tDCS, Exercise and BBB Permeability
Description
This study aimed to assess the unique and combined effect of tDCS and exercise on BBB permeability, as assessed through changes in salivary S100B. It was hypothesised that active tDCS would induce a significant increase in S100B when compared to sham stimulation and no stimulation. It was also hypothesised that the increase in S100B concentration would be greater following active tDCS and exercise compared to tDCS or exercise alone. Regarding the effects of exercise, a significant increase in S100B following the cycling task was hypothesised.
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Blood Brain Barrier Protein Biomarker Saliva Collection. Saliva samples were collected at the beginning of the session after initial questionnaires were completed; after tDCS; and after the TTE task. The first saliva sample was taken at least 10 minutes after participants first rinsed their mouths, as per recommendations for best saliva sampling practices.46 The second saliva sample was taken 5 minutes post tDCS, and the third saliva sample was taken 5 minutes post the TTE task. To collect saliva, participants were instructed to allow saliva to pool in the mouth, and then with their head tilted forward, asked to drool into a sterile, polypropylene microcentrifuge tube (1.7 mL, Axygen MaxyClear, USA). This method of collection adhered to recommended protocols.46 Approximately 1 mL of unstimulated whole saliva was obtained for each sample. Samples were transported on ice and stored in a − 20 °C freezer at the time of collection. Samples were then later transported to a − 80 °C freezer for long term storage until analyses were ready to be conducted. Samples were de-identified and assigned an internal ID to allow for the matching of repeat samples. S100B antibody measurement. At the time of analysis, saliva samples were thawed and protease inhibitor cocktail (1:500) was added (Pierce Protease Inhibitor Mini Tablets, EDTA-free, Thermo Scientific) before centrifugation at 10,000 rpm for 10 minutes at 4 °C to remove insoluble material and cellular debris. Supernatants were collected and used for all assays. S100B levels were quantified using a commercially available ELISA kit (Millipore, EZHS100B-33K SDS) according to the manufacturer’s instructions. Samples were diluted 1:1 with Assay Buffer containing lyophilized Matrix Solution. The recombinant S100B protein standard provided with the kit was used for generating the standard curve. Each measurement was performed in duplicate by researchers blinded to the intervention condition of the participant. The limit of detection was 2.7 pg/mL, with a lower-upper range of 2.7–2000 pg/mL. The precision was 4% or lower intra-assay and 4% or lower inter-assay. This was considered an acceptable range, with previous studies finding significant changes in S100B after BBB disruption in both blood17 and saliva14 in this range. The antibody pair used in this assay measures human S100B and has no cross-reactivity with S100A1, S100A6 and S100A13. S100B reading was done using a microplate reader (Multiskan GO, Thermo Fisher Scientific). Absorbance signals were converted into pg/ml as per standard curve concentrations.