Introduction: Recently, the role of danger-associated molecular pattern molecules (DAMPs, also known as alarmins) has been elucidated in the acute inflammatory response after traumatic brain injury (TBI). Previously, we reported that elevated cerebrospinal fluid (CSF) high mobility group box-1 (HMGB1) levels are associated with poor neurological outcome. Mitochondrial DNA (mtDNA), a novel DAMP, is released into the extracellular milieu after cell death and injury, and acts via Toll-like receptor 9 (TLR9), a pattern recognition receptor of the immune system that detects bacterial and viral DNA. Methods: All children with severe TBI who received CSF drainage for treatment of intracranial hypertension were included in this IRB approved study. CSF was continuously drained by gravity into a sterile buretrol and collected using sterile techniques. Using quantitative PCR, we measured free mtDNA in CSF samples from 36 children with TBI and 13 control subjects who had negative CSF from a lumbar puncture to rule out meningitis. We designed PCR primers that amplify bases between 421 and 781 encoding the mitochondrial gene cytochrome c oxidase I (RefSeq: NC_012920). The linearity of the quantitative assay was assessed by using a cloned plasmid DNA, which was serially diluted to prepare a series of calibrators with known concentrations (Chiu et al., 2003). All statistical analyses were performed using Mann-Whitney and Wilcoxon Rank-Sum test. Results: The mean age of the cohort was 6.3 yrs and 22 (61%) children were male. An initial Glasgow Coma Scale (GCS) score of <8 was documented in 70% of children with TBI; monitors were placed in the remaining 30% due to subsequent deterioration as GCS decreased to <= 8. CSF levels of mtDNA were significantly increased (log 2.38-fold change) in patients with TBI compared to the control population. In this preliminary data set, a positive correlation was noted between CSF levels of mtDNA, and another DAMP, HMGB1 at 24 h after TBI (rho = 0.5674, p<0.05), but there was no significant associations between mtDNA levels and age, sex and GCS or Glasgow Outcome Score. Conclusions: The elevated CSF concentrations of mtDNA, a novel DAMP, support the notion of mtDNA as an endogenous danger signal after TBI in children, and possible footprint of brain tissue necrosis given its association with CSF HMGB1. Our ongoing work is focused on identifying the pathways in the secondary injury response that are activated by this novel danger signal.
(C) 2013 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins