Although the risks associated with THC use in adolescence have been a topic of significant debate, substantial evidence has suggested that THC is a potent antioxidant with neuroprotective properties (Hampson et al., 1998, 2000). In this study, RmTBI affected 4/6 of the behaviours examined, with therapeutic administration of THC following RmTBI producing beneficial outcomes in three of these measures. In vehicle treated animals, RmTBI increased loss of consciousness, increased anxiety-like behaviour in both the EPM and the open field, reduced exploratory behaviour and produced sex-dependent effects on depressive-like behaviours (reducing depression in males and increasing depression in females). Interestingly, daily administration of THC reduced anxiety in the EPM and reduced depressive-like behaviour in the forced swim task. These findings are in line with previous literature demonstrating that THC (at lower doses) reduces anxiety and emotional responses to psychosocial stress (Crippa et al., 2009; Childs et al., 2017), and suggests that THC may offer therapeutic potential for the treatment of RmTBI-induced anxiety. In addition, daily administration of THC mitigated RmTBI-induced impairments in short-term working memory. This is consistent with prior studies demonstrating that THC administration improves cognitive performance (Suliman et al., 2018). In summary, results from the behavioural test battery suggest that daily administration of THC following RmTBI has potential therapeutic efficacy for the treatment of PCS.

Telomeres are evolutionarily conserved sequences of repetitive DNA at the ends of chromosomes that maintain the integrity of the genome and protect the DNA from oxidative stress (Zhu et al., 2011). While telomere shortening has been recognized as a marker of biological aging and neurodegeneration (Klapper et al., 2001), research in our laboratory has demonstrated that telomere shortening is a characteristic of mTBI and can be used as a prognostic tool in rodent models (Hehar and Mychasiuk, 2016; Wright et al., 2018). Consistent with our previous studies, RmTBI reduced telomere length but interestingly, there was no RmTBI-induced reduction in telomere length for animals who were administered THC providing further support for the therapeutic benefits of THC.

The analyses of mRNA expression changes across the brain regions revealed numerous important neurological alterations associated with THC and RmTBI at the molecular level. First, we did not see any THC-induced changes in hippocampal Bdnf expression. This is important as evidence suggests that exogenous THC can affect long-term synaptic plasticity and remodel neurocircuitry (Gerdeman and Lovinger, 2003), and Bdnf has recently been implicated as a critical moderator of cannabinoid-mediated neurogenesis (Ferreira et al., 2018). We did, however, see downregulation of Bdnf in the PFC and NAc of THC treated rats. This is contradictory to previous studies that have found an upregulation of Bdnf in the NAc and ventral tegmental area of adult rats who were chronically administered THC (Butovsky et al., 2005). As studies have found that serum Bdnf levels are associated with whether or not individuals have a history of cannabis use (D’Souza et al., 2009), the timing of sample collection, age of the rats and history of exposure likely contribute to the discrepant changes in Bdnf expression identified in this study.

The numerous interactions identified in mRNA expression changes of Cnr1 are of particular relevance given that chronic THC exposure is known to alter the expression of Cnr1 (Zhuang et al., 1998) and cannabinoid receptor activation is critical for attenuation of brain damage following injury (Mechoulam and Shohami, 2007; Fishbein-Kaminietsky et al., 2014). Exactly how the endocannabinoid system assists in neuroprotection and acute neuronal injury is not fully understood. However, the activation of CB1 receptors by THC causes an alteration in GABA/glutamergic neurotransmission, inhibition of acetylcholine release and modulates immune responses and the release of inflammatory mediators (Pacher et al., 2006; Boggs et al., 2018). Moreover, CB1 receptors located on post-synaptic neurons control N-methyl-D-aspartate ion channel activity and therefore, provide protection against acute excitotoxicity (Paloczi et al., 2018). Given these functions of CB1 receptors, THC appeared to provide therapeutic benefit with respect to expression of Cnr1 in the HPC of both males and females; while Cnr1 mRNA levels were reduced in RmTBI animals treated with vehicle, they were increased in RmTBI animals treated with THC. This therapeutic benefit was also identified in the PFC, but only for female rats treated with THC.

TBI is believed to causes disruption to the catecholaminergic system, specifically because these neurons in the PFC are vulnerable to axonal injury, metabolic stress and mechanical injury, which often results in cognitive impairment (Jenkins et al., 2016). In addition, and of importance for this study, Comt modulates the acute neuronal effects of THC and is involved in the release of striatal dopamine (Bossong et al., 2019). The changes in Comt expression in the PFC were consistent with both of these concepts; RmTBI reduced Comt expression in vehicle animals but not THC animals, suggesting that THC administration provided therapeutic restoration of TBI-induced reductions in Comt expression. THC also altered expression of Comt in the NAc, corroborating evidence that Comt modulates THC-induced dopamine release in the striatum.

We examined GR expression because it can be used as a measurable indicator of the stress response. The endocannabinoid system is intricately involved in regulating the stress response and numerous studies have demonstrated that THC can directly modulate the HPA axis and glucocorticoid release (Manzanares et al., 1999). While current literature indicates that endocannabinoid signalling suppresses HPA activity (Hill and Tasker, 2012), stressful stimuli such as a traumatic brain injuries do the opposite and activate this system (Grundy et al., 2001). In this study, we found that while sham animals exposed to THC had reductions in GR mRNA expression in the HPC, RmTBI animals exhibited GR expression levels equivalent to vehicle-sham animals, suggesting that THC may have provided neuroprotection. These alterations in GR expression may have also contributed to the reduced anxiety-like behaviour on the EPM and open field tasks. Interestingly, GR expression was upregulated in the NAc in response to THC in both sham and RmTBI. Given the NAc and HPC work synergistically in feedback loops involved in drug administration and stress (Piazza and Le Moal, 1998), the reductions of GR in the HPC may be associated with the increased expression in NAc. To our knowledge, this is the first study to examine changes in GR expression in the NAc following THC exposure and this finding warrants further investigation.

In addition, neuroinflammation, such as microglial activation, is hypothesized to play a significant role in the pathophysiology of mTBI (Patterson and Holahan, 2012). Microglial activation has been found to persist in areas remote from the area of focal damage after TBI and has been related to various cognitive and behavioural impairments (Ramlackhansingh et al., 2011). We examined Iba1 mRNA expression as an indicator of neuroinflammation and microglial activation and found that THC exposure actually increased Iba-1 mRNA expression in the PFC and had no effect in the HPC or NAc. While the expression of Iba-1 is typically upregulated in microglia following TBI (Ito et al., 1998), in this study, THC administration did not confer therapeutic benefit.

Finally, RmTBI is believed to pose a threat to Vegf-2R expression, angiogenesis and tissue repair, with inhibition of Vegf-2R being associated with worse TBI outcomes, including greater lesion size and decreased neurogenesis (Xiong et al., 2011). Given that the cerebral vasculature is crucial for the delivery of oxygen and other nutrients to the brain, functional impairment and dysregulation of this system following TBI may inhibit recovery (Wendel et al., 2019). In both the PFC and NAc, RmTBI significantly reduced Vegf-2R mRNA expression levels in vehicle-treated animals, but this RmTBI-induced reduction was absent in THC-treated animals. Similar to the results identified for Comt and Crn1, post-injury THC administration appears to have provided some therapeutic benefit.