Diffuse axonal injury (DAI) is a common feature of severe traumatic brain injury (TBI) and may also be a predominant pathology in mild TBI or “concussion”. The rapid deformation of white matter at the instant of trauma can lead to mechanical failure and calcium-dependent proteolysis of the axonal cytoskeleton in association with axonal transport interruption. Recently, a proteolytic fragment of alpha-II spectrin, “SNTF”, was detected in serum acutely following mild TBI in patients and was prognostic for poor clinical outcome. However, direct evidence that this fragment is a marker of DAI has yet to be demonstrated in either humans following TBI or in models of mild TBI. Here we used immunohistochemistry (IHC) to examine for SNTF in brain tissue following both severe and mild TBI. Human severe TBI cases (survival <7d; n=18) were compared to age-matched controls (n=16) from the Glasgow TBI archive. We also we examined brains from an established model of mild TBI at 6h, 48h and 72h post-injury versus shams. IHC specific for SNTF was compared to that of amyloid precursor protein (APP), the current standard for DAI diagnosis and other known markers of axonal pathology including non-phosphorylated neurofilament-H (SMI-32), neurofilament-68 (NF-68) and compacted neurofilament-medium (RMO-14) using double and triple immunofluorescent labelling. Supporting its use as a biomarker of DAI, SNTF immunoreactive axons were observed at all time-points following both human severe TBI and in the model of mild TBI. Interestingly, SNTF revealed a subpopulation of degenerating axons, undetected by the gold-standard marker of transport interruption, APP. While there was greater axonal co-localization between SNTF and APP after severe TBI in humans, a subset of SNTF positive axons displayed no APP accumulation. Notably, some co-localization was observed between SNTF and the less abundant neurofilament subtype markers. Other SNTF positive axons, however, did not co-localize with any other markers. Similarly, RMO-14 and NF-68 positive axonal pathology existed independent of SNTF and APP. These data demonstrate that multiple pathological axonal phenotypes exist post-TBI and provide insight into a more comprehensive approach to the neuropathological assessment of DAI.

Although concussion is now recognized as a major health issue, its non-lethal nature has limited characterization of the underlying pathophysiology. In particular, potential neuropathological changes have typically been inferred from non-invasive techniques or post-mortem examinations of severe traumatic brain injury (TBI). Here, we used a swine model of head rotational acceleration based on human concussion to examine blood-brain barrier (BBB) integrity after injury in association with diffuse axonal injury and glial responses. We then determined the potential clinical relevance of the swine concussion findings through comparisons with pathological changes in human severe TBI, where post-mortem examinations are possible. At 6-72 h post-injury in swine, we observed multifocal disruption of the BBB, demonstrated by extravasation of serum proteins, fibrinogen and immunoglobulin-G, in the absence of hemorrhage or other focal pathology. BBB disruption was observed in a stereotyped distribution consistent with biomechanical insult. Specifically, extravasated serum proteins were frequently observed at interfaces between regions of tissue with differing material properties, including the gray-white boundary, periventricular and subpial regions. In addition, there was substantial overlap of BBB disruption with regions of axonal pathology in the white matter. Acute perivascular cellular uptake of blood-borne proteins was observed to be prominent in astrocytes (GFAP-positive) and neurons (MAP-2-positive), but not microglia (IBA1-positive). Parallel examination of human severe TBI revealed similar patterns of serum extravasation and glial uptake of serum proteins, but to a much greater extent than in the swine model, attributed to the higher injury severity. These data suggest that BBB disruption represents a new and important pathological feature of concussion.

The hippocampus is integral to working and episodic memory and is a central region of interest in diseases affecting these processes. Pig models are widely used in translational research and may provide an excellent bridge between rodents and nonhuman primates for CNS disease models because of their gyrencephalic neuroanatomy and significant white matter composition. However, the laminar structure of the pig hippocampus has not been well characterized. Therefore, we histologically characterized the dorsal hippocampus of Yucatan miniature pigs and quantified the cytoarchitecture of the hippocampal layers. We then utilized stereotaxis combined with single-unit electrophysiological mapping to precisely place multichannel laminar silicon probes into the dorsal hippocampus without the need for image guidance. We used electrophysiological recordings of simultaneous laminar field potentials and single-unit activity in multiple layers of the dorsal hippocampus to physiologically identify and quantify these layers under anesthesia. Consistent with previous reports, we found the porcine hippocampus to have the expected archicortical laminar structure, with some anatomical and histological features comparable to the rodent and others to the primate hippocampus. Importantly, we found these distinct features to be reflected in the laminar electrophysiology. This characterization, as well as our electrophysiology-based methodology targeting the porcine hippocampal lamina combined with high-channel-count silicon probes, will allow for analysis of spike-field interactions during normal and disease states in both anesthetized and future awake behaving neurophysiology in this large animal.

While hippocampal-dependent learning and memory are particularly vulnerable to traumatic brain injury (TBI), the functional status of individual hippocampal neurons and their interactions with oscillations are unknown following injury. Using the most common rodent TBI model and laminar recordings in CA1, we found a significant reduction in oscillatory input into the radiatum layer of CA1 after TBI. Surprisingly, CA1 neurons maintained normal firing rates despite attenuated input, but did not maintain appropriate synchronization with this oscillatory input or with local high-frequency oscillations. Normal synchronization between these coordinating oscillations was also impaired. Simultaneous recordings of medial septal neurons known to participate in theta oscillations revealed increased GABAergic/glutamatergic firing rates postinjury under anesthesia, potentially because of a loss of modulating feedback from the hippocampus. These results suggest that TBI leads to a profound disruption of connectivity and oscillatory interactions, potentially disrupting the timing of CA1 neuronal ensembles that underlie aspects of learning and memory.

Diffuse axonal injury (DAI) is an important consequence of traumatic brain injury (TBI). At the moment of trauma, axons rarely disconnect, but undergo cytoskeletal disruption and transport interruption leading to protein accumulation within swellings. The amyloid precursor protein (APP) accumulates rapidly and the standard histological evaluation of axonal pathology relies upon its detection. APP+ swellings first appear as varicosities along intact axons, which can ultimately undergo secondary disconnection to leave a terminal “axon bulb” at the disconnected, proximal end. However, sites of disconnection are difficult to determine with certainty using standard, thin tissue sections, thus limiting the comprehensive evaluation of axon degeneration. The tissue‐clearing technique, CLARITY, permits three‐dimensional visualization of axons that would otherwise be out of plane in standard tissue sections. Here, we examined the morphology and connection status of APP+ swellings using CLARITY at 6 h, 24 h, 1 week and 1 month following the controlled cortical impact (CCI) model of TBI in mice. Remarkably, many APP+ swellings that appeared as terminal bulbs when viewed in standard 8‐µm‐thick regions of tissue were instead revealed to be varicose swellings along intact axons when three dimensions were fully visible. Moreover, the percentage of these potentially viable axon swellings differed with survival from injury and may represent the delayed onset of distinct mechanisms of degeneration. Even at 1‐month post‐CCI, ~10% of apparently terminal bulbs were revealed as connected by CLARITY and are thus potentially salvageable. Intriguingly, the diameter of swellings decreased with survival, including varicosities along intact axons, and may reflect reversal of, or reduced, axonal transport interruption in the chronic setting. These data indicate that APP immunohistochemistry on standard thickness tissue sections overestimates axon disconnection, particularly acutely post‐injury. Evaluating cleared tissue demonstrates a surprisingly delayed process of axon disconnection and thus longer window of therapeutic opportunity than previously appreciated. Intriguingly, a subset of axon swellings may also be capable of recovery.