BackgroundThe continuum of pro- and anti-inflammatory response elicited by traumatic brain injury (TBI) is suggested to play a key role in the outcome of TBI; however, the underlying mechanisms remain ill -defined.MethodsHere, we demonstrate that using bone marrow chimeric mice and systemic inhibition of EphA4 receptor shifts the pro-inflammatory milieu to pro-resolving following acute TBI.ResultsEphA4 expression is increased in the injured cortex as early as 2 h post-TBI and on CX3CR1gfp-positive cells in the peri-lesion. Systemic inhibition or genetic deletion of EphA4 significantly reduced cortical lesion volume and shifted the inflammatory profile of peripheral-derived immune cells to pro-resolving in the damaged cortex. These findings were consistent with in vitro studies showing EphA4 inhibition or deletion altered the inflammatory state of LPS-stimulated monocyte/macrophages towards anti-inflammatory. Phosphoarray analysis revealed that EphA4 may regulate pro-inflammatory gene expression by suppressing the mTOR, Akt, and NF-κB pathways. Our human metadata analysis further demonstrates increased EPHA4 and pro-inflammatory gene expression, which correlates with reduced AKT concurrent with increased brain injury severity in patients.ConclusionsOverall, these findings implicate EphA4 as a novel mediator of cortical tissue damage and neuroinflammation following TBI.

Traumatic and non-traumatic brain injury results from severe disruptions in the cellular microenvironment leading to massive loss of neuronal populations and increased neuroinflammation. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity and free radical release contribute to neural tissue damage. NLRX1 is a member of the NLR family of pattern recognition receptors and is a potent negative regulator of several pathways that significantly modulate many of these events. Thus, we hypothesized that NLRX1 limits immune system signaling in the brain following trauma. To evaluate this hypothesis, we utilized Nlrx1−/− mice in a controlled cortical impact (CCI) injury murine model of traumatic brain injury (TBI). Here, we show that the Nlrx1−/− mice exhibited significantly larger brain lesions and increased motor deficits following CCI injury. Mechanistically, our data indicate that the NF-κB signaling cascade is significantly up-regulated in the Nlrx1−/− animals. This up-regulation is associated with increased microglia and macrophage populations in the cortical lesion. Utilizing a mouse neuroblastoma cell line (N2A), we also found that NLRX1 significantly reduced apoptosis under hypoxic conditions. In human patients, we identify 15 NLRs that are significantly dysregulated, including significant downregulation of NLRX1 in brain injury following aneurysm. We further demonstrate a concurrent increase in NF-κB signaling that is correlated with aneurysm severity in these human subjects. Together, our data extend the function of NLRX1 beyond its currently characterized role in host-pathogen defense and identify this highly novel NLR as a significant modulator of brain injury progression.

Although age-at-injury influences chronic recovery from traumatic brain injury (TBI), the differential effects of age on early outcome remain understudied. Using a male murine model of moderate contusion injury, we investigated the underlying mechanism(s) regulating the distinct response between juvenile and adult TBI. We demonstrate similar biomechanical and physical properties of naive juvenile and adult brains. However, following controlled cortical impact (CCI), juvenile mice displayed reduced cortical lesion formation, cell death, and behavioral deficits at 4 and 14 d. Analysis of high-resolution laser Doppler imaging showed a similar loss of cerebral blood flow (CBF) in the ipsilateral cortex at 3 and 24 h post-CCI, whereas juvenile mice showed enhanced subsequent restoration at 2-4 d compared with adults. These findings correlated with reduced blood-brain barrier (BBB) disruption and increased perilesional vessel density. To address whether an age-dependent endothelial cell (EC) response affects vessel stability and tissue outcome, we magnetically isolated CD31 ϩ ECs from sham and injured cortices and evaluated mRNA expression. Interestingly, we found increased transcripts for BBB stability-related genes and reduced expression of BBB-disrupting genes in juveniles compared with adults. These differences were concomitant with significant changes in miRNA-21-5p and miR-148a levels. Accompanying these findings was robust GFAP immunoreactivity, which was not resolved by day 35. Importantly, pharmacological inhibition of EC-specific Tie2 signaling abolished the juvenile protective effects. These findings shed new mechanistic light on the divergent effects that age plays on acute TBI outcome that are both spatial and temporal dependent.

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