Laboratory Assays

Venous blood was collected in heparinized tubes (Sarstedt, Germany) at day 3 after stroke. To avoid diurnal variation, the blood was obtained between 7:00 and 7:30 AM. The whole blood was diluted by 1:5 in sterile RPMI 1640 medium supplemented with l-glutamine (Sigma-Aldrich, St. Louis, MO). The samples were then stimulated in sterile tubes (Lonza, Walkersville, MD) for 4 h at 37 °C in 5% CO₂ with LPS (10 ng/mL, Escherichia coli 0111:B4, Sigma-Aldrich, St. Louis, MO) or LPS and dexamethasone-21-phosphate (10^–6 mol/L, Sigma-Aldrich, St. Louis, MO). The supernatants were removed and stored at − 80 °C until further analysis.

Similarly to previous studies, TNFα was chosen as an indicator of GC sensitivity because this cytokine has the greatest sensitivity to GCs in comparison to IL-1β and IL-6 (DeRijk et al. 1997).

The GC sensitivity index was defined as the ratio of TNFα released after blood stimulation with LPS and dexamethasone to the amount of TNFα released after blood stimulation with LPS alone. A higher index indicates lower GC sensitivity (and higher GC resistance).

TNFα and IL-6 concentrations were measured using a commercially available ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The cytokine detection limits were 0.19 pg/mL for TNFα and 0.11 pg/mL for IL-6. For both cytokines, the intra-assay CVs were < 5%, and the inter-assay CVs were < 10%.