Comparative Analysis of Apoptosis Following Traumatic Brain Injury in a Closed Head Controlled Impact Model

Introduction: Mild traumatic brain injury (mTBI) is the most prevalent severity of TBI. Due to the pervasiveness of mTBI and the cognitive impairments following injury, it is necessary to study the effects on local cell populations to determine effective treatment strategies. Secondary injury cascades play an important role in determining the fate of cells after TBI. The goal of this study was to characterize parameters of a closed-head controlled impact (CCI) model in the context of apoptosis, or programmed cell death. Since the secondary injury cascades can ultimately lead cells to an apoptotic fate, quantification is important for further understanding the effects of injury beyond initial insult.

Previous work has shown that mTBI is typically not associated with significant cell death, although multiple injury mechanisms and methodologies are used to replicate injury. Several closed-head models are currently in use, however variability among methods remains an ongoing challenge in terms of injury characterization. Therefore, comparison of methods and apoptotic outcomes are important for further quantification and understanding of closed-head impact models and the secondary injury cascade.

The specific study objective was to analyze how varying protocol parameters affect the apoptosis outcome within the regions of interest. We assessed apoptosis across hippocampal, cortical, and subcortical regions utilizing immunohistochemistry techniques. By varying the depth of impact parameter used to induce mTBI, the trajectory of apoptosis in a temporal context at acute timepoints was determined. Further, we compare apoptosis regionally to understand the location and type of cells that are undergoing apoptotic processes following injury.

Materials and

Methods: All animal procedures were approved by the Institutional Care and Use Committee at Virginia Tech. Male Sprague Dawley rats weighing approximately 300 to 315 grams at the time of injury were randomly assigned to a group of unique timepoint and parameter (Table 1).

Anesthesia was induced using 4% isoflurane for 5 minutes, then maintained throughout the injury protocol. The head was shaved, and a skull template was used to mark the right parietal cortex as the site of injury. An electromagnetic impactor with a tip of 5mm was set to a velocity of 6m/s, dwell time of 300ms, and a depth of 3mm or 8mm. Animals received a single impact and were immediately removed from anesthesia and placed on a heating pad, before returning to their home cage. At seven or three days post injury, animals were anesthetized and transcardially perfused with saline and 4% paraformaldehyde. Brains were harvested and fixed in 4% paraformaldehyde for approximately 24 hours, then immersed in 30% sucrose. The tissues were embedded in OCT at -80°C until frozen and sliced coronally at 30μm on a cryostat. To assess apoptosis, tissues were stained with anti-caspase-3 and anti-NeuN or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and anti-NeuN. Images of the amygdala, cortex, cornu ammonis 1 (CA1), cornu ammonis 2 (CA2), cornu ammonis 3 (CA3), and dentate gyrus (DG) were analyzed in QuPath and ImageJ. The percentage of total cells that are dead (PCD) and percentage of dead cells that are neurons (PDCN) was calculated.

Results, Conclusions, and Discussions: PCD was determined for caspase-3 and TUNEL stains respectively. Caspase-3 staining in the ipsilateral cortex yielded the only significant difference between injury parameters, P1 and P2, for both caspase-3 and TUNEL staining (Figure 1).

To determine the effect of depth, PCD was calculated to compare P1 and P2 for each region in the ipsilateral hemisphere at seven days. In the cortex, a 3mm depth produced significantly greater PCD than 8mm for both stains. Similarly, the DG and amygdala experienced an increase in apoptosis at 3mm compared to 8mm for TUNEL assay (Figure 2). These results agree that regions closer to the site of impact may experience more significant cell death. TUNEL assay additionally showed significant increases in apoptosis among the DG and amygdala. However, PCD among each parameter remained insignificant when compared to shams, suggesting that both depths produce mTBI.

Timepoint analysis revealed no significant differences when comparing PCD at three and seven days in caspase-3 staining. However, TUNEL results showed that CA1 and CA3 saw conflicting conclusions. Seven days following injury showed an increase in PCD in CA1, while CA3 showed a decrease (Figure 3). Conversely, the cortex, amygdala, CA2, and DG did not experience significant differences in apoptosis between timepoints for TUNEL assay. However, all injury parameters did not see a significant increase in PCD following injury, suggesting that both timepoints reveal similar apoptotic outcomes.

Regionally, analysis of PCD in caspase-3 staining showed no significant differences when compared to shams. However, TUNEL staining showed significant increases in PDCN in the amygdala, CA1, and CA3 of the ipsilateral hemisphere and CA1 and CA3 in the contralateral (Figure 4). Previous studies have reported that hilar neurons, CA3 pyramidal cells, and newly born DG neurons are susceptible to injury. However, apoptosis was still minimal when comparing across groups and to shams for PCD, reinforcing that regional differences in cell death exist, but remain insignificant overall.

Overall, when comparing PCD and PDCN between parameters and shams, significant apoptosis was not observed. This study further defined a CCI model of mTBI through the consideration of the secondary injury cascade and resulting apoptosis.

Acknowledgements (Optional):