Cytokine Profiling for Vascular Remodeling

All serum samples, just after centrifugation at 3000 rpm for 10 min, and all CSF samples were immediately stored at − 80 °C until cytokine analysis, other than IL-6. The serum and CSF IL-6 levels were measured on a single detection immediately after centrifugation at room temperature using the electrochemiluminescence immunoassay according to the manufacturer’s instruction (Roche Diagnostics K.K., Tokyo, Japan). The serum cytokine concentrations relevant to the blood vessel regeneration were measured using the MILLIPLEX® (Merck Millipore, Darmstadt, Germany) human angiogenesis/growth factor magnetic bead panel 1 with a single detection, according to the manufacturer's instruction. Fluorescence intensity from the immunoassay was acquired and analyzed using xPONENT 4.2 Software (Luminex Corporation, Austin, TX, USA). The measured cytokines relevant to the vascular remodeling included epidermal growth factor, angiopoietin 2, granulocyte-colony stimulating factor (G-CSF), bone morphogenetic protein-9 (BMP-9), endoglin, leptin, hepatocyte growth factor (HGF), placental growth factor, vascular endothelial growth factor (VEGF)-C, VEGF-D, fibroblast growth factor-2 (FGF-2), and VEGF-A. Values under the dynamic range were replaced by half of the lower sensitivity limit.