BackgroundMild traumatic brain injury (mTBI) is an all too common occurrence that exacts significant personal and societal costs. The pathophysiology of mTBI is complex, with reports routinely correlating diffuse axonal injury (DAI) with prolonged morbidity. Progressive chronic neuroinflammation has also recently been correlated to morbidity, however, the potential association between neuroinflammatory microglia and DAI is not well understood. The majority of studies exploring neuroinflammatory responses to TBI have focused on more chronic phases of injury involving phagocytosis associated with Wallerian change. Little, however, is known regarding the neuroinflammatory response seen acutely following diffuse mTBI and its potential relationship to early DAI. Additionally, while inflammation is drastically different in rodents compared to humans, pigs and humans share very similar inflammatory profiles and responses.MethodsIn the current study, we employed a modified central fluid percussion model in micro pigs. Using this model of diffuse mTBI, paired with various immunohistological endpoints, we assessed the potential association between acute thalamic DAI and neuroinflammation 6 h following injury.ResultsInjured micro pigs displayed substantial axonal damage reflected in the presence of APP+ proximal axonal swellings, which were particularly prominent in the thalamus. In companion, the same thalamic sites displayed extensive neuroinflammation, which was observed using Iba-1 immunohistochemistry. The physical relationship between microglia and DAI, assessed via confocal 3D analysis, revealed a dramatic increase in the number of Iba-1+ microglial processes that contacted APP+ proximal axonal swellings compared to uninjured myelinated thalamic axons in sham animals.ConclusionsIn aggregate, these studies reveal acute microglial process convergence on proximal axonal swellings undergoing DAI, an interaction not previously recognized in the literature. These findings transform our understanding of acute neuroinflammation following mTBI and may suggest its potential as a diagnostic and/or a therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0405-6) contains supplementary material, which is available to authorized users.

Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.

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Traumatic axonal injury (TAI) following traumatic brain injury (TBI) contributes to morbidity and mortality. TAI involves intra-axonal changes assumed to progress to impaired axonal transport (IAT), disconnection, and axonal bulb formation. Immunocytochemical studies employing antibodies to amyloid precursor protein (APP), a marker of IAT and RMO14, a marker of neurofilament compaction (NFC), have shown that TAI involves both NFC and IAT, with the suggestion that NFC leads to IAT. Recently, new data has suggested that NFC may occur independently of IAT. The objective of this study was to determine quantitatively the precise relationship between NFC and IAT. Following TBI, rats were studied at 30 min, 3 h, and 24 h. Using single-label immunocytochemistry employing the antibodies RM014, APP, or a combined labeling strategy targeting APP/RMO14 in aggregate, the immunoreactive (IR) profiles were counted in the corticospinal tract (CSpT) and medial lemniscus (ML). In the CSpT, the number of axons demonstrating RMO14-IR approximated the number of axons showing APP-IR, with the APP-IR population showing a significant increase over 24 h (p < 0.05). The sum of both single-label counts equaled the aggregate APP/RMO14 numbers, demonstrating little relationship between NFC and IAT. In the ML, 75% of fibers demonstrated a separation of APP-IR and NFC-IR; however, 25% of the ML fibers showed co-localization of APP-IR and RMO14. The results of these studies indicate that, in the majority of damaged axons, NFC is not associated with IAT. Our findings argue for the use of multiple markers when evaluating the extent of TAI or the efficacy of therapies targeting the treatment of TAI.

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