Real-Time PCR Quantification of 87 miRNAs from Cerebrospinal Fluid: miRNA Dynamics and Association with Extracellular Vesicles after Severe Traumatic Brain Injury

Abstract

Severe traumatic brain injury (sTBI) is an intracranial damage triggered by external force, most commonly due to falls and traffic accidents. The initial brain injury can progress into a secondary injury involving numerous pathophysiological processes. The resulting sTBI dynamics makes the treatment challenging and prompts the improved understanding of underlying intracranial processes. Here, we analysed how extracellular microRNAs (miRNAs) are affected by sTBI. We collected thirty-five cerebrospinal fluids (CSF) from five sTBI patients during twelve days (d) after the injury and combined them into d1-2, d3-4, d5-6 and d7-12 CSF pools. After miRNA isolation and cDNA synthesis with added quantification spike-ins, we applied a real-time PCR-array targeting 87 miRNAs. We detected all of the targeted miRNAs, with totals ranging from several nanograms to less than a femtogram, with the highest levels found at d1-2 followed by decreasing levels in later CSF pools. The most abundant miRNAs were miR-451a, miR-16-5p, miR-144-3p, miR-20a-5p, let-7b-5p, miR-15a-5p, and miR-21-5p. After separating CSF by size-exclusion chromatography, most miRNAs were associated with free proteins, while miR-142-3p, miR-204-5p, and miR-223-3p were identified as the cargo of CD81-enriched extracellular vesicles, as characterised by immunodetection and tunable resistive pulse sensing. Our results indicate that miRNAs might be informative about both brain tissue damage and recovery after sTBI.