Identification of subacute behavioral deficits following repetitive moderate levels of blast overpressure

Repetitive high-energy blast exposure is a major medical concern among service members as neurological impairment and mental health status (including post-concussive symptoms, anxiety, depression, and PTSD) are substantially worsened by injury severity and repetitive exposure. Both military and sports-related concussion research has frequently demonstrated that serial neurotrauma has cumulative effects, and that initial injury may lower the impact threshold for subsequent injuries, particularly if the subsequent injury occurs prior to full neurologic recovery from the initial injury. While important in its own right, such efforts do not replicate the higher energy, moderate-to-severe blasts (e.g., such as those due to improvised explosive devices), servicemembers in combat unfortunately often experience. In this study we randomly assigned adult male rats to one of three experimental groups: sham (S), single blast injury (BOP), and repetitive blast injury (rBOP). Rats in the BOP group were exposed to a head-on whole-body blast overpressure (120 ± 7 kPa) on study day (SD) 16 to align all of the animals for behavioral testing on the same day. This moderate-level of BOP has been shown to induce micro-hemorrhaging and astrocyte/microglial activation in the brain with limited mortality (< 5%). Rats in the rBOP were exposed to a single BOP event on SD 0, 14, and 16. Rats in the S group received maximum isoflurane exposure on all three SDs. The latency to righting reflex was measured as the length of unconsciousness immediately after rats were removed from the anesthesia chamber until the animals regained consciousness and righted themselves to the prone position. After the final BOP exposure, all rats were evaluated on a battery of behavioral assessments: the open field test (OFT) on SD 21 to assess exploratory behavior, light-dark box test on SD 22 to examine anxiety-like behaviors, y-maze on SD 23 to test spatial-working memory, novel object recognition (NOR) on SD 26 and 27 to investigate recognition memory, and fear conditioning on SD 28 and 29 to detect changes in associative learning and memory. There was a significant main effect of injury type on mean righting reflex time (s) wherein rBOP rats exhibited an increased latency to righting reflex on SD 0 (p < 0.0001) and SD 16 (p = 0.0001) as compared to S rats. BOP injured rats also exhibited an increased latency to righting reflex as compared to S rats (p = 0.0349). While there were no significant differences in mean ambulatory time between BOP and rBOP rats in the OFT, there was a significant reduction in mean ambulatory time between those exposed to rBOP and S rats (F = 4.840, p = 0.0145). In the NOR test, BOP injured rats spent significantly less time exploring the novel object in comparison to S-treated rats (F = 3.523, p = 0.0411), as indicated by the reduction in novelty index. Most notably, as compared to S and rBOP injured rats, the BOP rats exhibited a significant reduction in percent time freezing during the cue test portion of the fear conditioning assessment (F = 97.39, p < 0.0001). Neither the y-maze or light-dark box test yielded any significant findings between all three groups. In y-maze, all three cohorts spent a majority of their testing time in the starting arm and almost all rats did not leave the dark box in the light-dark box test. Taken together, our results indicate that the rBOP group exhibited a physiological impairment following each BOP as the duration of unconsciousness increased from the first to third exposure. The majority of the deficits observed in the rBOP group were due to locomotor and anxiety-like behavior changes as compared to sham animals whereas rats in the BOP group exhibited greater deficits in learning-and memory-based behavioral tasks. Further work to link possible neuroinflammatory-neurodegenerative transcriptomic changes in distinct brain regions to the behavior deficits and potential neuroprotective effects observed is ongoing.