Simultaneous Detections of 27 Cytokines during Cerebral Wound Healing by Multiplexed Bead-Based Immunoassay for Wound Age Estimation

Quantification of 27 cytokines following cerebral wounding was performed for wound age estimation. The cytokines evaluated included interleukin (IL)–1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 p40, IL-12 p70, IL-15, IL-17, IL-18, basic fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), Interferon-γ (IFN-γ), keratinocyte derived cytokine (KC), leukemia inhibitory factor (LIF), macrophage-colony stimulating factor (M-CSF), monokine inducible by interferon gamma (MIG), macrophage inflammatory protein (MIP)–1 alpha, MIP 2, platelet-derived growth factor BB (PDGF BB), regulated upon activation, normal T-cell expressed, and secreted (Rantes), tumor necrosis factor–α (TNF-α), and vascular endothelial growth factor (VEGF). The proliferation of glial cells as well as the infiltration of inflammatory cells were also evaluated. Although astroglia proliferated from 72 hours post-injury, inflammatory cell dynamics were generally steady. Among cytokines analyzed in the present study, IL-1β, IL-5, IL-6, IL-12 p40, G-CSF, IFN-γ, KC, LIF, MIP2, and PDGF BB increased during the early phase of cerebral wound healing, and M-CSF increased during the middle phase, while IL-15, IL-18, and MIG increased during the late phase. In contrast, IL-1α, IL-10, IL-12 p70, and TNF-α were suppressed throughout the cerebral wound healing process. Based on our findings, quantitative cytokine analyses at the cerebral wound site may be a useful tool for wound age estimation. Further, this study suggests that multiplex data gained from the same sample using a single methodology demonstrates highly accurate cytokine interactions during the process of cerebral wound healing.