Traumatic brain injuries (TBIs) account for the majority of injury-related deaths in the United States with roughly two million TBIs occurring annually. Due to the spectrum of severity and heterogeneity in TBIs, investigation into the secondary injury is necessary in order to formulate an effective treatment. A mechanical consequence of trauma involves dysregulation of the blood–brain barrier (BBB) which contributes to secondary injury and exposure of peripheral components to the brain parenchyma. Recent studies have shed light on the mechanisms of BBB breakdown in TBI including novel intracellular signaling and cell–cell interactions within the BBB niche. The current review provides an overview of the BBB, novel detection methods for disruption, and the cellular and molecular mechanisms implicated in regulating its stability following TBI.

Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.

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⁺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.SIGNIFICANCE STATEMENTAlthough a clear “window of susceptibility” exists in the developing brain that could deter typical developmental trajectories if exposed to trauma, a number of preclinical models have demonstrated evidence of early recovery in younger patients. Our findings further demonstrate acute neuroprotection and improved restoration of cerebral blood flow in juvenile mice subjected to cortical contusion injury compared with adults. We also demonstrate a novel role for endothelial cell-specific Tie2 signaling in this age-related response, which is known to promote barrier stability, is heightened in the injured juvenile vasculature, and may be exploited for therapeutic interventions across the age spectrum following traumatic brain injury.

Traumatic brain injury (TBI) elicits the immediate production of proinflammatory cytokines which participate in regulating the immune response. While the mechanisms of adaptive immunity in secondary injury are well characterized, the role of the innate response is unclear. Recently, the NLR inflammasome has been shown to become activated following TBI, causing processing and release of interleukin-1β(IL-1β). The inflammasome is a multiprotein complex consisting of nucleotide-binding domain and leucine-rich repeat containing proteins (NLR), caspase-1, and apoptosis-associated speck-like protein (ASC). ASC is upregulated after TBI and is critical in coupling the proteins during complex formation resulting in IL-1βcleavage. To directly test whether inflammasome activation contributes to acute TBI-induced damage, we assessed IL-1β, IL-18, and IL-6 expression, contusion volume, hippocampal cell death, and motor behavior recovery inNlrp1^−/−,Asc^−/−, and wild type mice after moderate controlled cortical impact (CCI) injury. Although IL-1βexpression is significantly attenuated in the cortex ofNlrp1^−/−andAsc^−/−mice following CCI injury, no difference in motor recovery, cell death, or contusion volume is observed compared to wild type. These findings indicate that inflammasome activation does not significantly contribute to acute neural injury in the murine model of moderate CCI injury.

Background: Efferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation, prevents the release of inflammatory molecules, and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remain ill-defined. Methods: We used GFP bone marrow chimeric knockout (KO) mice to demonstrate that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing MERTK in the brain to restrict efferocytosis of resident microglia and peripheral-derived monocyte/macrophages. Results: Single-cell RNAseq identified MERTK expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis and overall protein expression of p-MERTK, p-ERK, and p-Stat6. The percentage of GFP⁺ monocyte/macrophages and resident microglia engulfing NeuN⁺ or TUNEL⁺ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to the wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with MERTK-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Selective inhibitors of ERK and Stat6 attenuated this effect, confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway. Conclusions: Our findings implicate the ERK/Stat6/MERTK axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.