A growing number of studies indicate that repeated mild traumatic brain injuries (RmTBI) are associated with significant and lasting neurological alterations and an increased risk of developing neurodegenerative diseases. Advanced Magnetic Resonance Imaging and blood-based protein analysis not only can provide a reliable method for the clinical assessment and treatment of RmTBI but also valuable insights into the mechanisms and the pathophysiological changes associated with this condition. In this publication, proteomic analysis of plasma, conducted in a microarray format, revealed abnormal levels of markers indicative of metabolic, neuronal, inflammatory and vascular changes in model animals. Plasma samples, collected from rats previously subjected to repeated mild lateral fluid percussion injury, were printed on ONCYTE AVID nitrocellulose film slides with an Aushon 2470 Arrayer. The slides were then incubated with primary antibodies against plasma proteins that have been linked to inflammatory and metabolic alterations and neuronal and vascular injury. Fluorescent detection revealed significantly elevated plasma levels of glial fibrillary acidic protein (GFAP), NFH (a marker of axonal injury), 4-NHE (4-hydroxynonenal Michael adducts) an oxidative stress indicator, and ceruloplasmin while levels of VEGF were found to be significantly reduced in RmTBI rats. Altered levels of blood biomarkers were accompanied by behavioral, cognitive and motor functions indicative of brain damage. Brain damage and structural changes in the sensory cortex, the hippocampus, and the corpus callosum were confirmed by MRI analysis. The Reverse Phase Protein Microarray method applied in this study provides a valid approach for the detection of blood biomarkers associated with RmTBI and has the potential to be extended to measuring changes in blood-protein concentration in a single mild fluid percussion injury.
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