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*This author wrote this paper as a senior thesis under the direction of Dr. Rebecca Delventhal.

1. Introduction

1.1 Traumatic brain injury in humans 

Traumatic brain injury (TBI) occurs when a mechanical force inflicts a sudden impact to the head which directly causes neuronal damage to brain tissue, resulting in neuropathology and dysfunction. Different classifications of TBI include closed head injury from acceleration and deceleration forces, penetration injury from a projectile, and blast injury from a blast wave. Closed head injury is the most prevalent type of TBI, and the force of impact can cause the brain to ricochet within the skull, which can lead to severe damage to neurons, glia, and blood vessels. The structural damage as a direct result of the impact is considered the primary injury. After the initial impact, damage to neuronal tissue can still occur hours or even days later through secondary injuries, which are caused by the cellular and molecular response to the damage brought about by the primary injury. Complications such as hemorrhage, inflammation, ischemia, hypoxia, cerebral edema, heightened intracranial pressure, and infection can occur from the primary injury and cause secondary injuries, such as oxidative damage and inadequate oxygenation (Mckee & Daneshvar, 2015). 

1.1.1 Classification and severity of traumatic brain injury 

Approximately 2.5 million people in the United States experience a TBI every year, and many injuries result in hospitalization (Friedan et al., 2015). While most people survive the direct consequences of the injury, between 3 and 5 million people in the U.S. are currently living with a disability that is a direct result of a TBI (Friedan et al., 2015; Zaloshnja et al., 2008), and within 5 years of rehabilitation, 1 in 5 patients will die (Corrigan et al., 2014).  

 Receiving a TBI often coincides with loss of consciousness, loss of memory before, after, or of the event itself, sensorimotor deficits, and states of confusion or trouble concentrating (Friedan et al., 2015). In humans, TBI is commonly quantified using the Glasgow Coma Scale (GCS), a 15-point scale measuring motor, verbal, and eye opening responses in patients potentially suffering from TBI (Sternbach, 2000). Some examples of responses we might expect a healthy individual to exhibit that may be affected after TBI include withdrawal from a painful stimulus, eye opening to a sound cue, and oriented, comprehensive speech (Sternbach, 2000). The GCS examines such responses and provides a score that can be used to diagnose injury severity, plan treatment, and signify a prognosis (Sternbach, 2000).  

In general, there are considered to be three levels of TBI severity, each diagnosed using a GCS score range: mild (13-15), moderate (9-12), and severe (8 or less) (Sternbach, 2000). Importantly, all severities of TBI have been associated with long-term physical, cognitive, and behavioral complications (Mckee & Daneshvar, 2015). Mild TBI, including concussions and some blast injuries, can be caused by activities in sports, military duty, and other conditions, including epilepsy. Complete neurological recovery is often expected after mild TBI. With moderate TBI, lethargy is initially present and after a severe TBI, such as from a car crash, individuals are often in a coma, unable to respond to stimuli. The risk of secondary injury, damage brought about by the cellular response to the primary injury, is high for patients with severe TBI (Mckee & Daneshvar, 2015). In a study of over 500 TBI patients, 89% of patients with severe TBI suffered from hypotension (a drop in blood pressure) as opposed to 11% of patients with moderate TBI (Andriessen et al., 2011). Eighty-five percent of patients with severe TBI also suffered from hypoxia (low blood oxygen levels) and had lower hemoglobin levels compared to moderate TBI patients, suggesting they were unable to transport oxygen through their blood as efficiently (Andriessen et al., 2011). Patients with severe TBI also had higher blood glucose levels, which have been associated with worse neurological outcomes after injury (Andriessen et al., 2011; Zhao et al., 2011). Such complications put patients with severe TBI at a much higher mortality risk. One study examining quality of life after TBI reported the mortality rate of severe TBI patients to be 35.7% compared to 17.2% and 4.1% of moderate and mild TBI patients, respectively. However, those who survive are almost always faced with long-term consequences as a result of such severe neurological damage.  

1.1.2 Long-term consequences of traumatic brain injury  

Apart from the damage brought about by secondary injuries, TBI can also lead to negative long-term health outcomes. Severe TBI has been linked with increased risk of dementia later in life, including increased risk of developing Parkinson's Disease and Alzheimer's Disease (Bower et al., 2003; Fleminger, 2003). Individuals diagnosed with Parkinson's Disease were four times more likely to have a history of head trauma, and males who experienced a head injury were 50% more likely to develop Alzheimer’s Disease (Bower et al., 2003; Fleminger, 2003). An earlier age of onset for Alzheimer's Disease has also been associated with TBI (Nemetz et al., 1999). Additionally, TBI has been linked with memory and cognitive deficits and an increase in depression diagnosis, with the risk of clinical depression being up to three times greater (Guskiewicz et al., 2005, 2007). 

TBI can also lead to changes in homeostatic mechanisms and behaviors. Homeostasis encompasses the processes designed to optimize function by maintaining stability and adapting to internal and external stimuli. An example of one such mechanism is metabolism, the breakdown of glucose into expendable energy. When blood glucose levels are high, a signal is released to bring glucose into cells to be metabolized. Once blood glucose levels become low again, another signal is sent to release stored glucose back into the blood. TBI can disrupt this homeostatic balance by causing hypermetabolism and catabolism, leading to increased energy expenditure and breakdown of important molecules such as fats and proteins (Lee & Oh, 2022). Such changes in TBI patients’ metabolic homeostasis can lead to malnourishment, and difficulties obtaining adequate nourishment can have a negative impact on recovery and mortality (Lee & Oh, 2022). In addition to metabolism, another homeostatic behavior that is disrupted by TBI is sleep.  

1.1.3 Traumatic brain injury causes long-term sleep disruption 

TBI is known to cause sleep disruption, which can further prevent proper function and rate of recovery after injury (Kalmbach et al., 2018). TBI can cause a range of sleep disruptions, including insomnia, excessive daytime sleepiness, and hypersomnia (Sandsmark et al., 2017). A meta-analysis of 21 studies reporting on sleep disturbances after TBI found that 50% of TBI patients experience some form of sleep disruption after injury, regardless of injury severity or type (Mathias & Alvaro, 2012). In TBI patients that were 3 to 24 months post injury, excessive daytime sleepiness due to nightly sleep disruptions was reported as the most common sleep-related symptom (Verma et al., 2007). Insomnia was the second most common symptom, where half of patients with insomnia suffered from sleep onset insomnia (problems falling asleep) and half from sleep maintenance insomnia (having difficulty staying asleep) (Verma et al., 2007). A separate study also found that within 6 months after TBI, rates of sleep onset insomnia were higher in patients with worse motor, vocal, and ocular function (Kalmbach et al., 2018). Similarly, rates of short sleep duration were higher in patients with impairment of such functions at 1 and 3 months after injury (Kalmbach et al., 2018). Other studies have shown that TBI can cause an increased need for sleep. Meta-analysis data reports that 28% of TBI patients are diagnosed with hypersomnia after injury (Mathias & Alvaro, 2012). Another study found that within six months after TBI, patients slept significantly more within 24 hours and had shorter sleep onset latency (Imbach et al., 2015). Over half the patients also experienced excessive daytime sleepiness, reported by a sleep latency less than 8 minutes (Imbach et al., 2015). Considering the prevalence of sleep disruptions after TBI, it is clear that homeostatic behaviors such as sleep are highly affected after injury. Studying how sleep may play a role in TBI recovery is important for improving injury outcomes.  

1.2 Negative impacts of sleep disruption  

Sleep is a natural, reversible state of unconsciousness that is characterized by immobilization and reduced responsiveness to stimuli (Rasch & Born, 2013)(Rasch & Born, 2013). Sleep is regulated by our circadian rhythm as well as homeostatic pressures. While the circadian clock is understood to control the timing of sleep, sleep homeostasis is considered to control the duration and consolidation of sleep (Dijk et al., 1999). Sleep homeostasis also regulates the response to sleep need or sleep pressure, which is built up during long periods of wakefulness and is relieved during sleep (Dijk et al., 1999).  

Sleep is a universal behavior found in nearly all organisms ranging from vertebrates like mammals, birds, and reptiles, to invertebrates like flies and worms (Cirelli & Tononi, 2008). Sleeping behavior leaves organisms vulnerable to predatory attacks and is not part of reproductive behavior necessary for species survival. Yet it continues to be conserved in nearly all organisms, so there must be an important evolutionary benefit to sleep.  

1.2.1 Sleep is required for proper neurological function and health maintenance  

Sleep in humans is frequently associated with learning and memory benefits, as sleep immediately after learning has been shown to improve memory while sleep deprivation impairs the ability to remember information (Rasch & Born, 2013). Slow-wave sleep in particular, characterized as stages 3 and 4 of non-REM sleep, has shown protective qualities towards memory (Alger et al., 2012). Participants allowed to enter slow-wave sleep when napping correctly recalled more words during a short-term test compared to those kept awake, and in the long-term they correctly recalled twice as many words than even participants who napped but were prevented from entering slow-wave sleep (Alger et al., 2012). In another study, when asked to learn and later recall a list of words, sleep-deprived participants forgot over 15% more words than those allowed to sleep normally (Gais et al., 2006). 

We need sleep for more than just learning and memory, however. Severe sleep deprivation in any organism for extended periods of time almost certainly leads to death (Cirelli & Tononi, 2008). Rats prevented from sleeping due to persistent physical stimulation died after 2-4 weeks of no sleep (Rechtschaffen & Bergmann, 1995). Hypothermia due to declining body temperature, catabolic tissue breakdown, and organ failure as a result of infection are all possible contributors to the certain death such severe sleep deprivation brings about (Rechtschaffen & Bergmann, 1995). Such fatal consequences are also seen in humans, particularly those with fatal familial insomnia (FFI). FFI is a rare genetic neurodegenerative disease where individuals eventually die after sleep disruptions worsen such that no sleep is acquired at all (Montagna, 2002). Cardiac complications, hypometabolism, severe loss of slow-wave sleep, and death of up to 90% of the neurons in certain thalamic regions of the brain are all characteristics of FFI (Montagna, 2002). Similar to severe sleep deprivation in rats, FFI is fatal and individuals with FFI typically die within 72 months of disease onset (Montagna, 2002). 

 Outside of the rare instances where sleep loss is fatal, chronic lack of sleep can still cause numerous adverse health outcomes, including dysregulation of body temperature and body weight and increased risk of infections (Garbarino et al., 2021). A meta-analysis of over 600,000 adults from across the world reported a significant association between sleep deprivation and obesity, with a similar association found even amongst children (Cappuccio et al., 2008). Another study investigating sleep-related infection risk showed that when exposed to a common cold virus, over twice as many participants who slept less than 5 hours per night became sick compared to participants who slept more than 7 hours (Prather et al., 2015). Interestingly, some evidence also suggests that sleep deprivation can even hinder our ability to develop immunity against certain pathogens (Fernandes et al., 2020). 

Sleep loss is also linked with the development of chronic disease. For example, individuals with sleep apnea are at a higher risk of developing cardiovascular disease (Kasasbeh et al., 2006). Repetitive surges in heart rate and blood pressure from the cycles of hyper- and hypoventilation characteristic of the disorder can often lead to hypertension (Kasasbeh et al., 2006). One study examining sleep and risk of Type-2 diabetes reported that sleeping less than 6 hours per night predicted worse glycemic control, an indicator of heightened diabetes risk (Knutson et al., 2006). Furthermore, poor sleep is even associated with higher depression risk, and over 80% of individuals with major depressive disorder reportedly experience some form of sleep disturbance (Zimmerman et al., 2006). 

Perhaps the most chronic disease of all is aging. Sleep consolidation, characterized by uninterrupted sleep without prolonged nightly awakenings, is generally high in adolescents and young adults, but sleep becomes much more fragmented in older adults (Dijk et al., 1999). For example, one study examining age-related reductions in homeostatic sleep drive found young adults, aged 20-30, slept about 7.5 hours every night on average with an average of 20 awakenings (Dijk et al., 2010). Older adults, aged 66 and above, only slept an average of about 6.5 hours with about 28 awakenings, indicating significantly more fragmented sleep in older adults (Dijk et al., 2010). The changes in sleep consolidation we see in older adults could be the result of a reduced homeostatic sleep drive as older adults have a reduced propensity, or sense of readiness, to fall asleep at night and spend less time in slow-wave sleep (Dijk et al., 2010).  

The age-induced gradual shift away from consolidated sleep and towards more fragmented sleep could be a contributing factor to the metabolic dysregulation, high infection risk, and development of neurodegenerative diseases that so frequently come with aging (Dijk et al., 1999). In addition to causing a higher risk for infection and obesity, prolonged sleep loss can reduce an individual’s resting metabolic rate (Buxton et al., 2012). Individuals with sleep complications are also at a higher risk for developing Parkinson’s Disease, and increases in amyloid-beta plaques, a defining characteristic of Alzheimer’s Disease, were found after only a single night of sleep deprivation (Hsiao et al., 2017; Shokri-Kojori et al., 2018).  

Clearly, sleep loss has many negative consequences not only on cognitive elements like learning and memory but also on our own health and survival. The natural decline in sleep duration and consolidation we see with age is just one of the many ways sleep problems can come about. Sleep problems are a common occurrence today, and sleep disorders put millions of people at high risk of suffering from the negative health outcomes seen as a result of chronic sleep loss (CDC, 2020; Stranges et al., 2012).   

Considering the vast array of negative consequences associated with acute and chronic sleep loss, it is vital that we receive an adequate amount of sleep each night. Human adults are recommended to get 7 to 9 hours of sleep every night (Watson et al., 2015). However, many adults do not routinely obtain enough sleep, and the prevalence of sleep disorders and other sleep disruptions today has brought about an epidemic of sleep loss (Chattu et al., 2018). 

1.2.2 Common sleep disorders and prevalence of sleep disruption 

There are various types of sleep disorders that occur at different frequencies and present different patterns of sleep disruption. Insomnia is the most common, affecting about 30% of adults worldwide, and is subdivided into two types: sleep onset (the inability to initiate sleep) and sleep maintenance (the inability maintain sleep) (Bhaskar et al., 2016; Stranges et al., 2012; Thorpy, 2012). Sleep apnea affects around 15% of individuals and occurs when breathing passages become constricted or closed during sleep, causing excessive snoring or gasping for air, disrupted sleep, and sleepiness during the day (Thorpy, 2012; Young et al., 2009). Hypersomnia is characterized by excessive daytime sleepiness and longer periods of nocturnal sleep (Dauvilliers & Buguet, 2005; Thorpy, 2012). Narcolepsy also includes excessive sleepiness during the day and often coincides with bouts of sudden loss of muscle tension (Thorpy, 2012). Restless legs syndrome causes sensations of “creeping” in the legs that causes an unpleasant feeling, along with aches and pains that can make falling asleep difficult. Prevalence of hypersomnia, narcolepsy, and restless legs syndrome are estimated to be below 10% of the general population (Dauvilliers & Buguet, 2005; Ohayon et al., 2012; Partinen et al., 2012). 

Perhaps not diagnosable, but another common, more recent form of sleep disruption occurs as a result of excessive use of electronic screens during the day and before sleeping, particularly in adolescents. A 2011 poll by the National Sleep Foundation found that 97% of young adults had one or more electronic device available for use in their bedroom (Gradisar et al., 2013). One study investigating how electronic use before bedtime affected sleep reported nearly all participants used some type of electronic device (or more than one) within 1 hour of their bedtime (Hysing et al., 2015). Excessive electronic use, characterized as over the recommended 2 hours per day, both during the day and in the hour before bedtime was positively correlated with higher sleep deficiency and longer sleep onset latency (American Academy of Pediatrics, 2001; Hysing et al., 2015). While an increase in sleep deficiency and sleep onset latency could likely be a result of sleep simply being replaced with time spent looking at a screen, there are other mechanisms that could explain why excessive electronic use can cause sleep disruptions. Exposure to bright light at night can cause the circadian rhythm to reset and delay the onset of sleep (Khalsa et al., 2003). Even more interesting is the possibility of persistent psychophysiologic (learned) insomnia, which can cause individuals to associate their bed or bedroom with electronic use, resulting in heightened arousal instead of relaxation once lying in bed (Hauri & Fisher, 1986; Hysing et al., 2015). Despite the differing mechanistic possibilities, it’s clear that excessive electronic use heightens the risk of sleep disruption. Due to the vast prevalence of electronics today, such disruptive effects pose a greater risk of spreading to far more people worldwide than diagnosable sleep disorders are capable of (Gradisar et al., 2013).  

With the prevalence of chronic sleep disorders and sleep problems such as those brought about by bedtime electronic use, sleep loss has become a frequent and widespread occurrence. An assessment by the CDC’s Behavioral Risk Factor Surveillance System found that 35-37% of adults in the United States have short sleep duration, defined by whether the recommended number of sleep hours is met, which for adults is a minimum of 7 (CDC, 2020). A study by the World Health Organization also found sleep problems in adults aged 50 and over from 7 countries across Africa and Asia (Stranges et al., 2012). It’s estimated that by 2030, over 260 million people will suffer from sleep difficulties (Stranges et al., 2012).   

It is clear that sleep disorders and general sleep disruptions are prevalent today. Considering the connection of sleep to TBI and injury-related impairment, understanding how sleep problems may affect recovery after TBI is important for developing preventative strategies and treatments to improve rehabilitation for TBI patients. 

1. Sleep disruptions affect injury recovery and outcomes 

TBI increases the risk of sleep problems after injury (see section 1.1). Given that sleep disruption is known to cause an array of negative health consequences, however, it is possible for sleep problems to have an adverse effect on TBI outcomes. One study examined the prevalence of impaired function in TBI patients with prior sleep problems (Kalmbach et al., 2018). Patients with TBI-induced disabilities ranging from severe, characterized by being conscious but unable to function independently, to upper-moderate, those struggling with work, leisure, and social activities but capable of being independent, were considered functionally impaired (Kalmbach et al., 2018; Wilson et al., 2021). Difficulty with both falling asleep and staying asleep prior to injury was associated with an increased risk of functional impairment (Kalmbach et al., 2018). Specifically, patients who had insomnia prior to injury were eight times more likely to have worse functional impairment after TBI than those without sleep problems before injury (Kalmbach et al., 2018). Another study looking at sleep disruptions as a predictor of TBI outcomes also found poor sleep to be associated with worse outcomes and longer rehabilitation (Sandsmark et al., 2017). TBI patients who did not suffer from sleep difficulties in the first 2 weeks post-injury were nearly 6 times more likely to have better functional outcomes after injury (Sandsmark et al., 2017).  

Altogether, there is significant evidence to suggest sleep disruption and sleep loss in humans can be detrimental to TBI recovery. TBI is also capable of causing more sleep difficulties, further perpetuating the negative effects of sleep problems on TBI outcomes. Considering this bidirectional relationship, understanding the physiological purpose of sleep may offer insight into why sleep loss can affect TBI outcomes and how TBI may interfere with sleep regulation. 

1.2.4 A physiological need for sleep 

Considering the universality of sleep behavior across the animal kingdom and the severe, sometimes fatal, consequences of sleep loss, there must be some physiological need for sleep beyond higher cognition. There have been many theories as to why such a need exists. One theory proposes sleep is required for energy conservation where, similar to hibernation, sleep offers a period of time where activity and energy expenditure are suppressed (Siegel, 2005). Another theory suggests sleep is needed for synaptic homeostasis, specifically the pruning of synapses formed during wakefulness to maximize the brain’s function and performance (Brown & Naidoo, 2010). Sleep has also been thought to regulate protein synthesis in the brain, as inhibition of neuronal protein synthesis has been found to increase sleep (Methippara et al., 2009). 

Another theory of a physiological need for sleep is the Free Radical Flux Theory, which suggests sleep is required for alleviating oxidative stress that builds up during wakefulness and causes cell damage (Reimund, 1994). The theory proposes that sleep is needed to maintain neural tissue stability, which is threatened by oxidative stress and the energy-demanding nature of the brain (Reimund, 1994). The brain is a very metabolically active organ, so it produces large amounts of reactive oxygen species (ROS), which are small molecular byproducts of metabolism that can cause cellular damage by reacting with DNA, proteins, and lipids (Phaniendra et al., 2015). The Free Radical Flux Theory proposes that sleep duration is directly correlated with the burden of ROS accumulation based on the metabolic rate of a certain species, where a higher metabolic rate increases ROS accumulation (Reimund, 1994). The theory suggests that endogenous antioxidant mechanisms in the brain that are meant to clear ROS cannot keep up with the amount of ROS generated throughout the day alone, so sleep is needed to reduce ROS levels before oxidative stress and cellular damage can occur (Reimund, 1994). Without sleep, it’s more likely that ROS, given its reactive nature, will begin to accumulate and cause cellular damage.  

1.3 Reactive oxygen species 

Reactive oxygen species (ROS) are free radicals that can occur naturally within an organism or be the result of environmental exposure. A free radical is an atom or molecule whose outermost valence shell has one or more unpaired electrons and is, therefore, very unstable and reactive (Figure 1). These radicals are able to remove electrons from other more stable molecules. As a result, the radical becomes more stable with its newly paired electrons, and the attacked molecule now has an unpaired electron and becomes a radical itself. This chain reaction can eventually cause damage to the cell by attacking and breaking bonds within important molecules such as DNA, lipids, and proteins, thereby damaging those molecules and preventing them from functioning properly (Phaniendra et al., 2015). 

ROS are classified into two types of compounds: radicals and nonradicals. As mentioned before, radicals have an unpaired valence electron and are highly reactive. Common examples of these include superoxide (O2-), hydroxyl (OH·), and nitric oxide (NO·). Nonradicals do not have an unpaired valence electron but can be easily made into radicals. Examples of nonradical ROS include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and nitroxyl (NO-). ROS can be acquired exogenously through sources such as chemicals, pollution, tobacco smoke, radiation, and some drugs. It can also be formed endogenously through different cellular processes, most commonly metabolic reactions (Phaniendra et al., 2015).   

Figure 1. Molecular structure of ROS and cellular damage. A. The chemical process behind ROS within cells. An unstable free radical with an unpaired electron steals an electron from a stable molecule. The stable molecule then loses that electron and becomes a radical itself. B. ROS-induced DNA damage and protein misfolding/aggregation. ROS can cause single- and double-stranded breaks in the phospholipid backbone of DNA and oxidize nucleotides. ROS can also cause proteins to misfold, resulting in protein aggregation and loss of proper protein function (Juan et al., 2021). 

The superoxide radical, one of the most common types of ROS, is formed in mitochondria when an extra electron is added to a dioxygen molecule (O2) (Figure 2) (Andrés et al., 2023; Phaniendra et al., 2015). Antioxidants like superoxide dismutase (SOD) 2 help neutralize and transform superoxide into hydrogen peroxide through a dismutation reaction. In this reaction, SOD catalyzes the reaction between two superoxide radicals and hydrogen ions to produce hydrogen peroxide and water, effectively neutralizing the superoxide radical. (Phaniendra et al., 2015; Wang et al., 2018). Hydrogen peroxide has no direct damaging effect on cells but can transform into a hydroxyl radical in the presence of ultraviolet light or a transition metal ion such as iron (Halliwell, Clement, et al., 2000). The resulting hydroxyl radical is reactive and capable of causing cell damage (Phaniendra et al., 2015). Since ROS naturally reside within organisms in radical and nonradical forms, their damaging abilities require antioxidant molecules to neutralize ROS and keep ROS from accumulating within the cell (Pham-Huy et al., 2008). In the instance where ROS levels become too high for our cellular systems to manage, accumulated ROS induce oxidative stress as cells are exposed to higher levels of cellular damage (Pham-Huy et al., 2008).  

Figure 2. The production and neutralization of superoxide in mitochondria. Normal function of the electron transport chain within the mitochondrial matrix and intermembrane space transfers electrons to help generate adenosine triphosphate (ATP), which the cell uses for energy. Most electrons react with oxygen and hydrogen molecules to produce water (H2O) at the end of the ETC. However, an estimated 0.2-2% of electrons leak from the ETC, most prominently from complexes I and III, and react with oxygen molecules to form superoxide radicals (Zhao et al., 2019). The antioxidant superoxide dismutase 2 (SOD2) catalyzes the reaction to neutralize superoxide radicals and produce hydrogen peroxide (H2O2) and water (Wang et al., 2018). The hydrogen peroxide nonradical can react with iron ions (Fe2+) to produce hydroxyl radicals (OH·) (Phaniendra et al., 2015). To prevent hydrogen peroxide from forming hydroxyl radicals, the antioxidant catalase helps degrade hydrogen peroxide into water and oxygen (Phaniendra et al., 2015).  

1.3.1 ROS-induced cellular damage  

ROS are capable of inflicting damage on many vital cellular components. Radicals can alter the molecular structure of DNA by causing single and double strand breaks in the phosphate backbone and oxidizing nucleotides (Juan et al., 2021; Sharma et al., 2016). They can also lead to lipid peroxidation, which inhibits normal membrane function and permeability (Wong-ekkabut et al., 2007). Additionally, proteins can be denatured by radicals, altering their structure and function (Butterfield et al., 1998). 

Most endogenous ROS are formed in the mitochondria since that is where the electron transport chain (ETC) takes place (Figure 2) (Phaniendra et al., 2015). The ETC is vital for creating the electrochemical gradient necessary to produce energy in the form of adenosine triphosphate (ATP) for the cell to use. Electron transfer is an important part of this process. However, electrons can leak from the complexes within the chain and react with other molecules, most notably oxygen, to produce ROS. Because of its proximity to the ETC and ROS production, mitochondrial DNA is much more vulnerable to oxidative damage than nuclear DNA (Phaniendra et al., 2015). Damage to mitochondrial DNA holds severe consequences for the survival of a cell as the genes encoded by mitochondrial DNA are vital for proper organelle function and metabolic processes (Habbane et al., 2021).  

In addition to the mitochondria, ROS can be found in the Endoplasmic Reticulum (ER). A main function of the ER is to fold proteins synthesized in the cell and secrete them in order for proteins to carry out their specific functions. ROS can be generated in the ER during the process of folding proteins using a mechanism called oxidative protein folding (Malhotra et al., 2008; Tu & Weissman, 2004). However, like with many locations within the cell where ROS is generated, the ER is susceptible to oxidative stress if too much ROS is accumulated. ROS and oxidative stress have been associated in the misfolding and inefficient secretion of proteins from the ER (Malhotra et al., 2008). When proteins processed by the ER are misfolded and are unable to be secreted, they aggregate within the ER and cause ER stress  (Brown & Naidoo, 2010). Cells under ER stress have shown higher levels of oxidative stress markers than cells with normal ER function (Malhotra et al., 2008). Additionally, reducing ROS levels using antioxidant treatment lessens ER stress by increasing protein secretion (Malhotra et al., 2008). Such evidence suggests that ER stress and oxidative stress go hand in hand.   

1.3.2 Nonpathological roles of ROS 

While there is overwhelming evidence that accumulation of ROS is detrimental, ROS at low levels does have a nonpathological purpose. Cells require a certain concentration of electrons, or redox state, in order to properly function (Valko et al., 2007). Similar to how pH is regulated in a cellular environment, cells tightly regulate their redox state, which is determined by the rate at which ROS are produced versus neutralized by antioxidants (Valko et al., 2007). When kept at low levels and within the range of the required redox state, ROS function as important signaling molecules (Valko et al., 2007). Regulated increases in ROS disrupt the balance of the redox state, and this imbalance in redox state acts as a signal to manage various important cellular functions (Valko et al., 2007). These include gene expression, pathogenic defense, cardiovascular growth and development, cell adhesion, and even sensation of blood-oxygen concentration (Galter et al., 1994; Griendling et al., 2000; Keisari et al., 1983; Valko et al., 2007).  

The innate immune response is also closely tied with redox signaling. Important components of the immune system, such as macrophages, are activated by ROS, and low ROS production during phagocytosis has been shown to cause hypersensitivity to infections (Morris et al., 2022; Pollock et al., 1995). ROS-induced damage also provides signals required for apoptosis (Valko et al., 2007). Apoptosis, or programmed cell death, is important for elimination of damaged cells. However, internally regulated apoptosis is also necessary for proper development and survival of cells, and changes in ROS levels help regulate this programmed death (Hengartner, 2000). Despite its nonpathological roles, ROS is mostly viewed as a pathological molecule due to its induction of oxidative stress at high levels. To avoid oxidative stress, cellular systems designed to neutralize ROS and prevent ROS accumulation are vital to cell health and survival.  

1.3.3 Antioxidants 

Antioxidants are molecules that help neutralize free radicals before cellular damage can be inflicted. Antioxidants give radicals a source of electrons to prevent them from taking electrons from more important molecules in the cell, such as the DNA phosphate backbone. We can acquire antioxidants exogenously through our diet and endogenously through sources within our bodies. Vitamin E, vitamin C, and beta-carotene are all examples of exogenous antioxidants that we obtain by consuming fresh fruits and vegetables, oils, and certain fatty foods (Pham-Huy et al., 2008). After we consume exogenous antioxidants, we absorb them through our gastrointestinal tract where they are transported to our cells (Halliwell, Zhao, et al., 2000; Okagu & Udenigwe, 2022).  

However, we get most of the antioxidants our bodies require endogenously (Pham-Huy et al., 2008). Endogenous antioxidants can come in the form of non-enzymes produced by our endocrine glands, like melatonin and uric acid, or as enzymes that are encoded in our DNA, like superoxide dismutase and catalase (Pham-Huy et al., 2008). There are three different types of superoxide dismutase (SOD) in particular, each encoded by a different gene (Flynn & Melov, 2013). Each type can be found in different parts of the cell: SOD1 is found mostly in the cytoplasm, SOD2 in the mitochondria, and SOD3 in the extra-cellular matrix (Flynn & Melov, 2013). The presence of SOD2 within the mitochondria puts it in close proximity to where the mitochondrial ETC produces free radicals (Flynn & Melov, 2013; Phaniendra et al., 2015). Because of this, SOD2 plays a very important role in neutralizing superoxide anions into their nonradical hydrogen peroxide counterparts before any damage can be done to important mitochondrial components and the surrounding the cell.  

Elimination or suppression of SOD2 is known to cause damage to many cellular components and tissues (Flynn & Melov, 2013). With the loss of SOD2, ROS production can go unchecked, leading to oxidative stress and further health problems. In humans, decreased SOD2 expression has been found to be correlated with cardiovascular complications in patients with sickle cell disease (Dosunmu-Ogunbi et al., 2022). Also, mutant mice that had a 48-55% decrease in SOD2 activity developed dilated cardiomyopathy, a condition that is characterized by enlarged heart cavities and thinner wall thickness and can be fatal (Li et al., 1995). Loss of SOD2 can also lead to metabolic dysfunction. Mice with deficient SOD2 activity showed an increase in susceptibility to neurotoxins, which can elicit mitochondrial dysfunction, leading to the generation of more ROS (Andreassen et al., 2001). With more ROS and less SOD2 readily available to neutralize radicals, chances of oxidative stress increase (Andreassen et al., 2001).  

Antioxidants and their role in preventing oxidative stress may also have an effect on neurodegenerative disease. For example, the progression of Alzheimer’s Disease can be worsened by oxidative stress (Flynn & Melov, 2013; Leuner et al., 2012). Parts of the ETC, specifically complex I, functionally deteriorate with age, and complex I-derived ROS can trigger formation of amyloid-beta plaques, key pathological features in Alzheimer’s Disease. One study found an increase in amyloid-beta plaques in vitro within just 2 hours of complex I dysfunction and ROS release (Leuner et al., 2012). Additionally, another study found that post-mortem samples of Alzheimer’s Disease patients had increased oxidative stress markers (Sultana et al., 2006). This evidence could imply that oxidative stress is a factor in inducing Alzheimer’s Disease, but it could also suggest that Alzheimer’s pathology itself leads to an increase in oxidative stress. Similarly, SOD2 is upregulated early on in the progression of Alzheimer’s Disease, but it is unknown if SOD2 is directly related to Alzheimer’s Disease progression or if it is upregulated in response to an increase in oxidative stress (Flynn & Melov, 2013). Still, antioxidants may play a role in preventing Alzheimer’s Disease progression. One study found amyloid-beta plaque formation was significantly suppressed after treating cells with deficient complex I using the antioxidant vitamin C (Leuner et al., 2012).  

While antioxidants are found throughout our bodies, the Free Radical Flux Theory posits that there may not be sufficient amounts to clear all the ROS that are produced during periods of high activity, like wakefulness, that then accumulate (Reimund, 1994). Depleted levels of antioxidants have been reported during periods of extended wakefulness, along with increases in oxidative stress (Trivedi et al., 2017). Considering this evidence, an additional process to clear ROS, characterized by periods of little activity, may be necessary to prevent oxidative stress. That process could be sleep (Reimund, 1994). 

1.4 Reactive oxygen species and sleep  

If sleep is important for clearing ROS, then it could be that with sleep loss comes the inability to protect against oxidative stress. A study examining sleep and oxidative stress found that women under mild sleep restriction for 6 weeks had a 78% increase in oxidative stress when measuring oxidative stress markers in endothelial cells (Shah et al., 2023). This protective effect of sleep against oxidative stress was also found in flies. Short-sleeping fruit flies exhibited an increase in sensitivity to oxidative stress such that when exposed to H2O2 and paraquat, a chemical known to cause oxidative stress, the flies had significantly shorter survival (Hill et al., 2018). Additionally, increasing sleep in flies through genetic and pharmacological methods caused a greater resistance to oxidative stress, resulting in higher survival rates when exposed to H2O2 and paraquat (Hill et al., 2018). Also, ROS accumulated only in the gut of the fly after sleep deprivation, suggesting the oxidative stress response may be tissue specific (Vaccaro et al., 2020). Increases in oxidative stress and lipid peroxidation, a consequence of oxidative stress, were also found in rats after sleep deprivation (Silva et al., 2004). Interestingly, sleep deprived rats who had increased oxidative stress also displayed deficits in memory, as shown by reduced avoidance of an environment with a learned association to an aversive shock stimulus (Silva et al., 2004). Such evidence suggests oxidative stress as a possible mechanism for how sleep deprivation impairs cognitive processes like memory.  

With the connection between sleep and oxidative stress, it is no surprise that sleep is also related to the expression of oxidative stress response genes. Short-sleeping flies showed an increase in expression of antioxidants and mitochondrial stress genes (Hill et al., 2018). The same findings were also seen in mice under sleep deprivation for 72 hours (Lungato et al., 2013). However, another study found antioxidant levels were decreased in rats after 5 days of sleep deprivation (Everson et al., 2005). This inconsistency illustrates the fact that expression of oxidative stress response genes may be time dependent, as opposing effects on expression can be seen after differing lengths of sleep deprivation.  

In addition to oxidative stress genes, sleep can also affect expression of ER stress genes, which respond to accumulation of misfolded proteins within the ER. During periods of sleep loss, ER stress genes, such as binding immunoglobulin protein (BiP), are upregulated (Naidoo et al., 2007). Additionally, transgenic flies that overexpressed BiP had increased rates of recovery sleep following sleep deprivation, suggesting expression of ER stress genes may play a role in regulating sleep (Naidoo et al., 2007). However, considering the role of ROS in generating ER stress, ROS may be the underlying mechanism behind this association between sleep and expression of ER stress genes (Brown & Naidoo, 2010; Malhotra et al., 2008). It is possible that during sleep deprivation, ER stress is activated by ROS that has not been cleared due to sleep loss, and the same ROS accumulation that activates ER stress and causes ER stress gene overexpression could increase recovery sleep.  

1.4.1 Reactive oxygen species as a regulator of sleep homeostasis 

Considering sleep is protective against oxidative stress, ROS can act as a regulator of sleep such that as ROS levels fluctuate, so does the need for sleep. In flies with neuronal overexpression of antioxidant genes SOD1 and SOD2, sleep duration was significantly shorter, suggesting a lesser need to clear ROS (Hill et al., 2018). Another study found rats injected with low levels of tert-butyl hydroperoxide (TBHP), a reagent known to promote ROS production, had significantly increased sleep (Ikeda et al., 2005). Interestingly, the levels of TBHP used were enough to induce sleep without causing oxidative damage (Ikeda et al., 2005). This finding supports the concept that ROS, as a molecule found naturally within organisms, can be nonpathological at low levels and may play an important role in signaling when and how much sleep is needed. Considering ROS as a molecule to regulate sleep could offer a mechanistic explanation for how TBI and sleep impact each other. If ROS acts as a regulator of sleep and a consequence of sleep loss, and sleep can impact TBI recovery, it is possible that ROS is directly correlated with TBI outcomes. 

1.5 Reactive oxygen species and traumatic brain injury 

TBI has been found to induce oxidative stress. In one study examining humans with TBI, markers for oxidative stress found in cerebral spinal fluid were 9 times higher than that of uninjured patients (Bayir et al., 2002). Rats with TBI also exhibited increased oxidative stress markers at 6, 24, and 72 hours after TBI delivery (Tyurin et al., 2000), indicating the effect of TBI on ROS is conserved. Interestingly, another study in humans found the concentration of serum free thiols, an antioxidant and known biomarker of oxidative stress, to be reduced in TBI patients within 24 hours of receiving the injury (Visser et al., 2022). However, this reduction could be the result of depleted antioxidant stores due to more ROS needing to be cleared. Interestingly, serum free thiol concentration was also associated with worse recovery at 6 months post-injury, with higher oxidative stress levels in patients with incomplete recovery than those who recovered completely (Visser et al., 2022). This finding suggests higher ROS levels may be detrimental to TBI recovery.  

There are numerous ways that TBI can impact ROS production. TBI can disrupt metabolic homeostasis, increasing the metabolic rate and in turn producing more ROS (Fesharaki-Zadeh, 2022; Lee & Oh, 2022). With ischemia, a possible secondary injury of TBI, an inadequate blood supply prevents proper amounts of oxygen from reaching damaged parts of the brain. Lack of proper oxygen supply disrupts the ability for mitochondria to produce adenosine triphosphate (ATP), the primary energy source for cells, resulting in mitochondrial dysfunction and the excessive production of ROS (Andrabi et al., 2020). Widespread release of the excitatory neurotransmitter glutamate as a result of the physical impact of a TBI can also cause ROS production (Eastman et al., 2020). Excessive glutamate can induce neurotoxicity, causing a major influx of calcium ions into neurons and overloading neuronal mitochondria with calcium (Peng et al., 2019). This mitochondrial overload leads to the generation of excess amounts of ROS in an injured brain (Peng et al., 2019).  

TBI also has the potential of dysregulating endogenous antioxidant mechanisms as a way of inducing ROS increase. In fruit flies given TBIs, a known oxidative stress response gene, GstD2, was found to be upregulated (Katzenberger et al., 2016). However, antioxidant levels in human TBI patients were found to be depleted in the days after injuries occurred (Bayir et al., 2002). This finding was supported by another study done in rats, which found depleted antioxidant levels immediately after TBI, however normal antioxidant levels returned 72 hours after injury (Tyurin et al., 2000). It is possible that endogenous levels of antioxidants are decreased for a period of time as a result of neutralizing higher levels of ROS than normal. The excessive production of ROS after TBI along with possible temporary depletion of the antioxidant systems necessary to remove ROS can lead to further problems, such as the breakdown of the blood-brain barrier, sensorimotor deficits, and chronic inflammation as a result of cellular and tissue damage induced by oxidative stress (Fesharaki-Zadeh, 2022).   

Considering the cellular havoc that can be caused by ROS and oxidative stress – and the risks associated with both TBI and sleep disruptions – it is important to consider how ROS may play a mediating role in the relationship between TBI and sleep. Investigating this relationship could lead to a better understanding of how sleep impacts TBI outcomes and the importance of sleep in TBI prevention and recovery. However, studying these questions in humans poses a challenge as human TBIs can only be ethically studied after they occur naturally.  

Instead, we can use Drosophila melanogaster, also known as the common fruit fly, to experimentally address these questions in a controlled manner. Fruit flies have been well established as models of human diseases. About 65% of all known genes related to diseases in humans exist in flies as functional homologs (similar versions of a gene) (Ugur et al., 2016). Because of this, we can use flies to 2study human diseases more precisely than is possible in humans, by examining the effect of gene manipulation on survival and behavior. Decades of using flies as model organisms provides us with a large number of assays and tools to study correlates of human diseases and behaviors, including TBI and sleep (Hendricks et al., 2000; Katzenberger et al., 2013; Ugur et al., 2016).  

1.6 Modeling traumatic brain injury in D. melanogaster 

There are many different models that have been developed to deliver TBIs to fruit flies  (Barekat et al., 2016; Behnke et al., 2021; Katzenberger et al., 2013). Some models deliver a head-specific injury, which can be useful for ensuring that any injury related phenomenon is a result of injury to the brain specifically (Behnke et al., 2021). Other models deliver full body injuries, which may offer a more realistic injury paradigm similar to those experienced by humans, since forces that cause human TBI are rarely delivered to the head only (Barekat et al., 2016; Katzenberger et al., 2013).  

In this study, I use a modified model of the high-impact trauma (HIT) device developed by Katzenberger et al. (2013) as a way of delivering full body TBIs to many flies simultaneously (Figure 3). In this model, flies are subjected to a repeated number of strikes to achieve injury through rapid acceleration and deceleration within a vial.  

We can use fruit flies to model TBI in mammals because many injury outcomes seen in mammalian TBI models are reproducible in flies. Some of these characteristics include acute death, shortened lifespan, and decline in locomotor activity. In flies that received a TBI with the HIT device, acute mortality was increased and flies had a significantly shorter lifespan and worse locomotor impairment (Delventhal et al., 2022; Katzenberger et al., 2013). In other studies that looked at head-specific delivery of repeated mild TBI, injured flies showed a smaller increase in acute mortality, indicating TBI severity was indeed mild (Behnke et al., 2021). However, they still found significant deficits in lifespan and long-term locomotor ability in injured flies (Behnke et al., 2021). Additionally, other TBI-induced pathologies found in humans that are reproducible in flies include increased sensitivity to TBI in older flies, activation of the innate immune pathway, increased phosphorylation of Tau protein, and immediate ataxia (Barekat et al., 2016; Corrigan et al., 2014; Friedan et al., 2015; Kalmbach et al., 2018; Katzenberger et al., 2013). Injured flies also show increases in vacuolization, which is used as a measure of whole-brain neurodegeneration (Behnke et al., 2021; Bower et al., 2003; Fleminger, 2003). 

Like in humans, TBI has also been found to cause sleep disruption in flies. Mild TBI given to flies caused sleep-maintenance insomnia, or more fragmented sleep, one week after injury (Barekat et al., 2016). Also after injury, flies showed alterations in their circadian locomotor activity, where injured flies showed no rhythmic activity pattern (Barekat et al., 2016).  

1.7 Modeling sleep in D. melanogaster 

Like TBI, we can also use flies as a model to study sleep. Although sleep is found in nearly all organisms, the behavior can look different across the animal kingdom (Cirelli & Tononi, 2008). A study by Hendricks et al. (2000) proposed a list of criteria for an inactive state in flies to be considered sleep-like or synonymous to sleep in humans. The criteria included rhythmically controlled periods of uninterrupted immobility, a posture or resting place specific to a species, a higher difficulty becoming aroused or alert, and a homeostatic regulated period of rebound sleep in response to longer periods of wakefulness (Hendricks et al., 2000).  

These criteria can be seen fulfilled when considering the sleeping behavior of other animals, including humans. A circadian pacemaker rhythmically controls the timing and consolidation of our sleeping periods, sending signals to initiate sleep at night and to promote wakefulness during the day (Dijk et al., 1999). Humans take up a typical horizontal position when sleeping, with the most common position being sleeping on our sides (Skarpsno et al., 2017). While we sleep, we become less responsive to stimuli unless they reach above our arousal threshold (Berry & Gleeson, 1997). Arousal stimuli can range from external, like an alarm clock, to internal, such as lack of oxygen in individuals with sleep apnea (Berry & Gleeson, 1997). Finally, during long periods of wakefulness, our homeostatic regulator builds up a higher sleep pressure and we subsequently exhibit longer sleep duration in the form of rebound sleep to make up for the sleep loss (Dijk et al., 1999).  

Detailed observations of resting behavior in flies indicated that flies met these same criteria for a sleep-like state. The number of resting flies at a time followed a rhythmic pattern, with a peak during the subjective night and an increase in activity at the start of the subjective day. Resting flies took up a specific posture, usually turning away from their food supply in a prone or supported position, and had small, sporadic movements of their proboscis, abdomen, or extremities that were not recognized as a patterned motor behavior. Resting flies were not disturbed by awake flies that ran into them and were not awoken by light mechanical stimulus created by tapping on the vial, indicating a higher arousal threshold. Finally, after a period of sleep deprivation, the number of sleeping flies increased. Evidence of rebound sleep in flies after periods of sustained wakefulness suggest the presence of a homeostatic regulatory mechanism in flies (Hendricks et al., 2000).  

Such observation of flies’ resting behavior showed all criteria were fulfilled to consider rest in flies a sleep-like state. With a circadian sleep and activity pattern, characteristic resting posture, decreased responsiveness to stimuli, and rebound sleep after deprivation, sleep in flies can be considered analogous to that of mammals (Hendricks et al., 2000). In addition to meeting the criteria for a sleep-like state, flies also showed similar responses to stimulants as seen in humans (Hendricks et al., 2000). Sleep decreased after flies consumed caffeine, and feeding flies an adenosine agonist, cyclohexyladenosine (CHA), increased their sleep (Hendricks et al., 2000). Numerous studies have used flies to examine sleep behavior and sleep related phenomenon, solidifying flies as a model organism to study sleep (Allada et al., 2017; Koh et al., 2006; Robertson & Keene, 2013).  

1.7.1 A genetic basis for sleep in D. melanogaster 

Certain genes in the fly genome have been identified as sleep regulating genes. Forward genetic screens have identified a series of fly mutants that display shortened sleep duration while maintaining their circadian rhythm. For example, a loss-of-function mutation of the gene sleepless has been shown to decrease sleep in flies by dysregulating nicotinic acetylcholine receptors (nAChRs) and the potassium ion channel Shaker (Wu et al., 2014). Similarly, mutations of the genes fumin, an allele for the Drosophila dopamine transporter, and redeye, which encodes for a nAChR subunit, also result in shortened sleep in flies (Kume et al., 2005; Shi et al., 2014).  

Flies with a mutation of the gene insomniac (inc) also display a shorter sleep duration (Stavropoulos & Young, 2011). Inc encodes for an adaptor of the Cullin-3 ubiquitin ligase complex, which is an important part of the protein degradation process (Stavropoulos & Young, 2011). When using a knockout mutation, where the gene is eliminated from the genome, one study found inc mutant flies had a reduction of sleep to less than 350 minutes per day compared to more than 900 minutes per day in control flies (Stavropoulos & Young, 2011). The study also found that when using an RNAi knockdown of inc, where expression of the gene is just reduced, sleep was shortened to the same degree as knockout mutants only when inc was knocked down in the neurons of flies (Stavropoulos & Young, 2011). Additionally, restoring inc function in knockout mutants subsequently rescued normal sleep-wake behavior, suggesting proper inc function is necessary for normal sleep duration. Importantly, no other morphological or behavioral changes in inc knockdown mutants were found, indicating the inc gene is only involved in sleep regulation (Stavropoulos & Young, 2011). Manipulation of the expression of sleep regulating genes like inc has been used in previous literature to manipulate sleep and study the effect of sleep loss in flies (Hill et al., 2018).   

1.8 The current study 

The currently study aims to use D. melanogaster as a model to investigate the role of oxidative stress in the potential bidirectional relationship between TBI and sleep. This study also aims to examine how antioxidants could be protective or detrimental to TBI recovery and sleep following injury.  

To do this, I first looked at how TBI affected sleep homeostasis over time by measuring changes in sleep phenotypes across different time points after TBI delivery. I measured sleep duration (total time spent asleep) and sleep fragmentation (frequency and length of sleep bouts) in flies with and without TBI. Based on previous data in both humans and flies (Barekat et al., 2016; Imbach et al., 2015; Kalmbach et al., 2018; Verma et al., 2007), I expected to see significant changes in sleep homeostasis in injured flies, including sleep fragmentation and sleep duration. Changes in sleep after TBI would indicate that TBI-induced damage disrupts important homeostatic regulators of sleep. Indeed, I found that in the short-term, at 48 hours and 1 week post-TBI, flies’ sleep was more fragmented. However, in the long-term, 2 and 4 weeks post-TBI, flies progressed to seeping more, suggesting that sleep disruption after TBI changes over time.  

To further examine the potential bidirectional relationship between TBI and sleep, I looked at whether sleep has a protective or detrimental effect on TBI outcomes. To accomplish this, I gave a TBI to short-sleeping flies with a genetic neuronal knockdown of the sleep-regulator gene inc (Figure 6A) (Stavropoulos & Young, 2011). After TBI delivery, I measured acute mortality as a readout of TBI recovery. To our knowledge, no previous research has investigated how manipulating sleep prior to TBI affects recovery in flies. However, studies on humans show that sleep problems prior to TBI resulted in worse recovery after injury (Kalmbach et al., 2018). Therefore, I expected to see worse TBI recovery in short-sleeping flies, represented by higher acute mortality. Indeed, I found acute mortality after TBI to be significantly higher in short-sleeping flies. Higher acute mortality in short-sleeping flies suggests sleep has a protective effect against TBI consequences while sleep loss is detrimental to TBI recovery.  

Finally, to investigate how oxidative stress and ROS accumulation may play a role in TBI recovery and if TBI-related oxidative stress contributes to the changes in sleep homeostasis we see after injury, I manipulated the expression of the antioxidant gene SOD2 in the neurons of flies. Genetic knockdown and overexpression of SOD2 were used as a method of increasing and decreasing ROS levels, respectively. In using this method, we assume that a decrease in neuronal SOD2 expression would increase ROS levels in the brain and overexpression of neuronal SOD2 would decrease ROS (Figure 7A,8A). I hypothesized that flies with neuronal SOD2 knockdown would have worse TBI outcomes and would sleep more as a result of a heightened need to clear accumulated ROS. I also predicted that flies with neuronal SOD2 overexpression would have better recovery from TBI and would sleep less due to lower ROS levels. Data supporting these theories would suggest that oxidative stress is detrimental to TBI recovery and is the molecular basis for why TBI causes sleep disruption in injured flies. Such data would also show that antioxidants are protective against TBI and prevent sleep disruption from occurring after injury. However, I found that SOD2 overexpression was detrimental against TBI outcomes while SOD2 knockdown showed no significant effect. I also found that altering SOD2 expression had a disruptive effect on sleep. While these results did not align with what I predicted, they do suggest that neuronal expression of SOD2 and the levels of ROS in the brain have an influence on TBI outcomes and sleep.  

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.