Microwave and magnetic (M2) proteomics of a mouse model of mild traumatic brain injury

Abstract

Short-term increases in oxidative stress and decreases in motor function, including debilitating effects on balance and motor control, can occur following primary mild traumatic brain injuries (mTBI). However, the long-term effects on motor unit impairment and integrity as well as the molecular mechanisms underlying secondary injuries are poorly understood. We hypothesized that changes in central nervous system-specific protein (CSP) expression might correlate to these long-term effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury. Motor impairment was determined by rotarod and grip strength performance measures, while motor unit integrity was determined using electromyography. Relative protein expression was determined by microwave & magnetic (M2) proteomics of ipsilateral brain tissue, as previously described. Isoprostane measurements were performed to confirm a primary oxidative stress response. Decoding the relative expression of 476 ± 56 top-ranked proteins for each specimen revealed statistically significant changes in the expression of two well-known CSPs at 1, 7 and 30 days post-injury: P < 0.001 for myelin basic protein (MBP) and P < 0.05 for myelin associated glycoprotein (MAG). This was confirmed by Western blot. Moreover, MAG, αII-spectrin (SPNA2) and neurofilament light (NEFL) expression at 30 days post-injury were directly related to grip strength (P < 0.05). While higher-powered studies of larger cohorts merit further investigation, this study supports the proof-of-concept that M2 proteomics is a rapid method to quantify putative protein biomarkers and therapeutic targets of mTBI and suggests the feasibility of CSP expression correlations to long-term effects on motor impairment.

The effect of mild traumatic brain injury on peripheral nervous system pathology in wild-type mice and the G93A mutant mouse model of motor neuron disease

“…The initial loss of body weight after TBI in wild type mice has been reported previously (Evans et al, 2014) and could be caused by decreased food and water intake as a result of nausea, and/or dizziness experienced after mild TBI (O’Neil et al, 2013). G93A mice characteristically lose body weight after disease onset as a result of reduced food intake and wasting of muscle tissue in the later stages of disease progression (Gurney et al, 1994).…”

“…EMG is used clinically to diagnose peripheral nerve injury and motor neuron diseases, such as ALS. Abnormal EMG findings were compiled as a score of denervation potentials per muscle group per limb in each animal as described previously (Evans et al, 2014). Linear regression showed a significant difference between the curves of EMG abnormalities for each treatment group [Linear Regression, p<0.001, F87.08 (6,69) n=5–7, Figure 4].…”

“…(Evans et al, 2014) TBI was administered as a closed cortical injury using pneumatic force. Prior to surgery mice were anesthetized in a chamber using 2–4% isoflurane in 100% oxygen and anesthesia was maintained at 1% for the remaining procedures.…”

“…A subdermal needle (ground) was inserted subcutaneously into tendinous tissue posterior to and near the reference electrode. Abnormal spontaneous activity in the form of denervation potentials (positive sharp waves and fibrillations) were recorded as a score of denervation potentials per muscle group per limb in each animal at 1, 7, and 30 days post injury, as described previously (Evans et al, 2014). Notably, EMG was performed after behavioral testing at each time point in order to avoid complications.…”

Proteomics: in pursuit of effective traumatic brain injury therapeutics

“…Interestingly, CANB1 was then upregulated two weeks after TBI suggesting a post-acute neurotrophic function. Further support for TBI proteomic profiling can be found in a proteomic study by Evans et al where they identified an inverse temporal relationship between the loss of mature MBP and initiation of myelin-associated glycoprotein (MAG) over three month period after injury [39]. We further observed that MAG undergoes transient dephosphorylated at Y611 and Y620 between one and two weeks following TBI (2.8-fold relative to naïve control; p=8.3E-7, n=6 per group), two key L-MAG signaling motifs for initiating myelination [40].…”