Melatonin in Traumatic Brain Injury and Cognition

Traumatic brain injury (TBI) is a leading cause of long-term disability and mortality in young adults. The devastating effects of TBI on emotion regulation, executive functioning, and cognition have been well-established, and recent research links TBI as a risk factor for neurodegenerative diseases such as Alzheimer’s disease. Despite an increased focus on the long-term cognitive dysfunction associated with TBI, research into potential treatments has not yet generated consistent successful results in human subjects. Many foundational studies have analyzed the cellular and molecular events involved in the inflammatory and healing processes following TBI, enhancing our understanding of the mechanisms that may contribute to the progression of dementia and cognitive decline in these patients. In this review, we will discuss the emergent research on melatonin within the framework of neuroinflammation and oxidative stress resulting from TBI and possibly preventing further sequelae such as Alzheimer’s disease. A literature review was conducted using standard search strategies to query the PubMed database. The following search terms were used with qualifiers of various combinations: TBI, traumatic brain injury, melatonin, treatment, dementia, Alzheimer’s, cognition, and neurodegeneration. Selected studies included meta-analyses, literature reviews, and randomized controlled trials (RCT) that evaluated melatonin’s role as a potential therapy to prevent post-TBI neurodegeneration, specifically the development of dementia and deficits in memory and cognition. Three independent reviewers assessed all articles for eligibility. After assessment for eligibility, 11 total studies were included. Much of the available data on melatonin in TBI has highlighted its significant neuroprotective and antiinflammatory effects, which can be significant in fighting against the neuroinflammatory processes indicated in neurodegeneration. In animal models, immunohistochemistry and histopathology have allowed researchers to study measures of cell injury such as inflammatory cytokines, edema, and markers of oxidative stress. Though the effects of melatonin in TBI appear to be mediated through mostly indirect mechanisms on inflammatory processes, some research has explored potential mechanisms that could be specific to melatonin. Animal model studies support that melatonin treatment after TBI significantly improves cognition and behavioral outcomes. However, clinical studies with human subjects are scarce. Beyond the apparent general antiinflammatory and antioxidant actions of melatonin, a review of the evidence identified some preliminary research that has suggested the significance of melatonin receptors specifically in TBI. While there is some evidence to suggest that melatonin is able to reduce post-TBI cognitive decline as measured by subject performance on memory tasks, there is a lack of longitudinal data on whether melatonin decreases the risk of developing dementia after TBI. Considering melatonin therapy’s promising preclinical data, favorable safety profile, and accessibility, further studies are warranted to assess the effects of melatonin as a post-TBI therapy on human subjects.


Introduction And Background
Traumatic brain injury (TBI) is a leading cause of long-term disability and mortality in young adults. The devastating effects of TBI on emotion regulation, executive functioning, and cognition have been wellestablished [1,2]. Recent literature similarly suggests that TBI is a risk factor for neurodegenerative diseases such as Alzheimer's disease [3,4]. Despite an increased focus on the long-term cognitive dysfunction associated with TBI, research into potential treatments has not yet generated consistent successful results in human subjects. However, a plethora of studies has analyzed the cellular and molecular events involved in the inflammatory and healing processes following TBI, enhancing our understanding of the mechanisms that may contribute to the progression of dementia and cognitive decline in these patients. Studies have shown that persistent neuroinflammation with chronic microglial activation may contribute to the future 1 2 3 2 1 development of neurodegenerative disease following TBI. Moreover, axons are particularly vulnerable to injury due to TBI-induced white matter degeneration, and the damaged axons may serve as a reservoir for amyloid precursor protein (APP) and beta-amyloid (Aβ) [3,4]. In addition, chronic post-TBI neuroinflammation may result in impairment of the ubiquitin-proteasome pathway for protein degradation, resulting in increased Aβ and p-tau (phosphorylated tau) deposition [3]. It has also been found that TBI depletes intracellular energy stores, causing failure of ATP-dependent ion pumps, which results in membrane depolarization and subsequent glutamate release. Glutamate incites neuronal cell death via excitotoxicity, which involves the release of reactive oxygen species (ROS) [5,6]. Therefore, it is thought that therapies promoting a decrease in neuroinflammation and oxidative stress may be able to prevent the development of neurodegenerative diseases such as Alzheimer's disease after TBI.
There are currently no Food and Drug Administration (FDA)-approved therapies for treating TBI. Due to the risk of post-TBI neurodegenerative disease, it is critical to investigate potential therapies that may prevent adverse outcomes in these patients. Melatonin, a naturally occurring hormone that crosses the blood-brain barrier and binds receptors in the central nervous system has been found to have neuroprotective effects that may reduce symptom burden after TBI [7]. Various studies have shown that melatonin exerts its neuroprotective action through its antiinflammatory and antioxidant capabilities, which may contribute to reduced risk of post-TBI secondary injury and subsequent functional deficits. Because such neuroprotection has been theorized to reduce the cognitive deficits apparent after TBI, recent research has investigated the effect of melatonin supplementation in post-TBI cognitive processes. In an attempt to more clearly characterize this supplement's specific role in cognition, we have examined the most recent literature on this nascent and developing topic. In this review, we will summarize the role of melatonin in preventing post-TBI neurodegeneration, particularly focusing on melatonin's potential to reduce the risk of cognitive impairment after TBI.
Melatonin, or 5-methoxy-N-acetyltryptamine, is most colloquially known as a sleep aid due to its availability as an over-the-counter supplement for sleep. Synthesized at night and secreted by the pineal gland, melatonin configures the circadian rhythm and has many pleiotropic effects, including antiinflammatory, antioxidant, and cell cycle-modulating properties [8]. The nycthemeral rhythm of endogenous melatonin secretion is based on the sun. Light is relayed from photosensitive ganglion cells in the retina via the retinohypothalamic tract as an inhibitory signal to the hypothalamus, suppressing the suprachiasmatic nuclei (SCN) and melatonin production during the day. Thus, melatonin is known as "the darkness hormone" and mediates entrainment to our external environmental cues. When administered at night, artificial light of sufficient intensity and duration suppresses melatonin production and disrupts melatonin rhythm [9].
The pathway continues as the SCN synapses with preganglionic cell bodies of the superior cervical ganglia (SCG) of the sympathetic chain in the upper part of the cervical spinal cord. The SCG therein send projections to the pineal gland and release nocturnal norepinephrine, the major regulatory neurotransmitter of the pineal gland. Melatonin production in the pineal gland is dependent on circulatory tryptophan, where it is taken up and transformed to serotonin. The binding of norepinephrine to adrenergic β1 receptors activates pineal adenylate cyclase, increasing cyclic AMP (cAMP) and de novo synthesis of serotonin-Nacetyl transferase (NAT), the rate-limiting enzyme for melatonin synthesis. Drugs that increase synaptic catecholamine availability, such as MAO inhibitors or tricyclic antidepressants, reinforce melatonin secretion. Conversely, drugs that decrease synaptic catecholamine availability, clonidine, and a-methylpara-tyrosine, and β1-adrenergic receptor blockers suppress nightly melatonin secretion [10].
There is no pineal storage of melatonin. As it is released in the circulation, it gains access to various fluids, tissues, and cellular compartments by means of its high lipid and water solubility (octanol/water coefficient of partition≈13). The liver primarily metabolizes melatonin, where it is first hydroxylated, then excreted in urine as sulfate and glucuronide conjugates. The maximum plasma level is around 03:00-04:00 a.m. for most chronotypes, with undetectable or low diurnal levels, reflecting the greatest nycthemeral amplitude change observed for a hormone [10].
Melatonin provides the body with night information and is considered to be the organizer of circadian rhythm, facilitating core temperature and cortisol cycles. Two high-affinity G protein-coupled membrane receptors, MT1 and MT2, have been presently identified in humans throughout the body, including the brain, retina, cardiovascular system, and skin. Melatonin facilitates sleep predisposition through vasodilation via MT2 receptor [11]. Physiological doses of melatonin inhibit the in vitro ACTH-stimulated cortisol production [12]. It also theorized that melatonin is a stabilizing factor within other physiological functions, including immune, antioxidant defenses, hemostasis, and glucose regulation. Beyond its membrane receptor activation, the small lipophilic melatonin can pass through biological membranes, functioning as a ligand for the orphan nuclear hormone receptor superfamily RZR/RO and influencing its pro-apoptotic effect on cancer cells [13]. As an antioxidant, melatonin is more potent than vitamin E and directly scavenges radicals. It further elevates its antioxidant role by raising levels of several antioxidative enzymes, including superoxide dismutase, glutathione peroxidase, and glutathione reductase, and inhibiting pro-oxidative enzyme nitric oxide synthase [14]. Melatonin scavenging of oxidative radicals in lymphoid cells is thought to have an immunomodulatory impact on inflammation and other inflammatory disease processes, such as traumatic brain injury and its sequelae.

Review Methods
A literature review was conducted using standard search strategies to query the PubMed database. The following search terms were used with qualifiers of various combinations: TBI, traumatic brain injury, melatonin, treatment, dementia, Alzheimer's, cognition, and neurodegeneration. Selected studies included meta-analyses, literature reviews, and randomized controlled trials (RCT) that evaluated melatonin's role as a potential therapy to prevent post-TBI neurodegeneration, specifically the development of dementia and deficits in memory and cognition. Three independent reviewers assessed all articles for eligibility. After assessment for eligibility, 11 total studies were included.

Results and discussion
The present study on the role of melatonin for dementia in TBI is sparse. A PubMed database keyword search for studies containing all three keywords of "TBI," "melatonin," and "dementia" returned zero results, while a search with the keywords "TBI," "melatonin," and "Alzheimer's" returned just one study. However, broader searches including keywords such as "cognition" and "neurodegeneration" yielded more results that suggested the presence of some foundational data about melatonin's potential roles in cognitive processes post-TBI. The studies are presented in Tables 1, 2. The neuroprotective functions of melatonin in TBI and SCI include its ability to reduce brain edema, decrease late-phase activation of NFkB, decrease AP-1 to basal levels, regulate inducible NOS, and increase the activity of superoxide dismutase and glutathione peroxidase, which protect against oxidative stress Both endogenous and exogenous melatonin have been found to provide neuroprotection. The level of endogenous melatonin in the CSF increases after TBI. There is limited data available on the adverse effects of melatonin, but recent studies indicate that melatonin may generate intracellular ROS via a reduction of intracellular glutathione activity in U937 cells   Much of the available data on melatonin in TBI has highlighted its significant neuroprotective and antiinflammatory effects, which are critical in fighting against the neuroinflammatory processes indicated in neurodegeneration and injury. As a physiological protective mechanism, the level of endogenous cerebrospinal fluid (CSF) melatonin increases in TBI patients to suppress the level of oxidants, and both endogenous and exogenous melatonin have been found to combat oxidative stress in the brain [17]. Immunohistochemistry and histopathology in animal models have allowed researchers to study measures of cell injury such as inflammatory cytokines, edema, and markers of oxidative stress. In one review of melatonin's roles in central nervous system (CNS) injuries, melatonin was reported to reduce neuroinflammation and edema, decrease late-phase activation of nuclear factor-kappa light chain enhancer of activated B cells (NFkB), decrease activator protein 1 (AP-1) to the basal level, and increase the activity of superoxide dismutase and glutathione peroxidase, which protect cerebral tissue against oxidative stress [17]. Cerebrovascular injury may also be a major contributor to neuroinflammatory damage [5,6].
Persistent neuroinflammation may provide a link between a past TBI and the future development of neurodegenerative diseases [3,4]. This connection offers a strong basis for establishing the role of melatonin in cognitive decline after TBI due to its roles in inflammation and brain injury. As suggested by experiments investigating patterns of positron emission tomography (PET) ligand binding in TBI patients, increased microglial activity may persist long after TBI. Increased binding is correlated with more severe cognitive impairment, suggesting chronic inflammatory response, especially in subcortical regions [26]. In the only study specifically looking at melatonin's role in neurodegeneration after TBI, researchers saw increased levels of ROS and malondialdehyde (a marker of lipid peroxidation) and reduced expression of the antioxidant protein nuclear factor erythroid 2-related factor 2 (Nrf2) in the brains of mice following repetitive mild TBI [18]. They also found treatment with melatonin reduced these effects, indicating the anti-inflammatory and antioxidant roles of melatonin. In the study, melatonin had further injury-reducing effects through decreased apoptotic cell death and lesion volume and a significant reduction in levels of expression of the Alzheimer's disease marker proteins beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), APP, and Aβ proteins. Following melatonin treatment, these mice also had improved performance in the Y-maze and beam walking tests, which are clinical models for measuring cognition and motor function, respectively. Other performance tests of cognition used in animal studies of melatonin post-TBI included the Morris water maze and a battery of behavioral assessments including novel context mismatch and swim force tests. Measures of motor function and coordination include time on a wire grip, balance beam, and Rotarod apparatus [15]. Performance measures have all been shown to improve after melatonin administration post-TBI, suggesting a role of melatonin in preventing the neurodegenerative changes associated with TBI.
Studies on adult mice have shown that melatonin administered at specific doses decreased lipid peroxidation levels and promoted antioxidant activity following TBI [7]. Melatonin appears to demonstrate similar neuroprotective effects in the pediatric population. In one study evaluating the effect of melatonin on oxidative damage induced by TBI in seven-day-old rat pups, results show that a single dose of melatonin at 5 mg/kg prevented an increase in levels of thiobarbituric acid reactive substances (TBARS), byproducts of lipid peroxidation [21]. A similar study by Ozdemir et al. in 2005 utilizing seven-day-old rats found that melatonin preserved hippocampal neurons following TBI and decreased deficits in spatial memory as identified by performance on a water maze task [7]. These results reinforce the idea that melatonin protects against secondary brain injury induced by ROS after TBI, which could attenuate the development of post-TBI cognitive decline in the future.
Though the effects of melatonin in TBI appear to be mediated through mostly indirect mechanisms on inflammatory and oxidative processes, some research has explored potential mechanisms that could be specific to melatonin and its receptors. One study showed that melatonin receptors (MT1 and MT2) were less abundant in the frontal cortex and hippocampus of rats 24 hours following controlled impact TBI compared to after sham surgery [25]. The authors note that this finding, though it requires more elucidation, could potentially provide an explanation behind the wide range in the efficacy of melatonin therapy after TBI.
Furthermore, in addition to generalized effects on inflammatory processes following TBI, melatonin appears to indirectly affect cognition through its other known physiological effects on sleep-wake cycles. For example, poor sleep patterns can disrupt normal processes of metabolite clearance that occur during sleep, leading to the accumulation of metabolites such as beta-amyloid and other neurotoxic waste products. Thus, some researchers have explored the effects of transcranial photobiomodulation (tPBM) for TBI patients as red (633 nm) intranasal light-emitting diode (iLED) is believed to increase melatonin [19]. NIR (near-infrared) photons are also used in tPBM, and photons delivered into the nose can reach medial temporal lobe structures, including the hippocampus and the adjacent perirhinal, entorhinal, and parahippocampal areas [19]. Preliminary results of tPBM in TBI patients have shown significant improvements in executive function, verbal memory, attention, verbal fluency, which were sustained at 12 weeks post-treatment. Predictably, sleep efficiency and average total sleep time also increased, which may have played a role in performance improvements. However, 5 mg melatonin supplementation (or 25 mg amitriptyline, to which it was compared) did not contribute to significantly improved sleep or neuropsychiatric parameters when compared to baseline in a double-blind randomized crossover trial with seven adult males who had suffered from post-TBI sleep disorders [15]. Interestingly, patients did self-report increased alertness when on melatonin, and effect sizes revealed improved sleep alertness, duration, quality, and latency (the same effect size patterns were seen with the amitriptyline treatment, in addition to patient self-reports of improved sleep duration and latency). While this study's small sample size precludes definitive conclusions, it highlights the complexity of identifying the potential roles and physiological mechanisms of melatonin in restoring cognition following TBI.
Moreover, most of the studies highlighted in this review focus on mice and rat models, which were sacrificed within a short period of time after the induction of TBI and subsequently did not produce adequate data to draw conclusions on melatonin's long-term effects after TBI. In addition, animal models may not be adequate predictors of outcomes in human subjects. Existing melatonin supplementation studies in human TBI patients are sparse, and those that exist are limited by small sample sizes and unclear significance. Therefore, there is a need for longitudinal retrospective studies on human subjects to determine whether melatonin therapy can produce significant differences in functional outcomes after TBI. Longitudinal studies can also be used to better characterize the progress of neuroinflammatory damage and recovery with the use of serial biomarkers and neuroimaging. Biomarkers such as levels of tau, p-tau, amyloid-beta, matrix metalloproteinases (MMPs), glial fibrillary acidic protein (GFAP), nerve fiber layer (NFL), and miRNAs offer some useful indicators of inflammation that can be measured in biofluids, while cerebrovascular dynamics, damage, and impaired metabolic clearance can be monitored over neuroimaging. Useful methods that will be invaluable to the longitudinal characterization of neurodegeneration include MRI to monitor vasoreactivity, blood flow, blood-brain barrier (BBB) permeability, and hypoperfusion; and PET to monitor hypometabolism and impaired metabolite clearance. Animal model studies support that melatonin treatment after TBI significantly improves cognition and behavioral outcomes. However, clinical studies with human subjects are scarce. Considering melatonin therapy's promising preclinical data, favorable safety profile, and accessibility, further studies are warranted to assess the effects of melatonin as a post-TBI therapy on human subjects.

Conclusions
The majority of findings from the studies gathered in this review demonstrate in murine models that melatonin exhibits neuroprotective effects through its antiinflammatory and antioxidant function, making it possibly beneficial in reducing the reactive processes that occur after TBI in the human brain. The proposed mechanisms by which melatonin exerts neuroprotection involve its ability to attenuate proinflammatory NFkB signaling, scavenge free radicals, decrease apoptotic cell death, and reduce the expression of abnormal proteins such as Aβ and p-tau. A reduction in such secondary injury processes may result in decreased risk of developing neurodegenerative diseases such as Alzheimer's disease following TBI. Beyond the apparent general antiinflammatory and antioxidant actions of melatonin, a review of the evidence identified some preliminary research that has suggested the significance of melatonin receptors specifically in TBI. While there is some evidence to suggest that melatonin is able to reduce post-TBI cognitive decline as measured by subject performance on memory tasks, there is a lack of longitudinal data on whether melatonin decreases the risk of developing dementia after TBI and further human clinical research is warranted.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.