Traumatic Brain Injury (TBI) is a global public health problem, with an estimated 69 million (95% CI 64–74 million) TBIs occurring worldwide each year (Dewan et al., 2018). The leading causes of TBI are falls, wars, transport accidents, and sports (Langlois et al., 2006). Mild TBI (mTBI) accounts for between 75%–90% of total TBI cases (Åhman et al., 2013) and can result in persistent cognitive dysfunction (Silver et al., 2009; Strain et al., 2015). Additionally, it is now recognized that individuals who are exposed to repeated mTBI (rmTBI) have increased susceptibility to the development of neurodegenerative disorders including chronic traumatic encephalopathy (CTE) (Lehman et al., 2012; Nordström et al., 2014; Mez et al., 2017; Montenigro et al., 2017).

TBI pathology involves primary injury which induces secondary injury pathways. The primary injury encompasses the physical damage resulting from external impact transmitted to the brain. In contrast, secondary injury involves concurrent and self-exacerbating molecular and chemical changes that occur over time that amplify the damage caused by the primary injury (Kabadi and Faden, 2014). These secondary injury mechanisms include cellular excitotoxicity, oxidative stress, and dysregulated neuroinflammation by chronic activation of astrocytes and microglia, among others (Abdul-Muneer et al., 2015). Excitotoxicity occurs through the overactivation of glutamate receptors, leading to an influx of calcium ions which can generate further neuronal damage and trigger apoptosis (Morimoto et al., 2002). In addition, this leads to excessive mitochondrial uptake of calcium resulting in oxidative stress (Fehily and Fitzgerald, 2017) and induces other pathological mechanisms including cytoskeletal damage (Deng et al., 2007). Dysregulated inflammation through the chronic release of pro-inflammatory cytokines and chemokines also contributes to the progression of secondary injury (Simon et al., 2017). Understanding these secondary injury pathways is critical for developing effective neuroprotective strategies for TBI, as there are currently no Food and Drug Administration (FDA) approved therapeutic compounds for treatment (Hiskens, 2022).

Acetyl-L-carnitine (ALC) is an endogenously produced carnitine metabolite present in tissue and plasma, and readily crosses the blood brain barrier (BBB) (Jones et al., 2010) unlike its unacetylated form. ALC is also a commonly available nutritional supplement, with a known safety profile, and had been well-studied for the role in aiding β-oxidation of long chain fatty acids in the mitochondria (Bigford and Del Rossi, 2014). ALC demonstrates a wide therapeutic potential in chronic conditions which affect the cardiovascular and nervous system such as diabetes and hypertension (Malaguarnera, 2012). Recently, it has also gained attention as a possible neuroprotective agent, displaying therapeutic potential for neurodegenerative conditions in both pre-clinical and clinical studies (Mancuso et al., 2012). In animal models, ALC has exhibited neuroprotective properties including anti-inflammatory and antioxidant effects (Bogaert et al., 1994; Zanelli et al., 2005; Bagetta et al., 2008; Xu et al., 2015), as well as protection following glutamate-induced neurotoxicity in neurodegenerative states (Nagesh Babu et al., 2011). ALC was able to prevent excitotoxicity-induced N-methyl-D-aspartate (NMDA) reduction in the hippocampus, striatum, and frontal cortex of aged rats (Castorina et al., 1994), regulate synaptic plasticity by counteracting the loss of NMDA receptors in the neuron membrane (Jones et al., 2010), increase the production and activity of neurotrophins (Chiechio et al., 2006), and enhance production of nerve growth factor in brain tissue through increased choline acetyltransferase activity (Piovesan et al., 1994). Studies investigating the therapeutic administration of ALC in rat models of moderate TBI also found beneficial effects. In these studies, ALC administration resulted in smaller lesion volumes, improved sensory and motor function, as well as non-spatial learning compared to controls (Scafidi et al., 2011; Chen et al., 2018).

In human studies, ALC has shown the ability to improve cognitive performance and slow decline in the early stages of neurodegenerative diseases (Spagnoli et al., 1991), as well as reduce neurodegenerative oxidative and nitrosative stress (Calabrese et al., 2003). Taken in conjunction with other nutritional supplements and lifestyle interventions, ALC has also demonstrated cognitive improvements and increased cerebral blood flow in retired American football players with a history of concussion (Amen et al., 2011). Furthermore, a human trial of L-carnitine on patient with severe TBI showed preservation of neurological function after just 1 week of daily administration (Mahmoodpoor et al., 2018). This finding was impressive given the decreased ability of L-carnitine to cross the BBB compared to ALC. While some studies have shown promise for improving clinical and psychometric outcomes in individuals with probable Alzheimer’s disease (AD) and mild cognitive impairment, other studies that included participants with moderate AD progression were less conclusive (Onofrj et al., 2013). It may be that this lack of improvement is related to a therapeutic “window of opportunity”; once the neurodegenerative mechanisms have commenced, a reversal of these processes is not attainable (Bigford and Del Rossi, 2014). Therefore, prophylactic ALC treatment in a paradigm of neurotrauma may be a way to maximize its therapeutic potential.

The translational application of ALC is attractive given that it is an over-the-counter supplement that can be safely administered long-term (Traina, 2016) and is often part of a typical athletic supplement profile. Despite this, there are few studies investigating the therapeutic efficacy of ALC in the context of rmTBI. This study aims to determine whether improvement in genetic markers of inflammation, excitotoxicity, neuronal and glial injury occur as a result of preventative administration of ALC in a mouse model of rmTBI. Measures of neurological function post-injury should also show improvement in learning and motor skill analyses.

Mice in the rmTBI and the ALC + rmTBI groups showed no signs of physical stress following impacts, and no mice were withdrawn from the study. Mice demonstrated no adverse effects from the administration of ALC.

There is currently a lack of evidence for safe therapeutics that can be administered long-term to reduce the risk of individuals developing cognitive and neuropsychological deficits after rmTBIs. This study evaluated the role of prophylactic ALC administration on reducing the secondary injury mechanisms of axonal neurodegeneration, reactive astrogliosis and microgliosis, inflammation and glutamate excitotoxicity in the hippocampus and cortex resulting from rmTBI. The dosing profile followed a modified prevention paradigm to fully supplement the anti-inflammatory and metabolic cellular protective pathways targeted by ALC supplementation. The mTBI model in this study has been characterized to deliver repetitive, biomechanically relevant impacts, to induce CTE-like neuropathology and cognitive impairment (Hiskens et al., 2021c). Through the evaluation of TDP43, MAPT, AIF1, GFAP, TNF, CCL11 and GRIA mRNA gene expression in the cortex and hippocampus, we show that ALC mitigated damage inflicted in these secondary neurodegenerative cascades. This was also confirmed by improvements in behavioral and cognitive function.

TDP-43 is an important protein for stabilization of RNA processes such as transcription, mRNA stability and alternative splicing (Jo et al., 2020). Recent discoveries have implicated the protein in several neurodegenerative diseases such as Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis, Lewy body dementia and frontotemporal dementia and CTE (Jaiswal et al., 2022). Thus, TPD-43 was an important marker to confirm the presence of chronic neurodegeneration in our study. While our results indicated that the TDP-43 gene expression showed no significant changes in the acute phase in both the cortex and hippocampus, within the chronic arm (at 3 months), TDP-43 was significantly elevated in the impact group relative to the ALC + rmTBI treatment groups in both the cortex and the hippocampus. This is consistent with TDP-43 protein aggregation being a key feature of chronic neurodegenerative conditions (McKee et al., 2016), but rarely seen in the acute phase of injury or disease (McKee et al., 2010). It is believed its aggregation in the cytoplasm is mostly due to factors such as mislocation of the protein from the nucleus in states of cellular stress, as well as defective clearance mechanisms from the cytoplasm (Meneses et al., 2021). Incorrect TDP-43 signaling induced by mTBI damage may also contribute to the impaired transport of proteins and organelles within neurons, leading to the aggregation of axonal debris that contributes to neurofibrillary tangles (NFTs) commonly found in CTE (Cherry et al., 2016).

A key constituent of these NFTs is tau, a microtubule-associated protein involved in axon construction (Moreno-Gonz et al., 2011). In our study, we observed upregulation of MAPT (microtubule associated protein tau) mRNA levels in the cortex of the rmTBI group at 48 h, which was significantly different from the non-impacted sham and ALC + rmTBI groups. Although MAPT expression change was less pronounced in the cortex of the rmTBI group at 3 months, significant differences were observed compared to the ALC-treated impact group, suggesting a neuroprotective mechanism that may preserve tau function and organization within the axon. ALC has been found to have protective effects in other tau-dependent neurodegenerative disease (Yin et al., 2010), but the tau-mediating involvement of ALC in mTBI has not been previously demonstrated.

In the acute phase following rmTBI, microglia activate inflammation as a key secondary injury response, aimed at providing immediate neuroprotection. The response induces cytokine production, arachidonic acid conversion to prostaglandins, and excitotoxin release, intended to repair damage and return homeostasis (Corps et al., 2015). However, repetitive or severe trauma brings prolonged microglial activation and chronic inflammation, damaging neurons and glia and ultimately inducing neurodegeneration (De Beaumont et al., 2009; Loane and Kumar, 2016). ALC has been shown to reduce microglial activation and protect against neuroinflammation in a Parkinson’s Disease model (Singh et al., 2018; Burks et al., 2019). In the present study microglial activation was assessed by allograft inflammatory factor 1 (AIF1) gene expression. Following rmTBI, AIF1 significantly increased in the cortex at both 48 h and 3 months, and in the hippocampus at 3 months, indicating the increased inflammation and microgliosis typically observed following TBI. As proposed, ALC administration showed significant attenuation of AIF1 in both tissues in the chronic phase compared to the rmTBI group in this study. This is an interesting distinction, since microglia are known to shift towards destructive functions in the chronic phase of injury, while the acute response may deliver beneficial pro-regenerative and neuroprotective substrates (Loane and Kumar, 2016).

Astroglial cells provide another mechanism for inflammation after mTBI, as they communicate with microglia to respond to the demands of the central nervous system (CNS) (Loane and Kumar, 2016). Glial fibrillary associated protein (GFAP) is an established indicator of astrocyte activity in human head trauma, and in this study an increase in GFAP mRNA expression was observed in the impact group in acute cortex and chronic hippocampus tissues, consistent with previous animal mTBI studies (Mychasiuk et al., 2016). In the ALC + rmTBI groups, the effects of impact were modulated by ALC, with levels not significantly different from the non-impacted control group. This data indicates that ALC can reverse the astrocytosis in the hippocampus that ensues following rmTBI. This effect is consistent with previous findings showing decreased astrocytic reactivity following ALC administration in models of neurodegeneration (Burks et al., 2019) and epilepsy (Tashakori-Miyanroudi et al., 2022).

The inflammatory cascade driven by glial cell response to injury involves the release of products such as cytokines, and our study assessed the levels of pro-inflammatory cytokine, TNF. ALC administration has been shown to decrease TNF activation in an epilepsy model (Tashakori-Miyanroudi et al., 2022). In our study, TNF increased in the cortex tissue of mice 48 h following mTBI. In the ALC + rmTBI group there was no difference in TNF mRNA expression compared to the sham control group at this, or any timepoint. TNF plays a critical role in initiating and increasing the mobilization of microglia and astrocytes following CNS injury (Woodcock and Morganti-Kossmann, 2013), and the relationship between TNF, AIF1, and GFAP expression change in response to mTBI and treatment is valuable for further exploration.

We sought further evidence of the neuroinflammatory response to rmTBI and ALC treatment by examining the expression of CCL11, a chemokine that has received increased attention in TBI (Kovac et al., 2011). CCL11 can have neuroreparative mechanisms, however excessive release can lead to oxidative stress and excitotoxic processes that cause synaptic dysfunction and neuronal death (Parajuli et al., 2015). Studies in mice have linked high levels of CCL11 to cognitive and memory impairment (Villeda et al., 2011), while elevated levels in the cortex of former American football players with CTE correlated with tau pathology and the number of years playing football (Cherry et al., 2017). The present study found increased mRNA expression of CCL11 in rmTBI mice in the cortex at chronic measurement, and in the hippocampus at 48 h post-injury. In both conditions, rmTBI groups were also significantly different to ALC + rmTBI treatment groups, whose CCL11 expression was no different to control. Understanding the potential role of CCL11 as a biomarker of mTBI and in chronic neuropathology is an area for future investigation.

Another important secondary injury mechanism following mTBI is excitotoxicity, involving impaired neuronal calcium regulation and glutamate release resulting in dendrite damage (Lewerenz and Maher, 2015). Upregulation of N-methyl-D-aspartic acid (NMDA) GluA1 receptors is triggered to counteract the damage caused by glutamate build-up in the synaptic cleft, indicated by GRIA1 mRNA expression. The effect of ALC in correcting dysfunctional glutamatergic neurotransmission has previously been demonstrated in a model of depression (Nasca et al., 2013). The hippocampus is especially vulnerable to excitotoxic insult (S et al., 2008), and in the present study rmTBI mice displayed significant hippocampal excitotoxicity at 48 h, with increased GRIA1 expression indicating the attempts to clear excess glutamate. Conversely, ALC + rmTBI mice had significantly lower GRIA1 levels than rmTBI mice and were more similar to those expressed in sham control. This GRIA1 dysregulation may be a contributor to reduced functional outcomes, as spatial working memory relies on the activation GluA1 receptors in the hippocampus (Freudenberg et al., 2013).

We further investigated whether the neuroprotective effects of ALC at the molecular level would translate into mitigation of behavioral and cognitive dysfunction seen following rmTBI. Time to regain RR in the rmTBI group were within the range of previous studies (Dewitt et al., 2013). Importantly, ALC was able to negate the rmTBI-induced RR dysfunction to levels seen in the sham group. Similarly, when undertaking NSS assessment to determine motor, sensory, coordination, and reflex function, rmTBI mice scored poorly while mice treated with ALC performed to the same level as uninjured controls. These data suggest that while the mild impacts resulted in no macroscopic pathology, there was still measurable neurological perturbation in the untreated group that was alleviated by ALC pre-treatment. These interactions may be related to the secondary injury pathways that were assessed via gene expression changes, however further mechanistic studies are required to investigate causation of this effect. Secondary injury to the hippocampus leads to impaired function, which we assessed using the MWM task, to include elements of learning and memory. The rmTBI group displayed impaired performance in the MWM in the acute and chronic testing, indicating the presence of spatial learning and memory deficits, which is consistent with findings from other studies in mice receiving a mTBI (Meehan et al., 2012; Tabet et al., 2022). This test provides an insight into cognitive performance following impact that is relevant in modelling the detriment seen in clinical studies. Evidence from populations such as retired professional football players indicates that repetitive concussive and subconcussive impacts may have long-term implications for cognitive and executive functions, such as working memory and decision making (Hart et al., 2013). At both acute and chronic measurement in our study, treatment with ALC showed protection in later testing sessions, indicating that ALC may contribute to the alleviation of spatial learning deficits that result from rmTBI. Our findings agree with Scafidi and others, who found ALC improved cognition and motor function following moderate TBI (Scafidi et al., 2011). In other studies of neurodegeneration, supplementation with ALC has been found to improve attention, learning and spatial working memory deficits induced by hypoxia (Meneses et al., 2021) and improved MWM performance in a model of Parkinson’s Disease (Singh et al., 2018). ALC has contributed to enhanced learning capacity and cognitive function in rats by providing acetyl groups for acetylcholine synthesis, thereby exerting an effect in cholinergic neurotransmission (Ando et al., 2001). Taken together, these data demonstrate the potential of ALC in eliciting behavioral benefit in the context of rmTBI, and future studies should investigate mechanistic determinants of this effect.