmth and pressure. Mice were stroked on the hairy skin found on the posterior dorsal thoracic and proximal hind limb22 in a cephalocaudal fashion (head to tail)20. Both brush and hand stroked mice received approximately 20 strokes every 5 minutes (0.5-1.0 Hz). Experiments were performed by 3 different experimenters trained to perform the same procedure. The reproducibility of the manual stimuli was tested and confirmed by similar application of pressure to a small balloon connected to a pressure gauge as previously reported 21.

Open field activity test

The open field test (OFT) is an ethologically based paradigm that provides objective measures of exploratory behaviour as well as a valid initial screen for anxiety-related behaviour in rodents. The test was carried out as previously described 23. The apparatus consisted of a white PVC arena (50 cm × 30 cm × 20 cm) divided into 10 cm × 10 cm squares (n = 15). The 3 central squares defined the “centre” region. Each mouse was placed in a corner square, facing the wall and observed and recorded for 3 minutes. The total number of squares crossed (all four paws in), total number of rears (defined as both front paws off the ground, but not as a part of grooming) and number of centre crossings was recorded. The walls and floor of the arena were thoroughly cleaned between each trial.

Light-dark shuttle box

In this test, exploratory activity reflects the combination of hazard and risk avoidance24. The apparatus consisted of a 45 cm × 20 cm × 21 cm box, divided into two distinct compartments: one-third (15 cm long) painted black, with a black lid on top, the remaining two thirds painted white and uncovered. A 2.5 cm × 2.5 cm opening joined the two compartments. One side of the bright box was transparent to enable behavioural assessment and the averseness of this compartment was increased by additional illumination supplied by a 50 W lamp placed 45 cm above the centre of the box floor. The test was performed in accordance with a previous published protocol 25. Each mouse was placed in the bright compartment, facing away from the opening and allowed to explore the box for 5 minutes. Dependent variables included the time spent in the light area, latency to cross to the dark area (all four paws in) and the total number of transitions between compartments. The apparatus was cleaned after each trial.

Fluorescence histochemistry and quantification of catecholamine-containing nerve fibres

Thymus and spleen were cut with a cryostat to obtain serial 16 μm thick sections and treated according to a modified version of the sucrose phosphate glyoxylic acid (SPG) method26 to identify cathecolamine-containing nerve fibres. Briefly, sections were first dipped in a solution containing 1% glyoxylic acid, 0.2 M sucrose and 0.236 M potassium phosphate monobasic (pH 7.4), then drained and finally covered with non-auto fluorescent immersion oil, heated at 95 °C for 2.5 min. To prevent diffusion and photodecomposition of fluorescence, the sections were analysed and photographed on the same day using an Olympus BH 2 fluorescence photomicroscope (Olympus Optical Co. Ltd, Tokyo, Japan) equipped with exciter filter BG 12 and barrier filter Y495, Color View III digital camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany) and AnalySIS FIVE software (Olympus Soft Imaging Solutions). The digital quantification of fluorescence intensity was performed using ImageJ software as described previously 27. Photomicrographs of 10 randomly chosen relevant test areas (×40 magnification) were taken in each of the 5 sections per thymus. After converting images to gray-scale mode, outlines of each fluorescent nerve profile were traced and intensities of fluorescent signal recorded (pixel intensity per unit area). To correct for the background fluorescence, the outlines were then placed on the closest adjacent areas not containing fluorescent material and the obtained values were subtracted from those corresponding to nerve profiles. The data were presented as fluorescence intensities of groups subjected to experimental treatment normalized to those of respective control groups.

Noradrenaline concentration

Noradrenaline concentrations in spleen and thymus were determined using ELISA. The analysis was performed using a commercial ELISA kit (Labor Diagnostika Nord GmbH & Co., Nordhorn, Germany), according to the manufacturer’s protocol.

Quantification of nerve fibre density

Fluorescent nerve fibre density in 10 randomly taken images (×40 magnification) from 5 thymic sections per animal was measured using a stereological grid point-counting approach28 and expressed as the percentage of field area occupied by the fluorescent nerve profiles.

Flow cytometric analysis

Lymphocytes collected from lymphatic organs (e.g. thymus, spleen) were stained in 100μl of FACS buffer (PBS containing 5% FCS and 0.02% of NaN 2 ). The antibodies used were anti-CD3 PE (clone 145-2C11, eBioscience), anti-CD4 FITC (clone GK 1.5, eBioscience), anti-CD8 Cy5 (clone 53-6.7, eBioscience), anti-CD25 FITC (clone PC61, BioLegend), anti-CD69 PE (clone H1.2F3, eBioscience). Cells were labeled with the appropriate concentration of conjugated antibodies for 1 h at 4 °C as previously described29. After labeling, cells were washed and analyzed. In all experiments stained cells were acquired with FACScalibur flow cytometer and analyzed using FlowJoTM software (Tree Star, Inc., Oregon Corporation).

T cell activation and cytokine production

Splenic T cells (1 × 105 cells/200 μl) were incubated with medium alone or stimulated with the indicated concentration of plate-bound anti-CD3 and anti-CD28 in 96-well plates. For CD25 and CD69 upregulation, lymph node T cells were stimulated for about 16 hr while the supernatants of 24 hr culture were used for cytokine production. Cytokine production was measured by cytometric bead assay using the mouse Th1/Th2 10plex kits (eBioscience). Each sample (25 μl of cell culture supernatant) was incubated with 50 μl bead mixture and 50 μl mix of antibodies conjugated with biotin for 2 h. After two washes, PE-conjugated streptavidin was added and samples were left rocking for 1 hour in dark. Finally, samples were washed and stored overnight at 4 °C. Standards diluted serially for 7 times were prepared and processed at the same time. Samples were analysed using BD LSR Fortessa and the FlowCytomix software (eBioscience).

Statistical analysis

Results were analysed as previously described30,31 using GraphPad. A one-way ANOVA followed by Bonferroni post-test or Newman-Keuls post-hoc comparisons was used for comparison between groups. Statistical significance was determined at p < 0.05. The results were expressed as mean ± S.E.M.