Traumatic brain injury (TBI) can be frustratingly difficult to diagnose, particularly for mild cases, which can result in inadequate or improper treatment. As a result, there has been considerable interest in identifying biochemical markers of TBI to aid in diagnosis and evaluation of treatment efficacy. CSF and serum alterations in several proteins have been investigated in both animal models and clinical studies, but these appear to be best suited for severe TBI cases. Urgently needed are biomarkers for mild TBI, which represents the majority of the cases and which are the most difficult to diagnose. To identify novel serum and CSF biomarkers for mild and moderate TBI, we propose an unbiased approach utilizing phage display. Phage display is a powerful tool for selecting peptides, proteins or antibodies with specific binding properties. It uses bacteriophages in which DNA encoding a peptide or protein is inserted into the gene encoding a coat protein of a filamentous phage such as M13 phage. The encoded protein or peptide is expressed on the cell surface of the phage and used to attract and bind proteins of interest. We propose to use engineered phage display peptide libraries in which >2 million independent clones express random sequences of 7 or 12 amino acid peptides.
In Aim 1, three different phage libraries will be screened to identify phages that bind to the serum and CSF obtained from rats with mild TBI, produced using the lateral fluid percussion model. The biofluids will be collected at 6h and 24h after injury. We will utilize a subtractive panning technique in which phage libraries are first bound to the biofluid (CSF or serum) from uninjured mice to remove non- specific phages, then incubated with injury biofluids to identify injury-selective phages. Preliminary data demonstrate the feasibility of this approach using serum from a mouse TBI model at 6h postinjury.
Aim 2 will use sequence and identify the proteins which bind to the phages selective for the injury biofluids. Together, the above results will identify novel biomarkers of mild TBI to assist in the diagnosis of TBI, determination of injury severity, evaluation of recovery and therapeutic efficacy, and prediction of outcomes.
Over 1 million individuals sustain a traumatic brain injury in a given year, with the majority being concussions or similar mild forms. However, diagnosis of such brain injury is difficult. Biomarkers with sufficient sensitivity and specificity would assistnot only with the diagnosis of brain injury, but also with determination of injury severity, evaluationof recovery and therapeutic efficacy, and prediction of outcomes.