We investigated the hypothesis that high Ca²⁺ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca²⁺ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca²⁺ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca²⁺ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca²⁺ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

Traumatic brain injury (TBI) can occur from physical trauma from a wide spectrum of insults ranging from explosions to falls. The biomechanics of the trauma can vary in key features, including the rate and magnitude of the insult. Although the effect of peak injury pressure on neurological outcome has been examined in the fluid percussion injury (FPI) model, it is unknown whether differences in rate of rise of the injury waveform modify cellular and physiological changes after TBI. Using a programmable FPI device, we examined juvenile rats subjected to a constant peak pressure at two rates of injury: a standard FPI rate of rise and a faster rate of rise to the same peak pressure. Immediate postinjury assessment identified fewer seizures and relatively brief loss of consciousness after fast-rise injuries than after standard-rise injuries at similar peak pressures. Compared with rats injured at standard rise, fewer silver-stained injured neuronal profiles and degenerating hilar neurons were observed 4-6 hr after fast-rise FPI. However, 1 week postinjury, both fast- and standard-rise FPI resulted in hilar cell loss and enhanced perforant path-evoked granule cell field excitability compared with sham controls. Notably, the extent of neuronal loss and increase in dentate excitability were not different between rats injured at fast and standard rates of rise to peak pressure. Our data indicate that reduced cellular damage and improved immediate neurological outcome after fast rising primary concussive injuries mask the severity of the subsequent cellular and neurophysiological pathology and may be unreliable as a predictor of prognosis.

Background

Injury to the brain can occur from a variety of physical insults and the degree of disability can greatly vary from person to person. It is likely that injury outcome is related to the biomechanical parameters of the traumatic event such as magnitude, direction and speed of the forces acting on the head.

New method

To model variations in the biomechanical injury parameters, a voice coil driven fluid percussion injury (FPI) system was designed and built to generate fluid percussion waveforms with adjustable rise times, peak pressures, and durations. Using this system, pathophysiological outcomes in the rat were investigated and compared to animals injured with the same biomechanical parameters using the pendulum based FPI system.

Results in comparison with existing methods

Immediate post-injury behavior shows similar rates of seizures and mortality in adolescent rats and similar righting times, toe pinch responses and mortality rates in adult rats. Interestingly, post injury mortality in adult rats was sensitive to changes in injury rate. Fluoro-Jade labeling of degenerating neurons in the hilus and CA2-3 hippocampus were consistent between injuries produced with the voice coil and pendulum operated systems. Granule cell population spike amplitude to afferent activation, a measure of dentate network excitability, also showed consistent enhancement 1 week after injury using either system.

Conclusions

Overall our results suggest that this new FPI device produces injury outcomes consistent with the commonly used pendulum FPI system and has the added capability to investigate pathophysiology associated with varying rates and durations of injury.

The changes of excitability in affected neural networks can be used as a marker to study the temporal course of traumatic brain injury (TBI). The cerebellum is an ideal platform to study brain injury mechanisms at the network level using the electrophysiological methods. Within its crystalline morphology, the cerebellar cortex contains highly organized topographical subunits that are defined by two main inputs, the climbing (CFs) and mossy fibers (MFs). Here we demonstrate the use of cerebellar evoked potentials (EPs) mediated through these afferent systems for monitoring the injury progression in a rat model of fluid percussion injury (FPI). A mechanical tap on the dorsal hand was used as a stimulus, and EPs were recorded from the paramedian lobule (PML) of the posterior cerebellum via multi-electrode arrays (MEAs). Post-injury evoked response amplitudes (EPAs) were analyzed on a daily basis for 1 week and compared with pre-injury values. We found a trend of consistently decreasing EPAs in all nine animals, losing as much as 72 ± 4% of baseline amplitudes measured before the injury. Notably, our results highlighted two particular time windows; the first 24 h of injury in the acute period and day-3 to day-7 in the delayed period where the largest drops (~50% and 24%) were observed in the EPAs. In addition, cross-correlations of spontaneous signals between electrode pairs declined (from 0.47 ± 0.1 to 0.35 ± 0.04, p < 0.001) along with the EPAs throughout the week of injury. In support of the electrophysiological findings, immunohistochemical analysis at day-7 post-injury showed detectable Purkinje cell loss at low FPI pressures and more with the largest pressures used. Our results suggest that sensory evoked potentials (SEPs) recorded from the cerebellar surface can be a useful technique to monitor the course of cerebellar injury and identify the phases of injury progression even at mild levels.