Intensity Specific Repetitive Mild Traumatic Brain Injury Evokes an Exacerbated Burden of Neocortical Axonal Injury.

Mild traumatic brain injury (mTBI) has been linked to enduring neurological damage following repetitive injury. Previously, we reported that intensity-specific, repetitive mTBI exacerbated microvascular and axonal damage in brainstem. For a more rigorous and global assessment, we assessed the burden of neocortical diffuse axonal injury (DAI) evoked by repetitive mTBI. Mice were subjected to mild central fluid percussion injuries at 1.4 and 1.6 atm with or without repetitive insult at a 3-hour interval and killed at 24 hours postinjury. Neocortical DAI within layer V was quantitatively assessed by double-labeling p-c-Jun and NeuN to identify both the axotomized and total neuronal population. Both confocal and electron microscopic findings revealed no apparent evidence of neuronal death. Repetitive mTBI of 1.6 atm group, but not of 1.4 atm group, demonstrated a significantly higher proportion of axotomized neurons. These results demonstrate that different intensities of mTBI induced different burdens of DAI after repetitive insult. Interestingly, the parallel loss of the righting reflex reflected differences in injury intensity, yet the duration of this reflex was not elongated by the repetitive insult. These data highlight some of the complex issues surrounding repetitive mTBI and its associated morbidity, mandating the need for continued exploration.

Mild Traumatic Brain Injury Induces Structural and Functional Disconnection of Local Neocortical Inhibitory Networks via Parvalbumin Interneuron Diffuse Axonal Injury.

Diffuse axonal injury (DAI) plays a major role in cortical network dysfunction posited to cause excitatory/inhibitory imbalance after mild traumatic brain injury (mTBI). Current thought holds that white matter (WM) is uniquely vulnerable to DAI. However, clinically diagnosed mTBI is not always associated with WM DAI. This suggests an undetected neocortical pathophysiology, implicating GABAergic interneurons. To evaluate this possibility, we used mild central fluid percussion injury to generate DAI in mice with Cre-driven tdTomato labeling of parvalbumin (PV) interneurons. We followed tdTomato+ profiles using confocal and electron microscopy, together with patch-clamp analysis to probe for DAI-mediated neocortical GABAergic interneuron disruption. Within 3 h post-mTBI tdTomato+ perisomatic axonal injury (PSAI) was found across somatosensory layers 2-6. The DAI marker amyloid precursor protein colocalized with GAD67 immunoreactivity within tdTomato+ PSAI, representing the majority of GABAergic interneuron DAI. At 24 h post-mTBI, we used phospho-c-Jun, a surrogate DAI marker, for retrograde assessments of sustaining somas. Via this approach, we estimated DAI occurs in ~9% of total tdTomato+ interneurons, representing ~14% of pan-neuronal DAI. Patch-clamp recordings of tdTomato+ interneurons revealed decreased inhibitory transmission. Overall, these data show that PV interneuron DAI is a consistent and significant feature of experimental mTBI with important implications for cortical network dysfunction.

Mild Traumatic Brain Injury Evokes Pyramidal Neuron Axon Initial Segment Plasticity and Diffuse Presynaptic Inhibitory Terminal Loss.

The axon initial segment (AIS) is the site of action potential (AP) initiation, thus a crucial regulator of neuronal activity. In excitatory pyramidal neurons, the high density of voltage-gated sodium channels (NaV1.6) at the distal AIS regulates AP initiation. A surrogate AIS marker, ankyrin-G (ankG) is a structural protein regulating neuronal functional via clustering voltage-gated ion channels. In neuronal circuits, changes in presynaptic input can alter postsynaptic output via AIS structural-functional plasticity. Recently, we showed experimental mild traumatic brain injury (mTBI) evokes neocortical circuit disruption via diffuse axonal injury (DAI) of excitatory and inhibitory neuronal systems. A key finding was that mTBI-induced neocortical electrophysiological changes involved non-DAI/ intact excitatory pyramidal neurons consistent with AIS-specific alterations. In the current study we employed Thy1-yellow fluorescent protein (YFP)-H mice to test if mTBI induces AIS structural and/or functional plasticity within intact pyramidal neurons 2 days after mTBI. We used confocal microscopy to assess intact YFP+ pyramidal neurons in layer 5 of primary somatosensory barrel field (S1BF), whose axons were continuous from the soma of origin to the subcortical white matter (SCWM). YFP+ axonal traces were superimposed on ankG and NaV1.6 immunofluorescent profiles to determine AIS position and length. We found that while mTBI had no effect on ankG start position, the length significantly decreased from the distal end, consistent with the site of AP initiation at the AIS. However, NaV1.6 structure did not change after mTBI, suggesting uncoupling from ankG. Parallel quantitative analysis of presynaptic inhibitory terminals along the postsynaptic perisomatic domain of these same intact YFP+ excitatory pyramidal neurons revealed a significant decrease in GABAergic bouton density. Also within this non-DAI population, patch-clamp recordings of intact YFP+ pyramidal neurons showed AP acceleration decreased 2 days post-mTBI, consistent with AIS functional plasticity. Simulations of realistic pyramidal neuron computational models using experimentally determined AIS lengths showed a subtle decrease is NaV1.6 density is sufficient to attenuate AP acceleration. Collectively, these findings highlight the complexity of mTBI-induced neocortical circuit disruption, involving changes in extrinsic/presynaptic inhibitory perisomatic input interfaced with intrinsic/postsynaptic intact excitatory neuron AIS output.