Investigating the Impact of Repetitive Mild Traumatic Brain Injury on Central Endocannabinoid Function in Adolescent Male and Female Rats

Abstract

Background: Mild traumatic brain injuries (mTBI) constitute up to 80% of all brain injury cases, with ~23% occurring in the adolescent population. mTBI are characterized by the rotational movement of the brain due to biomechanical forces. Following the primary impact, the mechanical force, the secondary injury occurs, with hallmarks of excitotoxicity and heightened neuroinflammation. While the primary injury cannot be prevented, the secondary injury may be mitigated, preventing lasting cognitive and behavioral deficits. The endocannabinoid (eCB) system, a widely expressed neuromodulatory system modulates various cognitive and neurobehavioral responses and exerts anti-inflammatory actions. The eCB system contains two primary ligands, Anandamide (AEA) and 2-Arachidonoyl glycerol (2-AG), which retrogradely bind to cannabinoid receptors located presynaptically on neurons (CB1R) and in glial cells (CB2R) working to modulate neural activity and neuroinflammatory processes, respectively. Previous work has found pharmacological manipulations of this system improves cognitive and neuroinflammatory outcomes following mTBI. However, it is not well understood how this system responds following repetitive mTBI in a regional manner. Overall, there is high heterogeneity in mTBI presentation, pathology, mixed models in literature and ultimately a lack of generalizability of results in the field. Therefore, this work aims to measure how repetitive mTBI alter eCB levels (AEA and 2-AG) with high regional and temporal specificity through a set of controlled and translational experiments in adolescent rats. Methods: Adolescent Male and female rats received 5 mTBIs, 1 mTBI every 72 hours, via the Lateral Impact Model (LIM), a well validated translational mTBI model. 3 primary endpoints were assessed: 1) eCB levels immediately after injury, 2) eCB levels 1-week post injury, and 3) neurobehavioral outcomes. AEA and 2-AG levels were measured from the prefrontal cortex, motor cortex, amygdala and hippocampus via mass spectrometry at both endpoints and neurobehavioral assessments began 24 hours after the last mTBI; the hanging bar task (motor), the light-dark box task (“anxiety-like” behavior), and the novel-object-context mismatch task (cognition). Results: Male and female RmTBI animals exhibited greater righting times (proxy of loss of consciousness) after injury than shams. RmTBI elevated AEA levels in the hippocampus immediately after injury and decreased 2-AG levels in the prefrontal cortex 1 week after injury in males. In females, RmTBI decreased 2-AG levels in the motor cortex 1 week after injury. Males exhibited motor deficits in the hanging bar task following RmTBI, however no other behavioral impairments were reported. Females did not demonstrate any motor, emotional or cognitive deficits. Conclusion: RmTBI caused a dynamic alteration of eCB levels in both male and female TBI animals across both time points. Additionally, RmTBI led to fine motor impairments in the hanging bar task in males. Further work should investigate how these eCB changes correlate to neuroinflammation and potential excitotoxicity following injury.