Abstracts

Rationale: Concussion is a form of mild traumatic brain injury (mTBI) caused by rapid rotational acceleration and deceleration of the head. Clinical evidence suggests an increased long-term risk of epilepsy following mTBI, and such risk is likely augmented in the setting of repetitive injury. Yet, the mechanism of post-mTBI epileptogenesis remains elusive. We aim to characterize chronic changes in neurochemical and cellular markers following repetitive mTBI (rmTBI) in mice that may render the brain more susceptible to seizure. Methods: Young adult (postnatal day 60) C57BL/6 male mice were randomized to rmTBI (N=102) or sham injury (N=100). For rmTBI, mice were anesthetized and underwent a single impact (54 g dropped from 106 cm above the head) daily for five consecutive days. Two cohorts (NrmTBI = 4 and 6, Nsham = 4 and 6) were implanted with microdialysis cannulae positioned in the somatosensory cortex on 5th injury day. Dialysate was analyzed by HPLC. The concentration of extracellular gamma aminobutyric acid (GABA) and glutamate (Glu) were measured weekly for six weeks after the last injury in the first cohort. Reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) was measured at one and six weeks in the second cohort. A third cohort (NrmTBI = 4 and 6, Nsham = 4 and 6) was systemically perfused with 4% paraformaldehyde at one week and six weeks, and brain tissues were processed for immunostaining of parvalbumin-expressing interneurons (PVIs). A fourth cohort, (NrmTBI = 6, Nsham = 6) implanted with wireless EEG transmitters, was recorded for 48 hours at one and six weeks. In a fifth cohort, seizure susceptibility was measured by pentylenetetrazol (PTZ) challenge test (50 mg/kg, i.p.) to record time to myoclonic jerk and generalized tonic clonic (GTC) seizure at one (NrmTBI=32 and 48, Nsham=30 and 48) or six weeks. Results: One week after the last injury, extracellular Glu concentration increased after rmTBI by 106% (p=0.0349), but returned to sham control levels from the second week post-rmTBI onward. Extracellular GABA concentration did not change at one week, but was reduced by 47% at six weeks (p<0.0001). GSH/GSSG ratios were lower in rmTBI animals at one (p=0.0083) and six weeks (p<0.0001). Parallel immunostaining revealed a 29% increase in PVI density in the somatosensory cortex one week after rmTBI (p=0.0115). However, PVI density declined to 60% of sham control at six weeks (p=0.0043). The change in PV expression in the somatosensory cortex is corroborated by a loss of power in the EEG gamma band (30-80 Hz) after rmTBI (p=0.0125) during circadian light cycle. PTZ challenge revealed shorter latency to (1) first myoclonic jerk at one week (p=0.0143) and six weeks (p=0.0057), and (2) GTC at six weeks (p=0.0017) after rmTBI. Conclusions: rmTBI results in reduced cortical GABAergic transmission, increased oxidative stress, and higher seizure susceptibility. Notably, an increased Glu:GABA ratio is driven by a Glu increase in the initial injury phase, followed by a reduced GABA phenotype, in the chronic post-rmTBI phase. These findings are consistent with observed increased epilepsy risk in human mTBI (Christensen et al., 2009). The initial increase in PVI density may reflect compensatory circuit changes at the early phase of disease progression. However, chronically, this compensation fails to sustain the injury, which ultimately leads to a loss of PVI function and increased vulnerability to seizures. Funding: This work was part of the Football Players Health Study at Harvard, funded by a grant from the National Football League Players Association (NFLPA). The content is solely the responsibility of the authors. It does not necessarily represent the official views of Harvard Medical School, Harvard University, its affiliates, Boston Children’s Hospital, or NFLPA.