This study performed a comprehensive analysis of cerebrospinal fluid (CSF) cytokine levels after severe traumatic brain injury (TBI) in children using a multiplex bead array assay and to evaluate the effects of moderate hypothermia on cytokine levels. To this end, samples were collected during two prospective randomized controlled trials of therapeutic moderate hypothermia in pediatric TBI. Thirty-six children with severe TBI (Glasgow Coma Scale [GCS] score of <or=8) and 10 children with negative diagnostic lumbar punctures. All children with TBI had continuous monitoring of intracranial pressure and CSF drainage via an intraventricular catheter. Moderate hypothermia (32-33 degrees C) was maintained for 48 h in 17 patients, and they were slowly re-warmed at 48-72 h. A multiplex bead array assay was used to analyze serial CSF samples (<18 h, 24 +/- 6 h, 48 +/- 6 h, and 72 +/- 6 h) for 21 pro-and anti-inflammatory cytokines and chemokines. Interleukin (IL)-8 and transforming growth factor beta were measured by enzyme-linked immunosorbant assay (ELISA). There was a strong correlation (Spearman correlation coefficient = 0.92, p < 0.001) between multiplex assay and ELISA for IL-8. Pro-inflammatory IL-1beta, -6 and -12p70, anti-inflammatory IL-10 and chemokines IL-8 and MIP-1alpha were increased after TBI compared to controls, p < 0.05; however, there was no association between cytokines and age, gender, initial GCS, or outcome. Hypothermia did not attenuate the increases in CSF cytokine levels after TBI versus normothermia. This investigation confirmed that the multiplex bead array assay is a useful method to measure CSF cytokine levels. Severe TBI in infants and children induces increases in pro- and anti-inflammatory cytokines and chemokines. It is the first clinical report of increased levels of MIP-1alpha after TBI in any patient population and the most comprehensive assessment of cytokines after TBI to date. Moderate therapeutic hypothermia did not attenuate the increase in CSF cytokine levels in children after TBI.